Tai lieu chia se tai: wWw.SinhHoc.edu.vn MINISTRY OF EDUCATION AND TRAINING THÁI NGUYÊN UNIVERSITY VŨ THỊ THU THỦY CREATING DROUGHT TOLERANCE LINES AND ISOLATING CYSTATIN GENES RELATED TO DROUGHT TOLERANCE IN PEANUT (Arachis hypogaea L.) Major: Genetics Code: 62.42.70.01 PHD THESIS ABTRACT Thai Nguyen - 2011 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn INTRODUCTION Reasons for choosing the topic Groundnut is a valuable crop in many aspects and many countries around the world wishing to expand and develop groundnut production Groundnut plant belongs to the bean group with low drought tolerance Compared to many other crops, groundnut specially needs water as its roots not have absorbing hair, groundnut fruit is shaped underground Statistical results show that groundnut production accounts for 40% of total cultivated land area of short-term industrial crops, of which 2/3 depends on the rain For many localities, groundnut is the main crop, investment in the development of groundnut production however has not yet been commensurate with its inherent potential There are many methods of improving plant varieties of which tissue culture of plant cells is an effective technique, allowing applications and improvement of many features of the plant During the culture, the cells can be genetically modified because of the influence of the environment If mutation generated agents are combined, the mutation generated frequencies will be significantly increased This is significant in creating the source material for breeding Effectiveness of application of plant cells technologies to improve the tolerance to externally adverse conditions is continuously confirmed In Vietnam, the birth of two rice varieties DR1 and DR2 which resist the drought and the cold primarily demonstrates the possibility Following is the research to improve drought tolerance and salt-resistance of sugarcane by Yadav et al (2006) and of wheat by Abdelsamad et al (2007), etc Drought tolerance of the plant is characteristics specified by multiple genes therefore the search and analysis of genes related to drought tolerance are studied by many scientists Several genes related to drought tolerance of plants has been isolated and published such as LEA genes in soybean and green beans, P5CS gene in soybean, cystatin genes in green bean plants, DREB genes in Arabidopsis, etc Cystatin gene (Cys) of vegetation was first published on rice by Abe et al (1987), so far Cys gene has been isolated in many species of higher plants in both monocots and dicotyledonous (Cys in green beans, Cys gene in potato gene, Cys genes in maize) Studies of the cystatin gene have been widely discussed focusing on its relationship to resistance to drought, cold and salt etc With the above mentioned reasons and from the practical needs of groundnut breeding optimism towards improving drought tolerance, we have carried out the thesis topic: Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn "Creating drought tolerance lines and isolating cystatin genes related to drought tolerance in peanut (Arachis hypogaea L.)” Research objectives - Create groundnut strain with drought tolerance higher than original breed using tissue culture of plant cells - Compare cystatin gene sequence of groundnut strain selected from scar tissue with that of the peanut cultivars Research contents - Screening scar tissue strain resistant to water loss affected by dry blowing and gamma irradiation combined with dry blowing - Analyzing the fluctuation of the number of selective lines through the generations - Making comparison and defining the discrepancy in genome of the line selected by RAPD technique - Amplifying, separating and defining the sequence of the cystatin gene of selective groundnut line and the original line New contribution of the topic i) The thesis is a systematic research on application of technologies in plant cells to improve and enhance drought tolerance of groundnut, from creating scar tissue, processing scar tissues that create somatic mutation, selecting cell lines resistant to dehydration, reviving plants, generating plants and analysis, assessment through generations, selecting lines of groundnuts preeminent by drought tolerance and some biological and agricultural features ii) Callus were treatment of scar tissue by gamma rays has been reduced in height and rate of regeneration plants, change colors and leaf shapes The findings are five indicators specific RAPD two lines of contact selectively RM47, RM48: RM48/OPA07750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348-200bp; RM47/OPH08-200bp Discovery of peanut cystatin genes of group I phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%) The gene contains an intron and two exons coding for 98 amino acid protein Cystatin of RM48 line derived from scar tissue is processed by gamma rays associated with the blower has positions of amino acid differences compared to the same original L18 iii) Determine the difference compared to the same original L18 on drought tolerance of three lines lost RM48, RM47, R46 can be derived from scar tissue dehydration is treated by gamma rays (2krad) with blower continuous hours in in vitro culture systems Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn The meaning of scientific and practical 5.1 Scientific Significance i) The results of the thesis provide scientific data to guide application of plant cells to improve drought tolerance of groundnut The technique selected cell lines under dehydration and treatment of scar tissue to increase the frequency of mutations arising The approach and methods to evaluate the difference of the selected line compared to just touch base in terms of morphology, expression yield component traits, drought tolerance, grain characteristics and biochemical differences in the genome ii) Provide information on the cystatin and cystatin genes in peanut Handling of scar tissue by radiation combined with water is blown causing agent appeared cystatin gene mutations as evidenced by differences in genetic sequences of the RM48 (derived from scar tissue to be irradiated with blow dry) than the R46 line (derived from scar tissue be blown) and like the original L18 5.2 Practical significance The results of a comprehensive review of drought tolerance of groundnut extent of scar tissue, germinating seeds and young plants as a basis to assess and apply measures to improve drought tolerance of groundnut Results bred three communication lines derived from scar tissue under treatment by irradiation and the blown advantage of drought tolerance Vietnamese and some characteristics of agricultural biology, biochemistry, can foster the seeds of new varieties or material for hybridization The structure of the thesis The thesis consists of 128 pages (including references), divided into sections: Introduction includes pages; Chapter 1: Overview document, 34 pages; Chapter 2: Materials and Methods, 14 pages; Chapter 3: Results and discussion Comment, 54 pages; The conclusions and proposals: pages; works were published concerning the thesis: page References: 15 pages; thesis has 22 tables, 20 pictures There are 145 references in Vietnamese and English Chapter DOCUMENT OVERVIEW The thesis has reference materials and review 34 domestic and 98 foreign documents with related content, including: (1) Groundnuts and drought-resistant properties of the peanut, (2) Advanced research drought tolerance of crops by plant cell technology, (3) Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn Analysis and evaluation of selected lines derived from callus culture (4) Genes related to drought tolerance in groundnut; (5) Cystatin and the role of cystatin in plants With the lead data collected, the analysis has confirmed that peanut valuable and important position in the economy of many countries around the world The trend of climate change that changed the elements of environmental conditions and a growing number of evaluation studies, breeding optimism towards higher resistance is made Like other crops, the methods used in breeding, including hybridization communication, breeding, mutant selection from the population and use of modern biotechnology The application of modern techniques of biotechnology to improve plant resistance was conducted in two directions, which are selected lines and transgenic somatic mutations The success of gene transfer techniques have been published on several crops and several properties related to the drought tolerance of plants has improved The technique selected cell lines in higher plants based application understanding of the omnipotence of the cell; heterogeneity of tissue or cultured cell populations; influence of culture medium in forming a complete body make cultured cell populations can be considered a population of plant cells, thereby screening the individual will be faster and more efficient than conventional breeding methods applied on intact plants Select the line by increasing resistance to the unfavorable factors of the external environment has been successful on several subjects such as rice, dry rice, wheat, tobacco These are our suggestions for choosing in vitro culture techniques to improve drought resistance in peanut Drought tolerance of plants is due to multiple gene traits