1 THAI NGUYEN UNIVERSITY COLLEGE OF EDUCATION Vu Thanh Tra RESEARCH ON THE GENETIC DIVERSITY OF SOME SOYBEAN VARIETIES THAT ARE RESISTANT TO RUST DISEASE IN DIFFERENT WAYS Speciality: Genetics Code: 62 42 70 01 ABBREVIATE BIOSCIENCE DOCTORAL THESIS Thai Nguyen - 2012 The study was fulfilled at Thai Nguyen University, College of Education Supervisor: A Prof Dr Chu Hoang Mau Dr Tran Thi Phuong Lien Judge 1: Judge 2: The thesis will be defended before Thesis Evaluation Committee at Thai Nguyen University level at : At: ., ……………… 20 PUBLICATIONS T.A Pham, C.B Hill, M.R Miles, B.T Nguyen, T.T Vu, T.D Vuong, T.T VanToai, H.T Nguyen, G.L Hartman “Evaluation of soybean for resistance to soybean rust in Vietnam” Elsevier – Field Crops Research 117, pp 131–138 Vu Thanh Tra, Tran Thi Phuong Lien, Chu Hoang Mau “Evaluation of genetic diversity of 50 Vietnam soybean varieties resists differently to soybean rust using SSR marker” Biological Journal 34(2):235-240 Vu Thanh Tra, Tran Thi Phuong Lien, Chu Hoang Mau “Study the genetic relationship of some Vietnamese soybean cultivars having different responses to rust” Technology and Science Journal – Thai Nguyen University 85(9)/2: 11-16 Vu Thanh Tra, Ha Hong Hanh, Tran Thi Phuong Lien, Chu Hoang Mau “Isolation and analysis of proteins from soybean leaves” Technology and Science Journal – Thai Nguyen University 85(01)/1: 303-310 Vu Thanh Tra, Ha Hong Hanh, Tran Thi Phuong Lien, Chu Hoang Mau “Study on leaf proteins infected soybean rust using two dimensional gel electrophoresis” (Submitted to Biotechnology journal) INTRODUCTION Preface Soybean (Glycine max (L.) Merrill) is a short-time industrial plant with high economic value and helpful to soil improvement It is easy to grow and particularly adaptable to various ecological areas Soybean seed contains 30-55% protein, and many types of nonreplaceable amino acids, 12-25% lipid and necessary vitamins for human body Soybean products are popularly used for different purposes such as food, cooking oil, functional food and materials for medicine and industries Besides high nutrition proportion, soybean is capable of fixing soybean nitrogen by the symbiosis of bacteria R Japonicum on root constituting nodules, improving land efficiently Therefore, soybean has been interested in and thrived in many countries around the world In Vietnam, soybean is the main plant encouraged to develop priorities, and to produce after the rice, maize and peanuts Vietnam used to be an exporter of soybean in the 1980s, but so far our country import millions of tons of soybean annually Although the cultivated area increases every year, it has low productivity and unstable yield, bad resistance to disease and stress Insects and in particular, the rust disease directly causes the effect of the planted area and reduction of productivity, quality of soybean seed, causing huge economic losses The rust disease of soybean are caused by fungus Phakopsora pachyrhizi and are regarded as one of the main threats on soybean plants, causing significant damage and reducing from 10-80% the yield and quality of soybean in many countries in the world, including Viet Nam In recent years, studies on the soybean rust have been carried out and obtained some significant results, but most researches have only focused on monitoring the process of disease development, epidemiological studies, assessing the loss of productivity or analyzing disease response They not pay attention to learning about resistance against rust disease The study of genetic diversity of soybean groups with different responses to the rust is not only significant in the conservation of the varieties that are resistant to disease but also have important implications in the work of selecting breeder with high quality Derived from the above reasons, we made the selection of topic: "Research on the genetic diversity of some soybean varieties that are resistant to rust disease in different ways" Objectives Evaluating the potential response of soybean varieties with rust disease in order to find out new resistant kinds, identifying the genetic diversity on the basis of DNA polymorphism analysis, comparing protein polymorphisms and mapping soybean leaf protein electrophoresis Activities - Assessing soybean’s resistance against rust disease - Identifying protein, lipid and amino acid composition in seeds of soybean - Analyzing