Dissertations / Theses on the topic 'Mungbean (Vigna radiata L.)'

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9800号
農博第1295号
新制||農||854(附属図書館)
学位論文||H14||N3722(農学部図書室)
UT51-2003-B340
京都大学大学院農学研究科地域環境科学専攻
(主査)教授 櫻谷 哲夫, 教授 堀江 武, 教授 泉井 桂
学位規則第4条第1項該当

Context: Approximately one billion people live in semi-arid and arid regions, and of these about 40% live on less than a dollar a day. Legumes crops are an important component of sustainable agriculture, but they are often grown under intermittent or terminal droughts. Thus, improving drought tolerance in legumes has the potential to improve food security and sustainability of agricultural systems. Objective: This study compares the response of two legume crops, common bean (Phaseolus vulgaris L.) and mungbean (Vigna radiata (L.) Wilczek), to water stress conditions under field and controlled-environment conditions. Methods: Field experiments were conducted in the Fergana valley, Uzbekistan, and controlled environment experiments were conducted at the Macdonald campus of McGill University, Ste-Anne-de-Bellevue, Canada, and at the Hermitage Research Station in Warwick, Australia. Results: Our results demonstrate that alternate furrow irrigation maintains yields, yet decreases water applied by 25%. In addition, mungbean showed the highest yields in the moderate deficit irrigation treatment in 2003 and severe deficit irrigation treatment in 2004 under field conditions in Uzbekistan. Common bean also showed a capacity to maintain yields under moderate deficit irrigation in both years. Further characterization of the legume responses to water deficit stress in controlled-environment experiments indicated that mungbean’s higher tolerance is attributable to higher transpiration efficiency, a more conservative water use in the vegetative stage, and a higher root-to-shoot ratio when compared to common bean. Root characteristics might also play an important role, although we have observed a large variability between genotypes. An additional field experiment in Uzbekistan demonstrated that an early maturing Canadian soybean cultivar could be grown after the harvest of winter wheat, and thus contribute to food security. It has also been demonstrated that i
Contexte : Environ un milliard de personnes vivent dans les régions arides et semi-arides, et 40% d’entre eux vivent avec moins d’un dollar par jour. Les cultures de légumineuses sont une part importante de l’agriculture durable, mais ces cultures sont souvent produites dans des conditions de sécheresse intermittente ou terminale. Améliorer la tolérance des légumineuses à la sécheresse peut donc augmenter la sécurité alimentaire et la durabilité des systèmes agricoles. Objectif : Cette étude compare la réponse de deux légumineuses, le haricot commun (Phaseolus vulgaris L.) et le haricot doré (Vigna radiata (L.) Wilczek), lors de conditions de stress hydrique imposées au champ et en environnement contrôlé. Méthodes : Les expériences au champ furent conduites dans la vallée de Fergana, en Ouzbékistan, et les expériences en environnement contrôlé furent conduites au campus Macdonald de l’Université McGill, à Ste-Anne-de-Bellevue, Canada, et à la station de recherche Hermitage, à Warwick, Australie. Résultats : Nos résultats démontrent que l’irrigation alternante maintient les rendements tout en diminunant de 25% l’apport en eau. De plus, le haricot doré a démontré les meilleurs rendements sous les traitements d’irrigation déficitaire moyen en 2003 et sévère en 2004 dans les expériences au champ. Le haricot commun a également démontré la capacité de maintenir ses rendements sous traitement d’irrigation déficitaire moyen les deux années. Une caractérisation plus poussée de la réponse des légumineuses au stress hydrique en milieu contrôlé a indiqué que la tolérance accrue du haricot doré est attribuable à une meilleure efficacité transpirationelle, une utilisation plus limitée de l’eau lors de la période végétative, et une proportion plus élevée de biomasse en racines lorsque comparé au haricot commun. Les caractéristiques racinaires semblent également jouer un rôle important,$

This research explores population genetics for the bacterial pathogen Pseudomonas savastanoi pv. phaseolicola, which causes halo blight disease in its host mungbeans (Vigna radiata). The pathogen and host populations were investigated at a board scale using field and glasshouse studies and in detail using molecular biology techniques including qPCR and next-generation sequencing. The study found both the bacterial pathogen and host (mungbean) to have highly conserved genetic backgrounds. This will make it easier for breeders to target critical resistance genes to prevent the infection of the halo blight pathogen in future cultivars.

Eight-day-old mung bean seedlings (Vigna radiata L.) grown in hydropondic medium were osmotically stressed by exposing roots to increasing concentrations of NaCl up to 4 bars. They were transferred after 16 to 18 hours to a similar solution containing tritiated water (THO). Periodic samples were taken of water transpiring from the leaves and of tissue water obtained from the same leaves, frozen and ground; specific radioactivity was determined by a scintillation counter. Proportional to increasing stress, the labelling of tissue water was increasingly delayed, the time for equilibration of the specific radioactivity in the two fractions lengthened, and equilibration occurred at higher concentrations of THO. Thus stress causes transpirational water to be increasingly restricted to extra-cellular pathways. Further investigations of stomatal function by leaf surface, of anatomy and of growth patterns were unsuccessful in finding an explanation for this behavior but did reveal a transpirational circadian rhythm and a continual layer of (air?) space between the palisade and spongy mesophyll, the latter organized into two compact rows.