decision Search trends drought gene research is a major concern of many authors The genes involved in drought tolerance of groundnut published in recent years such as LEA genes related to cellular dehydration Su's research et al (2010) on the same communication Luhua 14 detected at least eight genes LEA AhNCED gene encoding synthetic 9-cis epoxycarotenoid dioxygenase was also confirmed related to the resistance of plants, the peanut AhNCED isolated by Wan et al (2005) size 2486bp, encoding a protein consists of 610 amino acids PLD gene encoding phospholipase D was also confirmed related term was found in two types of communication is through AhPLD1 and AhPLD2 Nakazwa team (2006) Cystatin phytocystatin in plants is called, consists of two groups, different in size, mass and areas associated with the cysteine proteinase Since 1987, cystatin genes in rice were isolated, the cystatin gene has been isolated in many plants, but the peanut was little known Study the function of cystatin, many authors discuss the relationship of cystatin with tolerance of plants The complexity of the structure of the cystatin genes, the ability of gene expression in different growth stages of crops and in relation to the resistance of the disadvantages of Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn the external Some published on gene expression of cystatin-related drought tolerance has been demonstrated cove on bean plants, vegetables genes, sea mustard, winter wheat is the basis for our continued search and analysis on peanut cystatin genes Chapter MATERIAL AND METHODS 2.1 Plant materials The thesis using 10 peanuts cultivars as the materials research In particular, varieties (L05, L16, L18, L23, L24, V79, MD7, MD9) offered by the Center for research and development Legum, Institute of Food Crops Vietnam SD30 variety provided by Center of Agricultural Extention Nam Dinh province and red peanut (red BG) due to Bean Development Center Legum Viet Yen district, Bac Giang Province has to offer 2.2 Chemicals and equipment Using the chemical purity and dedicated derived from reputable companies such as the drug company's growth Sigma; Taq-polymerase, EDTA, SDS, agarose Invitrogen's 2.3 Research methodology 2.3.1 Cultured in vitro method To create the technical communications in vitro culture, the culture of the steps: creating scar tissue completely in the dark (10 days) to conduct the airflow caused by dehydration of the box sterile culture and irradiated and then combined to cause dehydration by blowing dry recycling plant, creating a complete tree as described by Nguyen Thi Tam et al (2006) 2.3.2 Field research methodology Mark individual plant lines regenerated from callus To make the planting and care as directed by the Ministry of Agriculture and Rural Development Track indexes and agronomic performance of plants at maturity 2.3.3 Method of physiological, biochemical Quantification of soluble protein by the method of Lowry; quantitative lipid Soxhlet amylase activity by the method of Heinkel; The methodαmethod; Define of sugar analysis Assessing dehydration tolerance of scar tissue by staining of cells et al Towill (1975) Assessing drought-resistant seed germination stage method caused due to sorbitol -amylase, sugar.α7% defined tolerance by determining the activity of Assessing drought tolerance at seedling stage by artificial methods to cause limited by Le Tran Binh et al (1998) Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn 2.3.3 Methods in molecular biology Total DNA extraction method Gawell et al (1991) DNA polymorphism analysis by RAPD technique Isolation of genes by PCR technique Separation of the gene by the method of Sambrook et al (2001) Gene sequence was determined on the ABI PRISM 3100 Avant automatic Analizer Genetic, Institute of Biotechnology 2.3.4 Methods of data analysis Analysis of statistics with Excel software as Chu Hoang Mau (2008) Data analysis by RAPD NTSYSpc software version 2.0; Results of genetic analysis using BioEdit and DNAstar software 2.4 Study location The experiment was conducted at the University of Pedagogy; University of Science; Institute of Life Sciences-University of Thai Nguyen Irradiation gamma ray irradiation at the National Center, Tu Liem, Hanoi DNA sequences were determined at the Institute of Biotechnology Field experiments are located at the Forest cane, Quang Vinh Ward-Thai Nguyen city Chapter RESULTS AND DISCUSSION 3.1 RESULTS CREATED THE DROUGHT TOLERANCE BY TECHNICAL CALLUS TREATMENT IN THE IN VITRO CULTURED SYSTEM 3.1.1 Screening lines callus subjject blow dry 3.1.1.1 Ability to create scar tissue and the growth of scar tissue