genetic diversity of soybean varieties by using molecular indicators RAPD and SSR - Mapping soybean leaf protein electrophoresis and protein identifier system by means of one-dimensional electrophoresis, two-way and identifying the protein on chromatography-mass spectrometry system Simultaneously analyzing and comparing the protein diverse components of soybean with rust infection and resistance Significant results - The study has classified the level of rust resistance of 50 varieties of soybean collected in Vietnam into groups: group which is susceptible to rust, the intermediate group and resistance group Especially, the study has discovered six new resistant varieties such as PMTQ, HSP1, HSP2, CNB, ZG and MTD65 At the same time, the biochemical compositions of 50 varieties of soybean have also been analyzed - Using molecular indicator RADP and SSR, the study analyzed genetic relationship of 50 kinds of soybean, the results showed that the polymorphism of SSR is at higher level - With the method of 2-dimensional electrophoresis, the study has established 2DE electrophoresis maps of soybean protein in resistant cultivars DT2000 with 119 points of protein on gene, including 35 proteins identified and classified Especially, three proteins related to disease resistance / defense, drought and stress tolerance, have been found These are the first data statistics of studying leaf protein system, mapping protein, identifying the protein in resistant cultivars This result plays an important role in understanding disease mechanisms and suggesting approaches the level of protein - With the method of comparing the change in protein expressed on the 2DE gene, the study also found that there are points of protein increasing and point decreasing compared to samples in two rust infected soybean to detect and identify 6/9 of protein Two protein samples in two susceptible cultivars (DT12, VMK) have points of protein higher than two rust-resistant cultivars (DT2000, CBU8325), have recognized and identified 4/6 of the protein These are the first figures published in Vietnam when comparing different expression levels of these proteins in infected and samples, among the infected and resistant cultivars by electrolysis 2DE combined identifications of mass spectrometry Scientific and practical meanings - The thesis has studied soybean, a meaningful plant to our agriculture Modern and highly reliable scientific methods have been used to study characteristics, biochemical components, DNA diversity and protein analysis of some kinds of soybean which have different reaction to rust The thesis has been seriously and carefully invested in, which has brought ample results The thesis will be reference to following studies on soybean Such scientific methods as DNA diversity analysis with RADP, SSR techniques or mass spectrum and electrophoresis analytical method are new approaches which are scientifically meaningful and bring various interesting information on disease infection and resistance as well - In fact, the thesis has classified different kinds of soybean which have different reactions with rust into groups Among them, new rust-resistance kinds are PMTQ, HSP1, HSP2, CNB, ZG and MTD65 The above information is very useful and can be applied in breed selection and creation The structure of the thesis The thesis comprises of 115 pages, which are divided into the following parts: Introduction including pages, Chapter 1: Theoretical overview, 29 pages; Chapter 2: Materials and methods, 20 pages, Chapter 3: Results and discussion, 46 pages; Conclusion and recommendations, pages; Announced works of the author: page Reference: 14 pages; The thesis uses 15 tables, 19 figures and 121 reference materials in Vietnamese and English Chapter OVERVIEW 1.1 Soybean and biochemistry of soybean 1.1.1 Soybean Soybean (Glycine max (L.) Merrill) is a plant of the legume (Fabaceae) The branch of Glycine has two sub-branches such as Glycine and Soja Originally, soybean comes from East Asia On morphological characteristics, soybeans are herbaceous, leaves of three types: cotyledon, leaf and double leaves The flowers of soybean are small without flavor, butterfly shaped Fruits are the adjacent fruit, difficult separation, slightly curved, at the young fruit is green, hairy when ripe with brown Seeds coming in many shapes: round, oval, flattened circles have high nutritional