Orientador: Aureluce Demonte
Banca: Rosiane Gomes Silva Oliveira
Banca: Thais Milena de Souza Bezerra
Banca: Tais Maia Bauab
Banca: Juliana Cristina Bassan
Resumo: A homologia entre as características estruturais, sequenciais e funcionais da β- conglicinina da soja em relação a outras vicilinas de leguminosas e a ação de seus peptídeos bioativos nos estimulou a investigar a atividade da globulina vicilina (8S) do feijão mungo verde como composto funcional. Objetivo: isolar e caracterizar a globulina 8S do feijão mungo verde e verificar a ação hipocolesterolêmica e antimicrobiana de seus hidrolisados em testes in vitro. Método: a proteína majoritária 8S do feijão mungo verde foi extraída a partir do método de isolamento, em seguida foi parcialmente purificada por cromatografia de permeação molecular, determinada sua massa molecular a partir da eluição em Coluna Sephadex G-200 e suas subunidades foram caracterizadas por SDS Page. A proteína purificada foi submetida à hidrólise enzimática sequencial com pepsina-pancreatina e o hidrolisado obtido caracterizado por permeação molecular e SDS Tricina. Diferentes frações provenientes da eluição do hidrolisado em Sephadex G-25 foram testadas quanto à inibição da HMG CoAr (3 - Hidróxi - 3 - metilglutaril - CoA redutase) e de microrganismos (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 e Helicobacter pylori ATCC 43504). Resultados: A 8S do feijão mungo verde é composta por polipeptídeos de 26, 29, 48 e 61 kDa e sua massa molecular é de 158,23±10 kDa, características condizentes com as vicilinas de outras leguminosas. As frações obtidas da eluição do hidrolisado em Sepahadex G-25... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The homology between the structural and functional characteristics of sequential β- conglycinin soy compared to other legume vicilin and the action of their bioactive peptides stimulated us to investigate the activity of globulin vicilin (8S) mung beans as functional compound. Objective: isolate and characterize the 8S globulin mung beans and check the hypocholesterolemic and antimicrobial activities their hydrolysates in in vitro tests. Method: The major protein 8S mung bean is extracted from the isolation method, then was partially purified by molecular permeation chromatography, its molecular weight determined from the elution Sephadex G- 200 and its subunits were characterized by SDSPAGE. The purified protein was subjected to sequential enzymatic hydrolysis with pepsin-pancreatin and the hydrolyzate characterized by molecular permeation and Tricine SDS. Different fractions from the hydrolyzate eluted on Sephadex G-25 were tested for inhibition of HMG CoAr (3 - hydroxy - 3 - methylglutaryl - CoA reductase) and microorganisms (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Helicobacter pylori ATCC 43504). Results: 8S mung bean is comprised of polypeptides of 26, 29, 48 and 61 kDa and its molecular weight is 158.23 ± 10 kDa, consistent with the characteristics vicilins from other legumes. The fractions obtained from the elution hydrolyzate in Sepahadex G-25 (10, 12, 14, 22 and 32) demonstrated significant in vitro inhibition of HMG-CoAr enzyme activity compared with the recognized hypocholesterolemic action drug (pravastatin), suggesting involvement of these peptide fractions in inhibiting an important step in cholesterol synthesis. The hydrolyzate obtained from sequential hydrolysis with pepsin-pancreatin presented antimicrobial activity against the tested microorganisms, particularly against H. pylori, is a promising advance in... (Complete abstract click electronic access below)
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O uso de adubos verdes constitui em alternativa viável para ser utilizado pelos agricultores que produzem em sistema orgânico, dado os grandes benefícios que a prática oferece. Dois experimentos foram conduzidos na Fazenda Experimental Rafael Fernandes, no distrito de Alagoinha, zona rural de Mossoró-RN, no período de agosto de 2016 a março de 2017, com o objetivo de estudar adubação verde com feijão mungo na viabilidade agroeconômica da hortelã. O delineamento experimental utilizado foi o de blocos completos casualizados em esquema fatorial 4 x 2, com três repetições. O primeiro fator foi constituído pelas diferentes densidades de semeadura do feijão mungo (50; 100; 150 e 200 plantas m-2) e o segundo fator pelas formas de manejo da biomassa do feijão mungo (incorporado e em cobertura). O cultivar da hortelã utilizada foi a Mentha piperita. As características avaliadas para esse vegetal em primeiro cultivo e na rebrota foram as seguintes: altura da biomassa, massa fresca, número de molhos, massa seca, teor e rendimento de óleo. Também foram utilizados indicadores econômicos, tais como: custo de produção, renda bruta, renda líquida, taxa de retorno e índice de lucratividade. No primeiro cultivo, a densidade de semeadura de 150 plantas m-2 contribuiu para o maior incremento na massa fresca e número de molhos com valores de 2.508,3 g m-2 e 30,8 unidades m-2 respectivamente, já para o rendimento de óleo a densidade de 200 plantas m-2 obteve um melhor resultado com valor e 1,24 g m-2. Em relação às formas de manejo (incorporado e em cobertura) não houve diferença estatística para massa fresca, número de molhos e rendimento de óleo. Para a rebrota da hortelã, a densidade de semeadura de 50 plantas m-2 de feijão mungo acarretou o maior incremento nas características de massa fresca e número de molhos, com valores de 922,6 g m-2 e 9,2 unidades m-2, mutuamente. Para o rendimento de óleo (0,78 g m-2), a melhor densidade de semeadura foi de 100 plantas m-2 de feijão mungo. Em relação às formas de manipulação (incorporado e em cobertura) observou superioridade da forma de manejo em cobertura para massa fresca, número de molhos e rendimento de óleo. A melhor eficiência econômica foi observada na densidade de semeadura 200 plantas m-2 com renda bruta (R$ 4.579,80), renda líquida (R$ 2.543.58) e índice de lucratividade (54,9 %). Para a taxa de retorno (R$ 2,70) na densidade de 150 plantas m-2. Nas formas de manejo da biomassa (incorporado e em cobertura), observou diferença estatística somente para a taxa de retorno na densidade de 150 plantas m-2, com valores de R$ 2,7 e 2,2, respectivamente. A utilização do feijão mungo mostrou eficiência econômica como adubo verde no cultivo da hortelã.
The use of green fertilize constituted in a viable alternative to be utilized by farmers that produce on organic system, due to the great benefits which the practice offers. Two experiments were conducted at experimental Rafael Fernandes Farm, in Alagoinha district, Mossoró-RN countryside, in the period from August 2016 to March 2017, with the aim to study green fertilizer with bean mung in agroeconomical of mint. The experimental delineatment utilized was complete blocks causalized on factorial scheme 4 x 2, with three repetitions. The first factor was constituted by differents sowing densities of bean mung (50; 100; 150 and 200 plants m-2) and the second factor by forms of management of biomass bean mung (incorporated and on covering). The cultivar of mint utilized was the Mentha piperita. The characteristics evaluated for the mint in the first cultivation and in the regrowth were as follow: biomass hight, fresh mass, number of sauces, dry mass, substance and income oil. We also utilized economic indicators, such as: cost production, gross income, net income, return rate and profitability index. In the first cultivation, the sowing density of 150 plants m-2 contributed for the major increase in the fresh mass and number of sauces, with values of 2,508.3 g m-2 and 30.8 units m-2 respectively, about the yield of oil, the density of 200 plants m-2, it got a best result with value of 1.24 g m-2. In concern the management forms (incorporated and in covering), there wasn’t statistical difference to fresh mass, number of sauces and income oil. For the regrowth of mint, the sowing density of 50 plants m-2 of bean mung caused the major increase in the characteristics dry mass and number sauces with values of 922.6 g m-2 and 9.2 units m-2 respectively. For oil income (0.78 g m-2), the best sowing density was 100 plants m-2 of bean mung. In concern management forms (incorporated and on covering) we observed superiority of management form on covering for fresh mass, number of sauces and oil income. The best economical efficiency was observed on sowing density 200 plants m-2 with gross income (R$ 4,519.80), net income (R$ 2,543.58) and profitability index (54.9 %). For rate return (R$ 2.70) on density of 150 plants m-2. In the management forms of biomass (incorporated and in covering), observed only statistical difference to a rate return on density of 150 plants m-2, with values of R$ 2.7 and 2.2, respectively. The utilization of bean mung showed economical efficiency like green fertilizer in the cultivation of mint.

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Uso de leguminosas como adubo verde é uma prática bastante consolidada, pelo fato de estar adicionando ao solo material vegetal rico em nitrogênio. O experimento foi conduzido na Fazenda Experimental Rafael Fernandes, no distrito de Alagoinha, zona rural de Mossoró-RN no período de agosto de 2015 a janeiro de 2016, com o objetivo de avaliar a Presença e ausência do feijão mungo sob doses de esterco bovino na viabilidade agroeconômica da alface. O delineamento experimental utilizado foi de blocos completos casualizados com os tratamentos arranjados em esquema fatorial 4 x 2, com quatro repetições. O primeiro fator foi constituído das doses de esterco bovino (1,0; 2,0; 3,0; 4,0 kg m-2 de canteiro) e o segundo fator foi constituído pela presença e ausência do adubo verde (feijão mungo). A cultivar da alface utilizada foi a “Regina”. Foram avaliadas as seguintes características: altura de planta, número de folhas planta-1, diâmetro da cabeça, produção de alface e massa seca de alface. Foram determinados alguns indicadores econômicos tais como: custo de produção, renda bruta, renda líquida, taxa de retorno e índice de lucratividade dos dois cultivos. Não houve interação entre os fatores-tratamentos para as características de produção, com produção de alface de 87,8 kg/100 m2. Houve diferença estatística no fator presença e ausência do feijão mungo com valores médios de 81,4 e 67 kg/100 m2 de alface, respectivamente. A maior eficiência econômica no cultivo da alface se deu na presença do feijão mungo na quantidade de 3,0 kg m-2, com renda bruta de 3343,75, renda líquida de 1582,40, taxa de retorno de 1,90 e índice de lucratividade de 43,42%. A utilização de feijão mungo no cultivo da alface constitui-se em uma opção viável para ser utilizado pelo agricultor.
The use of legumes as green manure is a well-established practice, because it is adding nitrogen-rich plant material to the soil. The experiment was conducted at the Fazenda Experimental Rafael Fernandes, in the district of Alagoinha, rural area of Mossoró-RN, from August 2015 to January 2016, with the objective of evaluating the Presence and absence of mung bean under doses of bovine manure in the agroeconomic viability of lettuce. The experimental design was a randomized complete block with treatments arranged in a 4 x 2 factorial scheme, with four replications. The first factor consisted of bovine manure (1.0, 2.0, 3.0, 4.0 kg m-2 of bed) and the second factor was the presence and absence of green mango (mung bean). The lettuce cultivar used was the "Regina". The following characteristics were evaluated: plant height, number of plant-1 leaves, head diameter, lettuce production and lettuce dry mass. Some economic indicators were determined such as: production cost, gross income, net income, rate of return and profitability index of the two crops. There was no interaction between the treatment factors for the production characteristics, with lettuce production of 87.8 kg / 100 m2. There was a statistical difference in the presence and absence of mung beans, with mean values of 81.4 and 67 kg / 100 m2 of lettuce, respectively. The highest economic efficiency in lettuce cultivation occurred in the presence of mung beans in the amount of 3.0 kg m-2, with gross income of 3343.75, net income of 1582.40, rate of return of 1.90 and index of profitability of 43.42%. The use of mung beans in lettuce cultivation is a viable option for the farmer to use.