studied 10 breeds optimism For the purpose of assessing scar tissue of the same communication in in vitro culture systems as the basis for the research to create drought-tolerant line of communication with the plant cell technology, we surveyed the ability to create scar tissue from the embryo of the seed cells and speed the growth of scar tissue in 10 varieties studied communications Research results show that rate of scar tissue from embryos of the 10 varieties ranged from 82.71% optimistic (L18) to 98.55% (V79) The same communication rate of scar tissue over the same 97% MD7, MD9, V79 Same can create scar tissue ratio lower than 90% include the same L18, L23, Red BG In particular, the proportion of scar tissue created at least the same L18 (82.71%) The volume of the same communication scar tissue is formed ranged Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn from 115.00 mg (L18) to 198.00 mg (V79) The same L05, L23, MD7, MD9, SD30, V79 mass of scar tissue over 180mg Four varieties L16, L18, L24 and Red BG mass of scar tissue formed by more than 160 mg The results showed the ability to meet the growth of scar tissue and scar tissue of the 10 varieties of communication, be sure to use for subsequent studies in selected cell lines 3.1.1.2 Dehydration tolerance of scar tissue the same communication research Dehydration tolerance of scar tissue is determined by the dehydration, the results of rapid assessment of scar tissue viability by TTC staining method and determine the survival of scar tissue after treatment by blowing dry The results determine the survival rate of scar tissue after treatment with blown shown the stamina of cells and this is the basis for screening cell lines The scar tissue dehydration under which we obtained the materials to make recycling plant for selecting drought-tolerant line of communication Results of studies of tree regeneration under dehydration scar tissue that, like us all participated in the study were able to regenerate plants from callus survived The models for survival rate from 83.33% tree reborn to 100.00% Based on research and results, we choose the time scale of 84% water loss from the initial fresh weight and tissue survival rate of about 10% to 20% is the threshold to filter the line Scar tissue processing and technical communications dry wind scar tissue, we identified peanut varieties L18 resistant dehydration and choose the best cream puff dry hours threshold to filter Results of assessment of dehydration tolerance of scar tissue, associated with evaluated tolerance of 10 seed lost in the period when the seed germinates and physiological drought period when young trees have shown that artificial limit , in 10 varieties studied communications, L18 resistant varieties dehydration least, have - amylase and sugar content in the lowest group inαthe activity of the day when the seed germinates term, the lowest drought index This result is consistent with published by the Center for beans, Institute of Food crops and trees provide food Vietnam 3.1.2 The effect of gamma rays associated with dry wind to the survival and regeneration of plants like communications L18 Like peanut varieties L18 is defined as strains resistant to drought, low water loss under dehydration among 10 varieties of research communications In addition, L18 is the same high-yield varieties So, with the goal of improving drought tolerance of the varieties have lost tolerance cream, we conducted surveys combined effects of gamma irradiation for the subject screening dehydration using the technique for blowing dry peanut varieties L18 with scar tissue Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn Results exploring the effects of gamma-rays are performed on the fifth dose level radiation: 0.5 krad; 1.0 krad; 2.0 krad; 3.0 krad; 4.0 krad blown with scar tissue in hours The results determine the rate of tissue regeneration of tree survival showed regeneration of scar tissue plants affected by irradiation combined with blown ranged from 21,30% to 88,87% Compared to the regeneration of plant tissue to survive only be blown, then the process of scar tissue by irradiation with blower reduces the rate of tree regeneration Significant difference of plant regeneration is affected by irradiation combined with the blower that is, tree regeneration of a change in morphology, particularly, variation in color and leaf morphology With low-dose irradiation (0.5 krad; 1krad; 2krad), the leaves are still green as the tree control But with high-dose irradiation as 4krad 3krad and greens of the leaves appear in about 1-2 weeks after the leaves begin to yellow UA Small leaf