value On genetic characteristics, soybean with diploid chromosomes 2n = 40 is a self-pollinated plant, is less cross pollination Haploid genome of soybean is from 1.29 -1.8 x 109bp Time of soybean growth is divided into three categories: early ripening, moderate and late 1.1.2 Biochemical characteristics of soybean 1.1.2.1 Proteins in soybean seed In soybean seeds, protein accounts for 12-55% Protein is stored in organelles with the function of providing acid amino and nitrogen sources or the enzymes involved in the process of seed germination Protein mainly stored in soybean seed is globulin 1.1.2.2 Lipids, vitamins and other substances in the soybean seed In soybean, lipid content accounted for 12-25% of dry weight The quality of lipids in soybean seed is very good, so it is widely used in food processing industry Soybean seeds have lipid-soluble vitamins, especially vitamin E In addition, the soybean seed also contains other substances such as carbohydrate, minerals, nucleic acids, growth stimulants 1.1.2.3 Leaf protein and soybean protein system The leaf soybean protein is the protein involved in metabolism and energy metabolism It is the protein involved in electron transport process, enzymes, the stored proteins, or proteins involved in the metabolism and metabolism of amino acids or proteins involved in resistance and stress 1.2 Rust disease and rust resistance in soybean 1.2.1 Rust disease in soybeans Rust disease caused by fungus Phakopsora pachyrhizi is one of the main diseases in soybean (Glycine max) in Asia and causing significant damage in the yield of soybeans in many countries Signs and symptoms of disease: Signs of the disease are the dots in the leaf blade about mm, growing gradually to turn yellow and then brown, the size reaching from 2-5mm with diverse, angular On the lower side of blade with the lesions, there appeared brown powder which is the lower of fungal spores Lifespan and infected process: The infected process begins when spores germinate to form a germ tube on the surface of rim (diameter of 5-400 micrometers) until the formation of fiber Lower spores usually develop for 5-8 days, maximum up to weeks after infection, the spore process can last weeks 1.2.2 Disease resistance of soybean rust The interaction between host and parasite is under the model of gene for gene Up to now, scientists have found that there are five major genes related to rust resistance in soybean, including gene Rpp1, gene Rpp2; gene Rpp3; gene Rpp4 and gene Rpp5 1.3 Methods of analysis of genetic diversity on soybean 1.3.1 The method of using form directives Form directive is a co-dominant directive, usually expressed as a characteristic controlled by a single locus such as the gene for flower color and shape of the seeds, skin 1.3.2 Method of using biological indicators The biochemical indicators appear in most polymorphic protein such as isozyme and stored proteins These isozymes can be separated by weight, molecular size and the carrier 1.3.3 The methods use DNA indicators 1.3.3.1 RAPD indicators In principle, the RAPD technique bases on PCR reaction by using short sequenced samples, pairs and randomly cloned DNA fragments with complementary sequences with the sequence of the samples The RAPD markers used in the study of diversity and genetic mapping of the typical locus include the following: (i) designing sample, (ii) Preparing DNA samples and run PCR, (iii) checking cloning products on agarose gene, (iv) Analyzing of results by specialized software (v) Determining genetic coefficients and setting the map of the genetic relationship of objects 1.3.3.2 SSR indicators Technical SSR (Simple Sequence Repeat) was first detected on the object by Litt and Luty (1989) The use of indicators SSR in genetic mapping of the typical locus include the following: (i) Isolating SSR from the genomic library, (ii) Determining the sequence of the SSR regions; (iii) Identifying typical sample pairs of DNA sequences limited by the ends of the SSR; (iv) Multiplying genomic regions corresponding to PCR using specific samples, (v) Analyzing the size of PCR products in order to determine the presence of SSR alleles 10 Chapter MATERIALS AND METHOD OF THE STUDY 2.1 Materials 2.1.1 Materials and methods for collecting infected leaves Using 50 soybean varieties derived from local Vietnam by the Center for Development and Research of beans and