碩士
國立臺灣海洋大學
食品科學系
95
Starch is the major carbohydrate reserve in higher plants. In the starch biosynthetic pathway, starch phosphorylase (EC 2.4.1.1, SP) catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Mungbean (Vigna radiata L.) starch is the most special that amylose content was 15-30% higher than normal level, which implied that SP plays an important role in amylose synthesis. Previous studies have identified the tryptic fragments of mungbean 105-kDa SP by MALDI-TOF and conducted immunological analysis. The objective of this study was to further clone the full-length cDNA of SP in order to investigate the characteristics and function of recombination SP in the future. Based on the internal amino acid sequence of N- and C-terminal of mungbean SP, degenerate primers were designed as gene-specific primers (GSP). The developing mungbean seed (Vigna radiata cv. Tainan no.5) was used as the material to extract its total RNA, and mRNA was purified and used as the template in RT-PCR. First, a 1819 bp sequence of a middle fragment was obtained, and then primers were designed from its internal sequence. The 5’-nucleotide conserved region sequences from fave bean, sweet potato and potato were used to design primers which were coupled with internal GSPs for RT-PCR amplification. Sequences containing the start codon ATG of 5’ 640 bp and 3’ sequences containing the stop codon TAG of 997 bp were obtained successfully. The full-length cDNA of the mungbean SP which possessed nucleotides of 2961 bp in length, containing complete open reading frame (ORF) that covers from start codon to stop codon was designated as Vrsp. Vrsp encodes a polypeptide of 987 amino acids with predicted molecular mass of 111 kDa and pI of 5.38. Putative protein sequences possess the motifs of starch binding site, L-78, catalytic site and pyridoxal phosphate (PLP) binding site. In addition, Vrsp includes the L-78 insertion sequence, it belongs to the known L-type isoform.

碩士
國立臺灣海洋大學
食品科學系
100
Starch phosphorylase (SP; EC 2.4.1.1) is one of the enzymes for starch metabolism. The aims of this study were to optimize the expression of a potential recombinant mungbean (Vigna radiata) sp clone (pMAL-c2X-Vrsp-modify) in the E. coli system, and to investigate the enzymatic characteristics of the recombinant SP (rVrSP) in the synthesis direction. Items include stable product formation condition, enzyme kinetics, and analysis of the enzyme products. Evaluation of the optimal expression condition for rVrSP showed that the cells grown at 37℃ until A600nm 0.6, then induced with 0.6 mM IPTG for 6 hrs at 20℃, the molecular size of the amount of the soluble MBP-rVrSP protein of 150-kDa was achieved. In situ zymogram staining of the MBP-rVrSP extract detected the activity of glucan synthesis, and the soluble enzyme was active at 4℃ for 30 days. Analysis of the 150-kDa band from either SDS-PAGE or from the activity gel slice of in situ staining, matched with gi|2506470 (α - 1,4 glucan phosphorylase L isozyme) protein sequences of Vicia faba by LC/MS/MS. The optimal synthesis activity of the MBP-rVrSP extract in gel or in vitro solution were tested at different conditions to react for 2 hrs, and to detect glucan content by iodine reagent. It showed that enzyme is most active at pH 6.0 under 30℃ when reaction solution containing 0.8% or when the gel matrix containing 1.2% glycogen. The stable product formation is observed at 20-30℃. The steady-state kinetic parameters were determined in the 50 μg soluble MBP-rVrSP extract that the apparent Km and Vmax values for G-1-P are 5.5 mM and 0.3 A600nm, respectively. When 50 mM G-1-P was reacted with 500 μg soluble MBP-rVrSP extract at 1, 2, 3, 4, 5 and 6 hrs at 30℃, and the chromatogram of the enzyme products by HPLC showed the peak eluted at an earlier time as reaction 0-3 hrs, and the major products molecular mass is about 4500-4000 Da (DP 28-25). However, as reaction 4-6 hrs, the peak profiles consist of two major products about 3800 Da and 1200 Da, indicating the soluble MBP-rVrSP extract can synthesize and degrade the glucans, and the reaction directions are determined by the balance between the available G-1-P and inorganic phosphorous concentrations.

碩士
中國醫藥大學
營養研究所
92
Abstract Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes in the starch biosynthetic pathway. It catalyzes the formation of branches, which plays a vital role in amylopectin synthesis. The objectives of this thesis are to clone full-length cDNA of SBE and analyze its structure and characteristics. Based on database search, the conserved regions from published SBE genes were obtained and used to design gene-specific primers for cloning. First, the mRNA from developing mungbean (Vigna radiata, cv. Tainan no.5) was extracted and used as template in RT-PCR. The cDNA sequences of the amplified RT-PCR products, after comparing with GCG nucleotide database, demonstrates that partial cDNA of two distinct mungbean SBE isoforms (SBEII and SBEI) were found. Then, primers designed from internal sequences of SBEII and SBEI were used in cloning their 5’ and 3’ cDNA ends by 5’/3’ RACE (rapid amplification of cDNA ends). The full-length cDNAs of the two SBE isoforms were obtained successfully, which possess sequences of 2571bp and 2208bp in length (designated VrsbeII and VrsbeI), respectively. VrsbeII and VrsbeI have also been registered in GenBank with accession numbers of AY622199 and AY667492. They both contain complete open reading frame (ORF) that covers from start codon to stop codon. VrsbeII encodes a polypeptide of 856 amino acids with predicted molecular mass of 97 kDa and pI of 5.47. Whereas, VrsbeI encodes a polypeptide of 735 amino acids with predicted molecular mass of 84 kDa and pI of 6.35. Besides, their putative protein sequences possess the properties of the α- amylase family, including four active conserved region- HSHS/A S, GFRFDG VT, G/AEDVS and AESHDQ, and catalytic (β/α)8-barrel domain. When compared in database, both VrsbeII and VrsbeI showed substantial similarity to the SBEs of kidney bean and pea. Furthermore, the identities between mungbean SBEII and SBEI at nucleic acid and protein levels are 59% and 56%, respectively. The deduced amino acid sequences from VrsbeII and VrsbeI via phylogenetic analysis is evident that they fall into two distinct gene families (family A and B). In conclusion, there are at least two different SBE isoforms involved in starch biosynthesis during the development of mungbean.

碩士
國立臺灣海洋大學
食品科學系
97
Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes in the starch biosynthetic pathway and it plays a vital role in the formation of amylopectin, the main component of the starch molecule. The chemical fine structure changed by SBE would affect the physical property and function of starch. The objective of the thesis was to optimize the expression of a potential recombinant SBEII clone and characterize the active recombinant enzyme to be used in modifying the starch molecular structure. The constructed pET21b-VrSBEⅡwas first transformed into BL21 (DE3) expression host and induced with 0.2 mM IPTG for 5 hours at 37℃, SDS-PAGE analysis showed the molecular size of the recombinant protein was 108 kDa. This rVrSBEⅡwas expressed as a soluble protein with crude enzyme activity of 0.25 U/mg and the activity was enriched 27 fold by Ni-NTA affinity chromatography. To increase the solubility and activity of the enzyme, the clone was further expressed in Origami B host and induced under the same induction condition. Improvement of rVrSBEⅡwas not only the solubility but the crude activity of 0.46 U/mg has increased to 14.9 U/mg after purification. The enzyme was most active at pH 7.0 at 30℃and relatively stable up to 30℃.More than 90% of the original activity remained after incubated at pH 7.0-9.5. When 1 mg/ml amylose substrate was used to react with 6�n�慊 of purified rVrSBEⅡat 30℃, there was a time-dependent decrease of the absorbance at OD660 and became steady after 90 minutes. GPC-HPLC analysis of the enzyme products showed the peak eluted at an earlier elution time as reaction proceeded, indicating the formation of branching and size enlargement by rVrSBEⅡin the product. The present study shows that the pET21b-VrSBEⅡclone was able to be expressed as a biologically functional enzyme in E. coli system and the protein solubility and activity were improved by expressing in Origami B host. Basic characteristics of rVrSBEⅡand the product were established.