morphology and leaf curling occurs Plant growth speed is inversely proportional to the intensity of the radiation dose At higher doses of radiation phenomena plants gradually died, relatives barren Regenerating tree height is much lower than the control plants Table 3.6 The effect of irradiation combined with blower am to survival and regeneration of tissue in the same communication tree L18 Radiation dose combination blower hours Rate of survived tissue (% ) Rate of regeneration (% ) 0,5krad 34,23 ± 0,37 47,63 ± 2,37 Normal 1,0krad 33,65 ± 0,52 88,87 ± 5,57 Normal 2,0krad 28,42 ± 0,43 38,87 ± 5,54 Normal 3,0krad 18,87 ± 0,59 26,17 ± 4,97 Small, yellow and fallen leaves, curly leaves edges 4,0krad 12,51 ± 0,42 21,30 ± 6,03 Small, yellow and fallen leaves, curly leaves edges Patterns of regenerated trees Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn A B C E D G Figure 3.3 Some pictures of the same tree regeneration touch L18 6-week-old stage A: Plant regeneration not blown; B: Plant reproduction under blower hours; C: irradiation dose 0.5 krad + blower hours D: irradiation dose 2.0 krad + blower hours; E: irradiation dose 3.0 krad + blower hours; G: irradiation dose 4.0 krad + blower hours Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn From the results of the evaluation survival and regeneration of scar tissue scar tissue after treatment with gamma irradiation we have identified the critical dose of scar tissue radiation dose 4krad la communication If you exceed the limit 4krad most dead scar tissue, can not produce enough of the line model for the further research, especially research on the selection line However, to obtain multiple mutations need to determine the appropriate dose of irradiation, to have created the beneficial genetic variation, both the rate of the tissue regeneration and high survival Therefore, we selected the dose 2krad blower with hours to screen the tissue and the entire plant Result of treatment of scar tissue in in vitro culture systems of peanut varieties L18 continuously blown by hours and radiation dose in combined blown 2krad continuous hours were obtained from 167 current and measuring 198 models communication lines are created 3.1.3 Agro-biological characteristics of the communication line in R0 and RM0 The study population lost R0 (plants regenerated from callus resistant blower), RM0 (Plant regeneration from callus under irradiation with blow dry) found that the communication line from the test tube to grow in the field (spring, 2008) that genetic variation is much larger than the original species in all study criteria (Table 3.7) Table 3.7 Agronomic characteristics of the population selected communication R0, RM0 ( X : Mean value; S X : average deviation form; Cv%: coefficient of variation) Target tracking Original var R0 Population RM0 Population Main body X ± S X height (cm) Cv % 37,00 ± 0,58 57,00 ± 4,61 18,50 ± 1,91 2,70 25,58 10,32 Number of branches/ plant X ± SX 8,33 ± 0,33 3,00 ± 0,39 6,75 ± 0,57 ( branches) Cv % 6,93 41,57 8,38 Number of X ± S X fruit/ tree plant Cv % ( fruit) 20,67 ± 0,67 22,80 ± 1,81 14,00 ± 1,96 5,59 25,05 13,98 X ± SX 16,33 ± 0,33 14,80 ± 1,50 8,17 ± 1,36 79,00 64,91 54,65 Số chắc/cây Tỷ lệ 10 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn As such the process of selecting the line lost drought include: (1) Create callusand scar tissue; (2) regeneration of trees and Tree Planting complete (3) track and analyze the genetic lines of selectivity Scar tissue in the peanut processing plant in vitro culture, selective survival of scar tissue, regenerating plants and monitoring populations and RM0 R0 can draw some comments below: (1) The line comes from scar tissue dry wind bear up tree height than the original species, while the line is derived from scar tissue in combination with radiation suffered blow dry reduced plant height compared to the same original (2) Populations of plants produced by tissue culture techniques of plant cells have some quick / plant than it resembles the original Military R0 can quickly reach / tree, populations reached 6,75 RM0 fast / tree, like the original with rapid 8,33 / plant (3) Population R0 is 22,80 fruit / tree, more than the same root (reached 110.30% from the same root); population RM0, with some troops may fruit / tree less like the original, only 14.00 fruit / tree (67.73% from the same root) (4) Coefficient of variation for genetic research targets of the two forces can be created by plant tissue culture techniques of plant cells larger than the original species From the evaluation results in generation of R0, RM0, we chose the line is derived from the