Agricultural Genetics Institute and Can Tho University is studying materials Including resistant cultivars against rust disease provided by the Institute of Plant Protection Some varieties were collected from local Cao Bang, Ha Giang and Thai Nguyen 2.1.2 Chemicals Chemicals were purchased from vendors such as Merck, Invitrogen, Amersham Pharmacia Biotech, New England Biolabs 2.2 Research methodology 2.2.1 Method of artificial infection 2.2.2 Methods of biochemical analysis 2.2.2.1 Determination of total lipid 2.2.2.2 Determination of total soluble protein 2.2.2.3 Determination of amino acid in soybean seed 2.2.3 The methods of analyzing genetic diversity in DNA 2.2.3.1 Method for extraction and purification of total DNA 2.2.3.2 Method for determination of DNA by spectral 2.2.3.4 Electrophoresis of DNA on polyacrylamide gene and silver staining 2.2.3.5 Polymorphic analysis by RAPD technique 2.2.3.6 Polymorphism analysis by SSR technique 2.2.3.7 Method of processing data 2.2.4 The formulated analytical methods of protein 2.2.4.1 Separation and extraction of protein from soybean leaves 2.2.4.2 Electrophoresis of SDS-PAGE 2.2.4.3 dimensional Electrophoresis - 2DE 2.2.4.4 Protein staining and image analysis of gene 2.2.4.5 Identify proteins on a 2DE electrophoresis with mass spectrometry 13 there are 14/20 samples with polymorphic fragments of 50%, sample M14 has polymorphic fragments with 50% (Fig 3.1), sample M18 has polymorphic fragments with 100% (Fig 3.2) (not showed) This result is consistent when analyzing polymorphic information content in the PIC value (table 3.2) Specifically, the value of sample M14 PIC was 0.27 (polymorphic rate of 50%) and PIC values of the sample M4 was 0.86 (high polymorphism), in which 13/20 samples for value PIC ≥ 0.5 Table 3.2 The ratio of the polymorphic fragments and value PIC of the samples Total Total % % No Sample PIC polymorphic polymorphic No Sample PIC polymorphic polymorphic fragments fragments fragments fragments M1 0.74 83.3 11 M11 0.80 83.3 M2 0.76 100.0 12 M12 0.00 0.0 M3 0.67 100.0 13 M13 0.80 100.0 M4 0.86 100.0 14 M14 0.27 50.0 M5 0.00 0.0 15 M15 0.35 83.3 M6 0.85 37.5 16 M16 0.00 0.0 M7 0.79 85.7 17 M17 0.00 0.0 M8 0.00 0.0 18 M18 0.50 100.0 M9 0.84 10 100 19 M19 0.77 87.5 10 M10 0.75 100 20 TRA4 0.82 10 80.0 Total 113 64.6 Genetic relationship of 50 soybean varieties Between 50 soybean varieties at the molecular level on the basis of analyzing RAPD with 20 random samples, we have established a tree diagram of the 50 soybean varieties (Figure 3.3) The result showed that 50 soybean varieties distributed into major branches (Branch I and Branch II) Branch I has only VK2 and DT12 with genetic distance compared to 48 remaining varieties of 21% (1 0.79) Branch II includes the remaining 48 soybean varieties with genetic distances ranging from 0% to 15% 14 Compared with the study of Nguyen Duc Thuan et al (2006) which was conducted on 30 varieties of soybean (from Mekong Delta Rice Institute) with 13 pairs of samples RAPD, the result showed that there are polymorphic samples, and four groups (groups A, B, C, D) are divided into similar coefficient from 0.8 to 0.98 Branch II Branch I Figure 3.3 Tree diagram of 50 soybean varieties bases on genetic similar coefficient and UPGMA clustering method 3.3.2 Analyzing DNA polymorphism of soybean varieties uses SSR molecular indicator In this study, we used 15 pairs of sample SSR in SSR PCR reaction to clone DNA fragments from the genomes of 50 soybean varieties, electrophoresis image of product SSR of 15 pairs (Figure 3.4A, 3.4B, 3.4C and 3.4D) Of the 15 indicators SSR used for analysis of genetic diversity in 50 soybean varieties there are 14 indicators for genetic polymorphisms (unless Satt460 without polymorphism result) Statistics of cloned DNA fragments, we detected 81 alleles at 14 locus, the number of polymorphic alleles at each locus varied from 15 to and six alleles on average (table 3.3) Indicator Satt489 has the most number of alleles with Value PIC (polymorphism information content) of the indicators SSR varied from 0.473 to 0.798, reaching 0.729 on average, and amplitude of the coefficient of diversity between the indicators is relatively narrow (0.473 to 0.798) (Table 3.3) This result for polymorphic is higher when comparing to a study by Tran Thi Phuong Lien et al on the same object (0.6326) Comparing with some other authors in the