碩士
國立臺灣海洋大學
食品科學系
97
Starch phosphorylase ( SP, EC 2.4.1.1 ) is commonly found in plants and is one of the vital enzymes in the starch metabolic pathway. It catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Previous studies have identified the fragments of mungbean 105-kDa SP by MALDI-TOF, conducted immunological analysis and amplified the full-length cDNA fragment of mungbean (Vigna radiata L.) SP (named VrSP). The objective of this study was to clone the sequence into an expression vector and further express into a biological-active recombinant protein in E. coli system. Its 3-D structure and functional features were predicted in silico by SWISS-Model. One matched template, rabbit muscle phosphorylase with potent inhibitor (IWW2A), predicted the N-terminal of VrSP from 92 residue to 411 residue, including partial starch-binding site. The other template, rabbit muscle phosphorylase (2GJ4A), predicted the C-terminal of VrSP from 585 residue to 984 residue, including catalytic site (711-727). VrSP insert was prepared by PCR using primers designed with EcoRI and SalI sites in the flanking region, and was ligated into the parallel sites on pMal-C2X vector, followed by transforming into Novablue cloning host and screening. Insert size of one recombinant plasmid was nearly closed to the expected 2.9 Kb, however after sequencing, 97% of its sequence is matching to the DNA sequences in E. coli cloning host. In addition, the other two major groups of recombinant plasmid had 2.2 kb and 1.4 kb insert fragments in pMal-C2X vector. It showed that the sequence of VrSP itself may cause recombination with the host E. coli chromosomal DNA during the growth of these recombinant clones.

碩士
國立臺灣大學
園藝學研究所
103
Two isogenic lines of mungbean, bruchid-resistant VC6089A and bruchid-susceptible VC1973A, and Sunright mungbean (Sunright Foods Co.) were used as experimental material to study the optimal environmental condition of cultivating mungbean sprout with high phytochemicals contents in the dark. Mungbean seeds were soaked at 20˚C, 26˚C and 32˚C for 0、4、8 and 12 h. Seed dry weight decreased as the soaking time extending to 12 h. Among 4 planting densities, 7 g·cm-2 treatment showed the highest harvest index, longer hypocotyl length and higher total flavonoids content in VC6089A and VC1973A sprouts. Total phenolics content in mungbean sprouts was highly positively related to harvest index (r=0.74***), so did the total phenolics content to total flavonoids content (r=0.77***). Among forcing pressures of 0, 100, 200, 300 and 400 g·51.84 cm-2, VC1973A mungbean sprout cultivated under 400 g·51.84 cm-2 showed shorter and thicker hypocotyl also shorter root. In forcing pressure experiment, correlation coefficient (r) of total flavonoids content to α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP) were 0.82*** and 0.64***, respectively. Among 6 combinations of temperatures based on 16 h/8 h cycle, fresh weights of VC6089A sprouts were the highest at 29˚C/23˚C and 26˚C/26˚C, while Sunright and VC1973A sprouts at 26˚C/26˚C and 29˚C/23˚C, respectively. Soluble solids and soluble protein contents of mungbean sprouts were both affected by the cultivating temperature combinations. VC1973A sprouts has the highest content of total flavonoids and total phenolics than other 2 entries. Comparing to 0, 10 and 25 mM NaCl solution, sprouts cultivated in 15 mM NaCl solution exhibited higher fresh weight and total flavonoids and total phenolics contents than under 0, 10, 20 and 50 mM NaCl treatments. VC1973A sprouts also showed higher Fe2+ chelating ability in 15 mM NaCl treatment. In NaCl experiment, sprout fresh weight was positively related to soluble sugar content (r=0.83***), total flavonoids content (r=0.84***), total phenolics content (r=0.85***) and DPPH scavenging ability (r=0.85***) in NaCl experiment. Contents of total phenolics and flavonoids contents could be reference values of DPPH radical scavenging activity and FRAP. Cultivation sprouts with 0, 10, 25, 50, 75 and 100mM 5 glucose solution, 50 mM treatment could increase ascorbic acid and total flavonoids in mungbean sprouts. Sprouts grown in 100 mM glucose solution had more soluble solid and protein. In conclusion, VC1973A sprouts had higher fresh weight and phytochemicals contents than VC6089A and Sunright sprouts. The mungbean sprout would have higher total flavonoids (0.38 mg·g-1DW) and total phenolics (11.1 mg·g-1DW) contents and better Fe2+ chelating ability (84.8 % of 50 mg DW·mL-1), DPPH scavenging ability (54.1 % of 50 mg DW·mL-1), and FRAP (150.9 µmol FeSO4·g-1DW), and higher fresh weight (54.5 g FW·51.84 cm-2). VC1973A seeds are suggested to be cultivated with 7 g·cm-2 sowing density, 400 g·51.84 cm-2 forcing pressure, 15 mM NaCl solution at 29˚C 16 h/23˚C 8 h in the dark.

博士
國立臺灣大學
植物學研究所
87
Mungbean ( Vigna radiata L. cv Tainan no. 5 ) is a chilling-sensitive plant. The growth of etiolated mungbean seedlings was very slowly at 10 oC in the light. The leaves continued to grow and finally expranded as those grown at 28oC in the light, but they did not turn to green. The development of plastids into chloroplasts in the etiolated leaves was retarded under chilling in the light condition when leaf ultrastructure was examined under T. E. M.. The synthesis of chlorophyll in the leaves were also retarded by 10 oC-chilling treatment. By SDS-PAGE and 2D-PAGE analyses of total leaf proteins, we found that some PS I and PS II proteins decreased dramatically especially in PSAA, PSAB and other thylakoidal proteins. We also have been screened 10 oC-chilling sensitive genes from vcd01 to vcd10. The length of DNA fragment in vcd01 to vcd10 were 4, 3, 2, 7, 4, 6.5,2.1, 5.6 and 6Kb, respectively. These genes included psaC, psbB, psbC, psbD, psbE, psbF, psbJ, psbN, psbH, psbT, ndhA, ndhB, ndhD, ndhE, ndhG, ndhI, ndhH, petA, petB, petG, rpoA, rpl14, rpl16, rps7, rps15 and ycf5 genes. And all of 10 vcd genes have been sequenced completely. By means of northern blot analysis, the expression of psaA-psaB, ndhB, petB, rpl14, rpl16, and rpoA in the leaves were retarded at 10 oC in the light. Time course studies of retardation gene expression in above genes, from the experimental results showed that the psaA-psaB gene expression reduced immediately after 1 h of 10 oC chilling treatment. The ndhB, petA, rpl14 and rpl16 reduced after 4 h of chilling treatment. But the rpoA gene only reduced slightly after 7 h of chilling treatment. The chilling stress also caused the water deficient in the mungbean seedlings. By means of northern blot analysis, the down-regulated RNA expression in water stress were significantly different from these genes in chilling-sensitive gene expression. The amino acid sequence deduced from the nucleotide sequences of vcd01 to vcd10 shared significant homonology with other plant chloroplast proteins. The base composition, PI value, hydropathy and molecular weight of PSAC, PSBB, PSBE, PSBF, PSBJ, PSBN, PSBT, NDHD, PETA, PETG and YCF5 were also disscused in this study. The availability of vcd01 to vcd10 will facilate the physiological and biological studies in roles of plastid development and the mechanism of these gene expression under 10 oC chilling in the light.

博士
國立清華大學
生命科學系
96
Mungbean (Vigna radiata (L.) Wilczek) is commonly used as human food. However, the genomic resources of this species available in databases are limited. This study has two sections, the expressed sequence tags (EST) related to early seedling development and chilling response are developed in the first part. Two mungbean varieties NM94 and VC1973A were obtained from the Asian Vegetable Research and Development Center-The World Vegetable Center (AVRDC). They differ in disease resistance and susceptibility to chilling temperatures. NM94 can maintain a better membrane integrity than VC1973A during seedling stage exposed to 4 degree C. To investigate the molecular mechanisms of the inherent chilling-susceptibility, the gene expression patterns in young seedlings of NM94 and VC1973A were compared in the second part of this thesis. A cDNA microarray containing 735 uniESTs was employed to profile the transcriptional changes during chilling stress and after recovery from chilling. The results show that a chilling exposure triggered the expression of a common set of CORs (cold regulated genes) in both NM94 and VC1973A. These CORs encode proteins that are involved in restructuring the protein synthesis apparatus, participating in protein trafficking and degradation, and synthesizing the stress protectants. The comparative expression profile indicates that the photosynthetic capacity and the cryoprotective proteins may be the key regulators of comparative toler55ance to chilling in NM94. Our data support the role of LTPs (lipid transfer proteins) in cryoprotection during chilling stress and resumption of plant growth after recovery from chilling.

碩士
國立臺灣海洋大學
食品科學系
95
Starch branching enzyme (SBE, EC 2.4.1.18) is a vital enzyme for amylopectin synthesis in the starch biosynthetic pathway. The aim of this thesis was to subclone VrsbeII of mungbean (Vigna radiata L. cv Tainan no. 5) into expression vector and produce active enzymes in the E. coli system. First, VrsbeII was constructed into pET-30 EK/LIC vector and transformed into E. coli NovaBlue host cells, hopefully full length sequence of the open reading frame could be obtained. However, there was only approx. 350 bp fragment could be stably maintained in pET-30 EK/LIC system and the unexpected outcome reoccurred. The sequence in the 350 bp was carefully examined and found astonishingly that two transposon-like 6-bp (CCAGTT) direct repeat sequences were in VrsbeII. Therefore, primers designed with Bam HІ and Not І sites which skipped one direct repeat sequence were used to prepare a 24 nucleotide shortened VrsbeII at the 3’-end by PCR. The redesigned insert fragment was ligated into the parallel sites on pET21b vector, followed by transforming into E. coli BL21 (DE3) cells, and expressed successfully as a soluble protein in the cytosol. The optimal expression condition for rVrSBEⅡwas evaluated that the cells were grown at 37℃ until OD600 to 0.6, then induced with 0.2 mM IPTG for 5 hrs, and the maximal crude enzyme activity of 0.25 U/mg was obtained. The crude enzyme was purified by HisTrapTM affinity column and the molecular size of rVrSBEⅡwas 108-kDa whose activity has enriched 25.5-fold (6.402 U/mg). The 108-kDa rVrSBEⅡwas also detected by anti-6x His-tag mouse monoclonal antibodies in Western blot. In summary, VrsbeⅡ has been expressed into a biologically functional protein in E. coli system and expected to further improve its enzymatic properties by genetic engineering for application in food use.

碩士
中國醫藥大學
營養研究所
93
The objectives of this study were to use proteomic approach to identify and analyze the expression of starch phosphorylase (SP) in mungbean (Vigna radiata L. cv KPS1) and proteins that might be related to SP. Besides, the proteomes in mungbean of different developing stages were analyzed in order to investigate what roles they might play in starch biosynthesis. Mungbeans from four stages DAF 11, 14, 18 and 21 (DAF, day after flowering) were collected and extracted as experimental materials. When MASS was cooperated with Western blotting using 55 kDa-SP antibody to detect SP related proteins, two forms of SP, L-SP and H-SP, were identified in the developing stages. A trend that L-SP expression decreased as mungbean grew was found. Some enzymes involved in starch biosynthesis were also identified such as sucrose synthase, phosphoenolpyruvate carboxylase and glucose-6-phosphate isomerase. MASS was also cooperated with in situ activity staining of branching enzyme (BE). The protein bands that synthesized amylopectin not only identified SP and sucrose synthase, but other metabolic enzymes (enolase, phosphoglycerate kinase, fructose-bisphosphate aldolase and malate dehydrogenase). However, no any known BE species has yet matched in the searched database. 2-D electrophoresis was cooperated with MASS to analyze mungbean proteomes in different developing stages that their 2-D mappings and the proteome database of DAF 18 mungbean were established. There were 61 protein spots identified, within which 12 protein spots were related with starch synthesis. The mungbean proteome database will be used for protein searching, matching and identification in the future.

碩士
國立臺灣海洋大學
食品科學系
98
Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes vital for amylopectin synthesis. The sequences of the previously obtained full-length cDNA of mungbean (Vigna radiata, cv. Tainan no. 5) sbeI (VrsbeI) and sbeII (VrsbeII’) were cloned. This study contains two parts: recombinant protein rVrSBEI expression analysis and chimeric gene design. First, pET-28a-VrsbeI plasmid DNA was transformed into BL21 (DE3) host cell and to find the optimal induction condition. The conditions for rVrSBEI induction were evaluated when the cells were grown at 37℃ until OD600 to 0.6 and then induced with 0.4 mM IPTG for 15 hrs; it showed seemingly a 117 kDa protein was induced. This recombinant protein was separated in and isolated from sodium dodecyl sulfate (SDS)- polyacrylamide gels for mass spectrometric identification. The induced protein band, however, is identified to be LacZ protein. The sequence feactures of the functional domain of VrsbeI and VrsbeII’ were used to design chimeric genes, then using specific primers that amplified two cDNA fragments: VrsbeI 1643 bp (VrsbeI 5’-1643) and VrsbeII 360 bp (VrsbeII 3’-360). In the future, we can ligate the two fragments into a chimeric sbe cDNA (cVrsbeI-II). Next, we can transform, express and analyze the chimeric enzyme activity to further use the enzyme in the modification of starch molecules.

碩士
國立臺灣海洋大學
食品科學系
95
Starch branching enzyme（SBE, EC 2.4.1.18）is one of the enzymes vital for amylopectin synthesis. The sequence of the previously obtained full-length cDNA of mungbean (Vigna radiate, cv. Tainan no. 5) SBEⅠ (named VrsbeⅠ) was confirmed and cloned. Its 3-D structures and functional features were predicted in silico with the only template, E. coli glycogen synthase (GS), 1m7x, by Swiss Model and REMUS. There is 27.4% amino acid sequence of VrSBEⅠ homologous to E. coli GS, in which 34.3% 3D structure of VrSBEⅠ was predicted. Six of the 8 conserved catalytic residues within the (α/β)8 domain of the α-amylase family in VrSBEⅠ were located in the structure. When using REMUS to compare amino acid sequences between two VrSBE isoforms, there were found to be potential epitope regions which are able to be bound specifically by antibodies. After confirming codon correctness by sequencing, VrsbeⅠ was cloned into pET-30 EK/LIC expression vector. The pET-30 EK/LIC-VrsbeI was expressed in BL21（DE3）pLysS cells in standard LB broth and the protein was induced by IPTG. The recombinant enzyme, rVrSBEⅠ, had His-tag and S-tag at the N terminal with an estimated molecular mass of 89 kDa. When Supplied with an extra 1% Glucose as carbon source during induction, it was able to decrease basal protein expression in the E. coli host. The crude cell extract possessing branching enzyme catalytic activities that decrease the A660 absorbance of amylose-iodine complex indicated that rVrSBEⅠ protein would be expressed as an active form. The crude extract was purified by HisTrapTM affinity chromatography. The activity of the purified rVrSBEI was also assayed by amylose branching assay. The decrease of the absorbance at A660 exhibited the specific activity of rVrSBEI was 314.6 U/mg and the purity has enriched 114-fold.