world, the result in genetic analysis of DNA polymorphism in our soybean is similar to the study of Abe et al (2003) on 131 varieties from 14 Asian countries (average coefficient diversity of 0.782), higher than the research of Narvel et al (2000) of 74 soybean varieties in North America (average coefficient diversity of 0.56) Table 3.3 The result of analyzing genetic diversity by indicator SSR Number PIC of Alleles Satt005 (ATT)19 D1b 0.770 Satt009 (ATT)14 N 0.690 Satt042 (ATT)27 A1 0.473 Sat_064 (AT)34 G 0.767 Satt146 (ATT)17 F 0.798 Sct_187 (CT)10 G 0.757 Satt150 (ATT)20 M 0.686 Satt173 (ATT)18 O 0.767 Satt175 (ATT)16 M 0.798 10 Satt373 (ATT)21 L 0.753 11 Satt431 (ATT)21 J 0.692 12 Satt489 (ATT)23(GTT) C2 0.784 13 Satt557 (ATT)17GAT C2 0.707 14 Satt567 (ATT)14 M 0.757 81 0.729 In the 15 pairs of sample SSR, pairs are often used to study genetic diversity are Satt_042, Satt_005, Satt_146, Satt_173, No SSR SSR Form Bond 16 Satt_175, Satt_009 and Satt_431, with many alleles: from 7-11 alleles The result of our study shows that these indicators for alleles range from 4-7 alleles The level of polymorphism corresponding to the genetic variation among soybean samples showed genetic diversity exists in the soybean, but not too large Highest similar coefficient was found between the SL and HG1 soybean of 0.97 This relationship is supported by the tree diagram through group analysis result (Figure 3.5) Analyzing tree diagram of Figure 3.5 shows that 50 soybean varieties were divided into two branches, the genetic distance between them was 29% (1-0.71): (1) The first branch, including varieties of rust infection and an intermediate cultivar VK2, can be divided further into two subbranches The first sub-branch (group I) includes varieties DBBT, CV, CSGL, DL, HG, QHCB, HG1, SL and VX93 with genetic distance close together, with similar coefficients ranging from 0.802 to 0.975 The second sub-branch divided into two small branches, including: (i) Small sub-branch I has varieties (group II) such as CSF, MH, DTBT, VMK, LVG, HN, CBD, ND, V79, DT84, DT12, and (ii) Small sub-branch II has varieties (group III): MD, MT2, CB7, VX92, TTHT and VK2, with similar coefficient of 0.65 to 0.95 (2) The second sub-branch includes resistant cultivars and intermediate varieties This branch also has two sub-branches: the first sub-branch consists of the varieties (group IV) such as DT96, CT2, HG2, VK3, MT1, CT1, PS, NS, M103, PT and PHCB with similar coefficient of , 75 to 0.95, meanwhile, the second subbranch is split into two small sub-branches Small sub-branch I has varieties (group V) such as CBU8325, ZG, CNB, HSP1, PI462312 with similar coefficient from 0.82-0.95 Small sub-branch II has varieties (group VI) such as MTD65, DT2000, HSP2, PI230970, 17 PI459025, PMTQ, DT95, PI200492 with genetic distances in the range from 0.68 to 0.95 Brand II Sub brand II Sub brand I Sub brand II Brand I Sub brand I Figure 3.5 Tree diagram of the relationship of 50 soybean varieties have different reactions to rust disease based on SSR molecular indicator The correlation between RAPD and SSR is in the analysis of soybean To compare the correlation of the two molecular indicators RAPD and SSR with the polymorphic analysis of 50 soybean, we noticed that using indicator RADP with 15/20 (75%) samples of polymorphism, meanwhile, indicator SSR of polymorphism is higher than 14/15 samples (count for 93.3%) This result can be explained by the short size and randomness of 20 samples RAPD, so they just start a random pair with a DNA genome Meanwhile, the sequence of SSR is the repeat sequence, studied and known in advance, they repeat many times in the body; therefore the level of polymorphism of SSR is higher In terms of value PIC, indicator RAPD has PIC ranging from 0.27 to 0.86 while the index of this indicator SSR varied from 0.473 to 0.798 Thus, using SSR makes the amplitude of coefficient relatively narrow diversity compared to RAPD On the other hand, 18 when using the tree diagram to analyze the genetic diversity, we noticed that in both RAPD and SSR of 50 soybean varieties are classified into two large branches with genetic distance, respectively 21 % and 29%, coefficient of genetic variation of SSR is more demonstrative than RAPD This result also reflected in large branches and sub-branch of the 50 soybean using two different indicators The result in RAPD is classified