碩士
國立臺灣大學
植物學系
85
The growth of etiolated mungbean seedlings (Vigna radiata L. cv. Tainan No.5), a chilling-sensitive crop, was significantly inhibited by low temperature (4℃) treatment of 12 hr, and a significant injury appeared 2 days after low temperature treatment followed by 1 day recovery at 28℃. Pretreatment of 40℃ could improve chilling tolerance of mungbean seedlings, and a set of isotope labeled HSPs was separated by 1-D gel electrophoresis. A group of 19-21kD class I low-molecular-weight HSPs was recognized by westerrn blotting using rabbit anti-soybean class I low-molecular-weight heat- shock protein antibody. This group of 19-21kD HSPs was only induced by high temperature at 40℃and reached the maximum level at 2 hr. The half life of these 19-21kD HSPs were about 2 days at 28℃, but the amount of proteins maintained at high level at 4℃ for 12 days. The more of the 19-21kD HSPs , the more chilling tolerance was observed. When these 19-21kD HSPs were degraded completely, the plant has lost its chilling tolerance. Using E.coli and yeast 60kD HSPs antibodies for western blot analysis, no HSPs ranging in 80kD to 40kD in mungbean seedlings were detected. The roles of each group of HSPs are still yet to be clearfied. Furthermore, the chilling tolerance observed in our mungbean system could be achieved by multiple HSPs but not only a single HSPs.

博士
國立清華大學
生物資訊與結構生物研究所
101
A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y2K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. VrDhn1 mRNA level was extremely high in mature seeds and decreased to ~30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous abscisic acid (ABA) markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. VrDhn1:GFP fusion protein is degraded in transgenic Arabidopsis under normal condition, but is more stable under dehydration. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity of non-specific interaction with DNA using electrophoretic mobility shift assays (EMSA), and the exogenous addition of Zn2+ or Ni2+ stimulated interaction. Phosphorylation occurs on threonine and tyrosine residues of VrDhn1 in mungbean seeds. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y2K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

Abstract Strong acids and chlorinated products are widely utilised for the maintenance of drip irrigation facilities worldwide. This research evaluated low concentration (10 ppm) of hydrogen peroxide (H2O2) products, in drip irrigation water, sourced from H2O2 containing high concentration (30,000 ppm) of an organic stabilizer (H2O2 High) and from H2O2 containing a low concentration (30 ppm) of the same stabiliser (H2O2 Low), evaluated in laboratory and field for effect on drip emitters, seed germination, seedling and crop growth, soil and water parameters. Findings from this research showed that seed germination was not impacted by H2O2 up to 5000 ppm. Positive effects on seed germination were noted for mungbean (Vigna radiata L.), egg plant (Solanum melongena L.), okra (Abelmoshus esculentus L.), chive (Allium porrum L.) and rocket (Eruca sativa L.) treated with 100 ppm H2O2. H2O2 High up to 1000 ppm did not negatively impact seed germination in other species tested. Root and shoot growth were enhanced in some species at lower concentration of H2O2 High, but were negatively impacted by higher H2O2 High concentrations for all crops except for corn. The negative effects on root and shoot growth were generally noted at higher concentrations (>1000 ppm). Therefore, continuous injection of H2O2 at low concentration (<100 ppm) in irrigation is unlikely to affect the seed germination and seedling growth. The impact of continuous injection of 10 ppm H2O2 on drip irrigation performance was evaluated in field trials. The emitter flowrates for surface drip single use tape on a chili (Capsicum frutescens L.) crop was not affected after 6 months of irrigation in either the control or H2O2 treatments. However, for above ground drip for table grapes (Vitis vinifera L.) and in subsurface drip for sugarcane (Saccharum officinarum L.) installations over four years, the emitter flow rate remained higher (2-16%) for H2O2 Low compared to H2O2 High and control. In aboveground drip, emitter clogging was reduced by 50% in H2O2 Low compared to the control and H2O2 High. The yield increases of 25, 10 and 4% in the sugarcane, chilli, and grape respectively probably due to H2O2 delivery proximal to root mass. H2O2 breakdown in soil was rapid, hence no residual H2O2 was found 10 minutes post irrigation. In tertiary treated wastewater (effluent), a single dose of H2O2 20 ppm was effective for suppression of algal blooms, whereas complete elimination was achieved by 2000 ppm by both products. In a circulating irrigation system, the emitter flow ceased after 900 hours due to biofouling in H2O2 High. In a bamboo (Bambusa spp. L) field trial using 20 ppm of products in non-circulating irrigation over 1966 hours, emitter flowrates were reduced by 50%. Drip emitter clogging was significantly reduced to19% for the H2O2 Low and 28% for H2O2 High compared to control (37%) of algae for effluent irrigation water. H2O2 High, unlike H2O2 Low, caused rapid emitter clogging in recirculating hard water irrigation, suggesting that HEDP in the presence of H2O2 caused Ca precipitation resulting in sudden emitter clogging. The degree of Ca precipitation in hard water increased with increasing HEDP inputs in irrigation. pH buffering of hard water delayed Ca precipitation. In the non-circulating irrigation higher concentration HEDP treatment in the hard water irrigation caused rapid emitters clogging. Low pH (≤6) increased solubility of Ca ions whereas higher pH led to precipitation causing crystal and amorphous Ca deposits. Continuous injection of H2O2 (10-20 ppm) in irrigation over a longer term did not show decline of soil biological functions (soil respiration, soil microbial biomass carbon and soil microbial diversity). H2O2 increased corn (Zea mays L.) yield by 9.2 and 70%, and coriander (Coriandrum sativum L.) yield by 2.3 and 15% for plants grown in vertisol and ferrosol, respectively, suggesting interaction effects, due to disproportionally greater decomposition of H2O2 to oxygen in ferrosol. In general, H2O2 Low injection in irrigation resulted not only in improved emitter performance, but also positive effects of crop growth, without noticeable negative impacts on soil. H2O2 High can acidify the rhizosphere, which may be of advantage in alkaline soil and/or irrigation water.

Mungbean (Vigna radiata (L.) Wilczek ssp. radiata) is an economically important crop in Asian countries where breeding research is being undertaken to improve varietal adaptation, yield and seed quality. The wild mungbean (ssp. sublobata) is a potentially useful adjunct to breeding, as the wild accessions possess traits that confer adaptation in their natural environments. The wild accession ACC 87 collected near Townsville has been identified as being perennial, a potentially useful trait for forage crop improvement. Accession ACC 1, from Mackay is very late flowering and was reported to possibly have a long juvenile (LJ) trait similar to that found in soybean. Before these and other potentially useful wild traits can be exploited, information is needed on their inheritance. Therefore, a study was conducted to examine the inheritance of traits in four hybrid cultivated X wild mungbean populations. The study examined the expression of traits in the parental plants, and the F1, F2, BCP1 and BCP2 progeny generations, when grown under controlled conditions in pots. The four genetic populations had been created by hybridizing using two cultivated mungbean varieties, Berken and Kiloga, with each of two wild parents, ACC 1 and ACC 87. Several morphological traits, including lobed leaflet shape, seed testa and hilum color, and plant habit were found to be under simple (qualitative) genetic control, with the wild type generally dominant. An exception was putative resistance to powdery mildew infection in the wild accessions, which appeared to be recessive. Many other traits like phenology, nodes per plant, seed yield and biomass were under quantitative genetic control. The perenniality trait in ACC 87 appeared to be under simple genetic control, with expression of perenniality due to two dominant complementary genes. In contrast, flowering in the two ACC 1 populations appeared to be quantitatively inherited, with no evidence of a LJ trait. There were many similarities in the genetic control of both qualitative and quantitative traits among the four hybrid populations, with only small differences due to the different cultivated parents. However, larger differences were apparent between the populations involving ACC 1 and ACC 87. Estimates of narrow sense heritability were high for many of the qualitatively inherited traits, indicating high additive genetic variance for those traits, and thus the capacity for genetic gain through selection. Transgressive segregation occurred for most of the quantitative traits in one or more of the four crosses, indicating the potential value of the wild germplasm in broadening the phenotypic range available to plant breeders. Several phenotypic and genotypic interrelations found between many of the wild traits among the four crosses. In particular, there were several significant genetic correlations among quantitative traits, indicating that selection for one of the traits should result in genetic advance in the other. The study confirmed earlier research that Australian accessions of the wild mungbean can be considered part of the primary gene pool of the cultivated mungbean. Consequently, the wild accessions provide an additional source of triats potentially useful for mungbean improvement. The study also established that traits of possible commercial interest, perenniality and powdery mildew resistance, were qualitatively inherited and thus should be readily transferrable into cultivated varieties. While the study failied to identify the presence of a LJ trait, it suggested that the wild germplasm could be a useful source of lateness genes for breeding vegetatively vigorous forage or cover crop varieties of mungbean.

博士
國立清華大學
生命科學系
91
ABSTRACT To explore the changes in response to environmental stresses, we isolated a lipid transfer protein (LTP) gene in rice (Oryza sativa) suspension cells in the presence or absence of sucrose by using mRNA differential display. LTPs are small and basic proteins, which consist of eight highly conserved cysteines forming four disulfide bonds. Two main families of LTPs, LTPI and LTPII, are identified with molecular mass of 9 kDa and 7 kDa. LTPs exist in various plants, and the expression of plant LTPs shows temporal and spatial patterns. We found this rice ltp (Osltp) mRNA expressed in developing and fresh seeds, as well as roots in mature plants; and the level of Osltp mRNA increased under water stress, such as high salt, dehydration, low temperature, and abscisic acid (ABA) treatments. Furthermore, we isolated two novel mungbean (Vigna radiata) ltp genes, Vrltp1 and Vrltp2, by screening cDNA library. Both Vrltp1 and Vrltp2 mRNAs expressed in floral buds and only Vrltp1 mRNA expressed in immature seeds. In the vegetative tissues, the Vrltp1 and Vrltp2 mRNAs display their shoot specificity. The levels of Vrltp1 and Vrltp2 mRNAs also increase in response to salt, dehydration and ABA treatments. We assume that these ltp genes participate in the protection from damage under water stress, and the regulatory mechanisms are needed to be elucidated.

This thesis reports the outcomes of research evaluating the application of Diversity Array Technology (DArT) to two important tropical legume crops, mungbean (Vigna radiata (L.) Wilczek) and soybean (Glycine max (L). Merrill.). Mungbean is comparatively under-studied, and is therefore likely to benefit from research to improve adaptation and productivity. Soybean has received more breeding attention than mungbean, but is still under-researched when compared with modern cereals like rice, wheat and maize. Drought remains an important constraint to soybean productivity in rainfed areas. DArT is a novel molecular marker technology that has been successfully applied in genetic studies in several plant species, but had not been previously applied to either of the crops of interest. A DArT marker library/ array for mungbean was created using two mungbean cultivars, Berken and Kiloga, and two wild accessions V. radiata. ssp. sublobata, ACC 1 and ACC 87, and one or more accessions of five other Vigna species, viz. V. lanceolata, V. mungo, V. mungo var. sylvestris, V. trilobata and V. vexillata. A DArT library/ array for soybean was developed using two oilseed type cultivars, CPI 26671 and Valder, a landrace G2120, a wild soybean (G. soja) accession and one or more accessions of two wild Glycine species, G. falcata and G. tomentella. For each crop, two genomic complexity reduction methods, utilizing PstI/TaqI and PstI/BstNI restriction digests, were selected for DNA clonal library development and for the isolation in each case of 7,680 DArT clones from genomic representations of pooled DNA samples. While the PstI/BstNI method produced more polymorphic clones than PstI/TaqI for the soybean library, there was no significant difference between the two methods for the mungbean library. In the initial library evaluation, there were nearly 1,500 polymorphic clones identified for soybean. Polymorphism frequencies in mungbean were around twice those in soybean, reflecting greater diversity in the mungbean germplasm samples. The DArT marker transferability from soybean to mungbean (13.6%) was nearly five times higher than that from mungbean to soybean (3.1%). The percentage of DArT marker transferability between mungbean and several other Vigna species ranged from 3.4 to 20.2%. The genetic similarities among 11 diverse Vigna spp. samples, evaluated using the DArT mungbean library, were consistent with published information on these taxa. These mungbean and soybean arrays were then used to evaluate the application of DArT markers for construction of genetic linkage maps and identifying putative qualitative and quantitative trait loci (QTLs). In mungbean, four F₅ recombinant inbred line (RIL) populations were derived from crosses between cultivars Berken and Kiloga and wild accessions ACC 1 and ACC 87. The F₅ RIL populations were evaluated for 54 qualitative and quantitative traits using plants grown in large pots on outdoor benches. There were large differences between the cultivated and wild parents and individual lines for all traits. Broad sense heritability estimates were moderate to high in most cases, with significant phenotypic correlations between many traits. A large number of polymorphic DArT markers were selected for the four RIL populations (1062 – 2013). The four mungbean linkage maps contained 672 to 981 DArT markers with segregation distortion levels higher in the ACC 87 (44.1 – 47.8%) than in the ACC 1 crosses (33.7 – 42.4%). Maps consisted of 15 – 19 linkage groups (LGs) and spanned lengths of 629.7 to 883.5 cM with average inter-marker distances of 0.9 – 1.2 cM. Various putative QTLs (77 – 122 QTLs) were identified for the vast majority of the 54 evaluated traits. In addition, the level of congruence across populations was reasonably strong. In soybean, one F₇ and two F₆ RIL populations derived from crosses between CPI 26671, Valder and G2120 were used in phenotypic evaluation and QTL mapping with DArT markers for physiological drought stress response traits. The RILs were grown in deep cylindrical pots in the glasshouse, and exposed to severe water deficit followed by re-watering. Traits recorded included relative water content (RWC), epidermal conductance (gₑ), and recovery in growth following re-watering. The drought stress responses in the parental plants and RIL populations were broadly consistent with prior studies: As plant available water (PAW) in the soil declined, both RWC and gₑ declined, with the relation between RWC and gₑ exponential rather than linear as in previous studies. Analysis of variance showed significant differences at both population and genotypic levels for all key traits. However, there were large environmental effects on most traits, which resulted in high coefficients of variation and low estimates of broad sense heritability. The three individual linkage maps contained 196 – 409 DArT markers and 15 – 22 LGs with the aggregate length ranging from 409.4 to 516.7 cM. An integrated soybean map was constructed consisting of 759 DArT markers, 27 LGs and an expanded length of 762.2 cM. Total numbers of putative QTLs identified in the CPI 26671 x G2120 (CG) and VG (Valder x G2120) populations were 106 and 34 respectively. In each of the population, 10 LGs harboured QTLs associated with RWC, gₑ and recovery ability, of which five similar LGs contributed to drought tolerance. A BLAST (Basic Local Alignment Search Tool) search for sequences of 19 selected DArT markers linked to QTLs conditioning drought response traits indicated that 18 DArT markers were unique and aligned to 12 soybean chromosomes. Comparison of these DArT markers with other markers associated with drought-related QTLs in previous studies confirmed that five of them overlapped whereas the remaining 13 had not been previously identified. However, except for chromosome 15, the chromosomes with which the DArT QTLs in the CG and VG populations were associated were ones that had been shown to harbour drought-related QTLs in previous studies. This study is the first showing DArT development in mungbean and soybean and its application for manipulating QTLs associated with wild physiological traits in mungbean and drought tolerance in soybean. DArT was successfully developed for both species, with more polymorphisms evident in the mungbean than in the soybean arrays. The study demonstrated that DArT provides high quality markers which can be used for diversity analyses, the construction of high-density genetic linkage maps and for QTL analysis. Meanwhile the marker transferability between arrays enabled plausible discrimination of genetic relationships between related taxa. In both mungbean and soybean, the large numbers of DArT markers that were generated contributed to relatively tight resolution in the genetic maps, enhancing the power for QTL detection. Potentially useful QTLs/ markers were identified for many traits in mungbean, including some potentially useful ones such as resistance to powdery mildew and thrips, late flowering, hardseededness and perenniality, and in soybean, for RWC, gₑ and recovery after drought stress. In mungbean, further research is needed to identify an appropriate approach for the construction of an integrated map from the four RIL populations used in this study.In both species, follow-up research is required to verify the QTLs detected in this study before they can be used for marker-assisted selection in mungbean and soybean breeding programs. Nonetheless, the QTL analyses based on DArT markers in this study have been shown to be useful in the genetic dissection of both qualitative and quantitative traits in both species, and it is apparent that DArT markers will offer advantages for a range of molecular breeding and genomics applications.