into two main groups, while SST of the 50 varieties are classified into five small groups (I, II, III, IV, V) with many similar coefficient (Figure 3.5) This result confirms that the method using SSR is higher and more reliable; this is the first base to search Vietnam soybean varieties carrying each specialized resistance gene 3.4 Analyzing polymorphism of leaf protein of several soybean varieties with different resistance to the rust To reach the soybean leaf proteome, we have chosen two pairs of soybean expressing for the antagonistic rust: imported soybean pairs are widely produced (DT12 and DT2000) and local soybean pairs (VMK and CBU8325) Soybean leaves infected with spores after 1, 3, 6, days are closely monitored On the leaves of 1, days, there are no lesions On the sixth day leaves, they begin to appear, but days have sporadic lesions blurred This is the time to find a powerful expression of proteins such as protein-related PR10 in soybean leaves by 2-DE electrophoresis and mass spectrometry Thus, we use leaves 6, days after infection to study 3.4.1 Mapping the proteome of soybean leaves by 2-dimensional electrophoresis technique 3.4.1.1 Separating soybean protein Soybean leaf protein of and days of VMK, DT12, CBU8325 and DT2000 (they are not infected sample) were separated by 2-DE and stained by coomassie G250 Figure 3.6 illustrates the 2DE electrophoresis images of leaves protein DT2000 at the time of days (Figure 3.6A) and days (Figure 3.6B) 19 Figure 3.6 2-DE Electrophoresis images compares to protein expression levels in leaves of soybean DT2000 at the time of days (A) and days (B) The magnified area expresses the comparison of different protein over the time The result shows that soybean DT2000 with 119 points of protein is detected on 2DE electrophoresis images in day sample while day sample only has about 103 proteins This result proved that leaf protein was expressed more in time of days Proteins separate similar positions very well, many proteins form streaks, or the corresponding position might be due to the same protein but have different isoform Soybean leaf proteins are separated on gradient strip bar with pH 3-10 and the length of 7cm, but electrophoresis images of proteins concentrated at about pH to This is quite reasonable, because most of the protein located in the range pH with slightly alkaline acidic or neutral On molecular weight, the protein is widely distributed, there are many proteins with molecular weights> 120 kDa, while some proteins are distributed in areas with low molecular weight ( 0.7 Genetic distance of 50 varieties of soybean was 29% 1.4 Has established 2DE electrophoresis map of soybean leaf proteins in DT2000 with 119 points of protein including 35 protein which were identified and classified according to nine functional groups Especially, there were proteins associated with disease resistance / defense, anti-stress, drought, found in soybean protein 1.5 In test samples of rust infected soybean (DT12, VMK), there are points of increasing protein and point of reducing protein compared to control sample, detection and identification have been 6/9 of protein Two protein samples in two varieties are susceptible to rust (DT12, VMK) with points higher than two rust-resistant varieties (DT2000, CBU8325), have been recognized and identified 4/6 of the protein Outlooks 2.1 Can use the rust resistance soybean to introduce for production or as raw materials in breeding 2.2 Can use information about genetic diversity at the DNA as the basis for breeding, but it is necessary to expand research and application techniques RAPD with the number of samples more specific and SSR samples screened in soybean 2.3 Research directions for these proteins rust resistant cultivars are promising approach with great potentials in detecting resistance mechanisms Therefore, we should continue to explore further research on the role and function of the protein changes of soybean varieties in order to understand the mechanism of infection, as well as rust leaf disease resistance of soybean plants [...]