碩士
國立清華大學
輻射生物研究所
84
Vacuoles of higher plant cells contain two primary electrogenic proton pumps, a vacuolar H+-ATPase (V-ATPase) and a H+- translocating inorganic pyrophosphatase (V-PPase), for the regulation of cell turgor, cytoplasmic homeostasis, and the storage of metabolites. In this work, the structure of the mung bean V-ATPase A subunit gene was investigated. In order to clone the cDNA, primers were synthesized according to the conserved sequences of cotton and carrot V-ATPase A subunit to conduct polymerase chain reaction (PCR) using the total DNA extracted from mung bean cDNA library as a template. The PCR product was then employed as a probe to screen the constructed mung bean cDNA library. The cloned V-ATPase A subunit cDNA exhibits 83.8, 83.4, and 81.1% nucleotide homology to those of cotton, carrot, and Brassica napus, respectively. The cDNA sequence encodes 623 amino acids with a predicted Mr of 68,664and a predicted isoelectric point of 5.17. The amino acid sequence of V-ATPase A subunit from mung bean seedlings shares 94.0~95.0% identity and 96.6~97.0% similarity to those from carrot, cotton, and Brassica napus. Expression of the A subunit gene was also investigated by Northern hybridization. It was found that the leave expressed the most abundance of the transcript, followed by hypocotyl and roots. Genomic Southern analyses reveal a simple reaction pattern of the gene, referring the lack of isoform for the A subunit. We also took further step to explore the genomic structure of the A subunit. To construct the DNA library, mung bean genomic DNA was partially digested by Sau3AI, and fractionated by sucrose gradient centrifugation. The 9 to 20 kb DNAs were ligated with l/Dash II DNA and packaged. The constructed library was screened using A subunit cDNA as a probe, and several positive clones were obtained. Some of these clones contain both 5?and 3?regions of the gene.

碩士
國立清華大學
輻射生物研究所
84
Membrane-bound inorganic pyrophosphatase (PPase), which couples PPi hydrolysis with proton pumping, has been found on membranes of the tonoplast, chloroplast, mitochondria, and some species of bacteria. Here, we further demonstrated the existence of a H+-pumping PPase from mung bean plasma membrane. Enzyme activity of PPase was observed on the plasma membrane and possessed an optimum alkaline pH at 8.0-8.5. PPi-dependent proton translocation was concomitantly found on highly purified plasma membrane vesicles. A successful protocol including plasma membrane preparation, detergent solubilization, gel filtration, and anion exchange chromatographies was established to purify the enzyme. Analysis of size exclusion gel filtration chromatography and SDS-PAGE revealed that plasma membrane H+- PPase was probably in a heterodimer form consisting of two subunits of 65 and 67 kDa. The PPase activity was fluoride- sensitive, but could be stimulated by K+ and phospholipid using Mg2+ as a cofactor at Mg2+ / PPi ratio of 2 : 1. Taken together, we believed that plasma membrane H+-PPase was a novel new type of membrane-bound alkaline inorganic pyrophosphatase.

Trabajo Final Integrador (Área de Consolidación Sistemas Agrícolas de Producción Extensivos) -- UNC- Facultad de Ciencias Agropecuarias, 2017
El objetivo de este trabajo fue analizar tres factores: fecha de siembra (dos niveles), distancia entre hileras (dos niveles) e inoculación (dos niveles) sobre Poroto Mung (Vigna radiata L.Wilczek) conducidos en secano durante la campaña 2016/17 en el Área Experimental del Campo Escuela de la Facultad de Ciencias Agropecuarias UNC (31°28’43’’S 64°00’28’’ W). El diseño utilizado fue en bloques completos al azar con tres repeticiones y dos distancias entre hileras, con el agregado de inoculante en parcelas designadas y lotes testigos sin inocular en el resto, sembradas en dos fechas distintas, separadas por 60 días aproximadamente. Se registró el rendimiento (kg/ha), el Nº semillas/m2, el peso de las semillas (g), la biomasa aérea (g), la humedad de los granos cosechados y el índice de cosecha. No se encontraron diferencias significativas, desde el punto de vista estadístico, para ambas fechas de siembra, agregado de inoculante específico para el cultivo y espaciamiento entre hileras (exceptuando la FS1 a 0,26 m donde si se encontró diferencia). Sin embargo, estadísticamente hablando, a nivel de medias los tratamientos con fecha de siembra tardía, menor espaciamiento entre hileras (0.26 m) y con inoculante fueron superiores, pero esas diferencias no fueron estadísticamente significativas.

Dissertação de mestrado em Biotecnologia e Bioempreendedorismo de Plantas Aromáticas e Medicinais
As leguminosas são uma classe de plantas muito utilizada na alimentação, sendo já 150 as espécies usadas como alimento. Além de possuírem um baixo conteúdo de lípidos, representam uma boa fonte de proteína e fibra e possuem numerosos compostos bioactivos na sua constituição, que podem produzir efeitos metabólicos e fisiológicos de interesse. Alguns deles exercem um papel relevante na prevenção de algumas doenças, tais como diabetes, doenças cardiovasculares e cancro. Habitualmente estas plantas são consumidas na forma de sementes fisiologicamente maduras ou germinadas sob a forma de rebentos. Neste trabalho foi avaliado o potencial químico e biológico de rebentos de Glycine max (L.) Merr., Vigna radiata (L.) R. Wilczek e Medicago sativa L., tendo-se identificado onze compostos fenólicos, incluindo quatro isoflavonas, quatro fitosteróis, dezasseis ácidos gordos, sete ácidos orgânicos e trinta e um compostos voláteis, demonstrando que os rebentos são uma boa fonte de compostos bioactivos da dieta. O potencial antioxidante foi avaliado contra três espécies reactivas, tendo-se verificado que os rebentos de G. max foram os mais activos contra o radical DPPH• (IC50 = 1,337 mg/mL), enquanto os de M. sativa foram os mais activos contra os radicais superóxido e óxido nítrico (IC50 = 0,067 mg/mL e IC50 = 0,426 mg/mL, respectivamente). Posteriormente, efectuou-se o estudo da influência da inoculação de sementes de G. max com Bradyrhizobium japonicum na síntese de compostos bioactivos e na sua actividade biológica. Verificou-se que a amostra controlo continha maior quantidade de fitosteróis, de compostos voláteis, de compostos fenólicos e, paralelamente, a amostra inoculada mostrou ser mais vantajosa relativamente ao teor de ácidos orgânicos e de ácidos gordos. Relativamente ao efeito no potencial biológico de sementes G. max, a inoculação não teve efeito ao nível das propriedades antioxidantes, sendo a amostra controlo a mais activa. De todas as amostras analisadas, os rebentos apresentaram um potencial antioxidante superior.
Pulses are a class of plants widely used in the diet, 150 species being already consumed as food. Besides their low lipids content, they represent a good source of protein and fiber and have numerous bioactive compounds, which can produce interest metabolic and physiological effects. Some of them have a relevant role in the prevention of diseases, like diabetes, cardiovascular disease and cancer. These plants are usually consumed in the form of physiologically mature seeds or germinated as sprouts. In this work the biological and chemical profiles of Glycine max (L.) Merr., Vigna radiata (L.) R. Wilczek and Medicago sativa L. were studied, and eleven phenolic compounds, including four isoflavones, four phytosterols, sixteen fatty acids, seven organic acids and thirty one volatile compounds were identified, demonstrating the potential of each of these sprouts as dietary sources of bioactive compounds. The biological activities against three different reactive species were also studied. From the results obtained it was concluded that G. max is more active against DPPH• radical (IC50 = 1.337 mg/mL), and M. sativa is more active against superoxide and nitric oxide radicals (IC50 = 0.067 mg/mL and IC50 = 0.426 mg/mL, respectively). In addition, the study of the influence of G. max seeds inoculation with Bradyrhizobium japonicum on the synthesis of bioactive compounds and on their biological activity was performed. It was found that the control sample contained higher amount of phytosterols, volatile compounds, phenolic compounds, while the inoculated sample was more advantageous in terms of organic acids and fatty acids. Regarding the biological potential of G. max seeds, the inoculation didn‘t produce effect on its antioxidant capacity, being the control sample the most active. The sprouts showed a higher antioxidant potential among all the analyzed samples.