... systems of PSI, PSII, protein and other chloroplast proteins involved in glycolysis process at high percentage in the leaves These proteins closely related to the function of the leaf that is receiving sunlight and converted into sugar during photosynthesis In addition to proteins involved in 22 photosynthesis, in this result, we also recognize that the group of storage proteins (9%), proteins involved in. .. diagram of the relationship of 50 soybean varieties have different reactions to rust disease based on SSR molecular indicator The correlation between RAPD and SSR is in the analysis of soybean To compare the correlation of the two molecular indicators RAPD and SSR with the polymorphic analysis of 50 soybean, we noticed that using indicator RADP with 15/20 (75%) samples of polymorphism, meanwhile, indicator... infected varieties of 9-day DT12 and VMK 24 In soybean leaves, the concentration of large proteins, mostly proteins involved in photosynthesis (RuBisCO and the photochemical system protein) or protein reserves in the leaves The increase (or decrease or even disappear) the concentration of protein in some samples compared to controls was infected by rust (DT12, VMK) proved in the same transformation has... research has supplied the initial population data simultaneously hint system protein research interests is to be able to approach closer to the disease mechanism, in addition to studies of the genetic instructions 3.4.2.2 Comparing protein expression levels between infected and soybean rust resistance To find out the changes of the protein between some soybean varieties infected and resistant, we have... times), including 8 points (points 1, 2, 3, 4, 5, 6, 7 , 9) with increasing concentration and a point (point 8) with reduced expression levels when compared to sample (Figure 3.9) Notably, in addition to four points (2, 6, 8 and 9) are large protein remaining 5 points (1, 3, 4, 5, 7) are relatively low in protein Figure 3.9 Comparing the diversity of soybean leaf protein and the experiment in the infected... had on metabolic level, the change of gene activity leads to protein content of some process of change as infection occurs The change of the content of these proteins is related to the disease process or other physiological responses of plants by the consequences of infection Chromatography with mass spectrometry ESI-Q-TRAP, differential protein spots were identified as Table 3.5 The recognition result... Function Increasing/ Decreasing Increasing 1 gi|5902586 212 15.4 Photosynthesis 2 gi|5606709 179 16.3 Photosynthesis 3, 4, 5 6 gi|169898 128 29.0 Storage 7 gi|170088 122 32 Storage 8 gi|15285625 89 20.1 Metabolism 9 gi|3114769 235 54.2 Increasing Increasing Increasing Increasing Increasing Creating energy in process of Decreasing fixing carbon 25 Currently, there have been some studies of the protein in. .. participated in the study analyzed amino acid composition in seeds Of these, there are seven resistant varieties, 4 intermediate varieties and 9 infected varieties including hybrids, local and imported varieties The result showed that the presence of 17 types of amino acid and amino acid content of each type in each soybean sample was studied; at the same time it showed that the three groups (resistant, intermediate,... map of soybean leaf proteins in DT2000 with 119 points of protein including 35 protein which were identified and classified according to nine functional groups Especially, there were 3 proteins associated with disease resistance / defense, anti-stress, drought, found in soybean protein 1.5 In test samples of rust infected soybean (DT12, VMK), there are 8 points of increasing protein and 1 point of reducing... EBI and Swiss-Prot, of the 35 protein in soybean leaves is consistent with the most basic components in the leaves, in which protein have photosynthetic function with the highest proportion of 29%, then the energy metabolism proteins of photosynthesis cycle (17%), and the proteins involved in glycolysis system (11%) This result is consistent, so the leaf protein groups involved in complex optical systems ... proteins are common as a basis for comparison, the study of disease related symptoms Our result is one of the first statistics, in which protein on the gene with 119 points, we identified the 35... in Asia and causing significant damage in the yield of soybeans in many countries Signs and symptoms of disease: Signs of the disease are the dots in the leaf blade about mm, growing gradually... there are 14 indicators for genetic polymorphisms (unless Satt460 without polymorphism result) Statistics of cloned DNA fragments, we detected 81 alleles at 14 locus, the number of polymorphic