same communication L18, in which three lines originating from the scar tissue is influenced by the blower is R44, R46, R48 and lines derived from scar tissue affected by radiation combined with blower is RM46, RM47, RM48, RM49 a starting material to review and filtering in the next generation 3.2 RESULT ANALYSIS OF SELECTED LINES THROUGH THE GENERATION 3.2.1 Assess the stability of agricultural biological characteristics of the selected contact in the first generation, the third generation Compared to other crops, peanuts are difficult maintenance, and low germination when stored last time Number of fruit trees in the first generation touch is not large enough to carry out research related to tolerance Therefore, the whole grains in the military can communicate selectively R0, RM0 we look into the service next year looks Results assessing the stability of agronomic traits on the main stem height, number of branches / plant, number of fruits / plant, number of fruits make / tree generation R1, RM1 look at winter harvest in 2008 (Table 3.8 ); generation R3, RM3 look at winter harvest in 2009 (Table 3.9) showed the stability of agricultural biological characteristics of the communication line is derived from the scar tissue has contributed to confirm the value of carefully Arts tissue culture plant cells could create new strains and shortening breeding, increasing the efficiency of the process of breeding 11 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn 3.2.2 Evaluation of selected lines in the fifth generation We assess the current selection in Thursday generation through the analysis of a number of state and other biochemical indicators; assess drought tolerance in seed germination stage, seedling stage and differences in genome by RAPD technique 3.2.2.1 Agronomic traits, grain yield and quality of the communication line choice in the fifth generation Results evaluation of stability of agronomic traits of selective line in the fifth generation Fall Winter crops 2010 shows contrition biological agriculture norms are the fluctuations in the low range 1,66% to 7,23% We conduct assessments and analyses of the characteristics quality grain yield of lines selected fifth generation through identifying elements such as productivity mass volume 100pods; 100 seeds; quality assessment on aspects of Biochemistry through identifying a protein, lipid content Results of research on biological characteristics of agriculture the quality of the contacts selected derived from callusis dehydrated and callusinfluenced irradiation combined with handle causing dehydration we selected lines has outstanding features, including: (1) The R46 has a mass of 100 fruit and seeds; (2) Lines of RM47 protein content higher; (3) Line of RM48 lipid content higher 3.2.2.2 Results evaluated tolerance of the selective in the fifth generation Drought tolerance of plants in general and in particular peanut is a trait difficult to control in the field Therefore, we evaluated the tolerance of the current generation of communication selected fifth in seed germination stage and seedling stage Causing the current term optimism fifth selective generation seed germination stage by treating the seeds with 7% sorbitol, and then α-amylase and determine drought tolerance through the activity of sugar The results showed that the -amylase and sugar content of the seeds germinated andαactivity of tended to increase from to days old days old and then decreased in stage nine days of age (Figure 3.5 and 3.6) Analysis of correlation between the variation of α-amylase activity and sugar content of communication like L18, L23 and the line selected in the period when the seed germinates physiological limits, the results presented in table 3.11 Table 3.11 shows that the α-amylase activity, sugar depends linearly on correlation coefficient (R) ranged from 98,11% to 99,99% The α -amylaseα activity of high resolution will make the process of starch to sugar occurs powerful, guaranteed to provide nutrients for the germination of seeds, especially the adjustment of osmotic pressure in cells extreme 12 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn conditions The -amylase and the way of the line, likeαdifference in the activity of us at the time of seed germination vary regarding their drought tolerance 0,7 1,8 1,6 0,5 % hạt nảy mầm ĐVH Đ/m g h ạt nảy m ầm 0,6 0,4 0,3 0,2 0,1 1,4 1,2 0,8 0,6 0,4 0,2 Ngày tuổi nảy m ầm R44 R44 R46 R48 RM46 RM48 RM49 L18 L23 R46 R48 RM46 RM47 RM48 RM49 L18 L23 RM47 ngày Figure 3.5 Variations on α-amylase activity of the touch like L18, L23, and the selection of limited physiological conditions ngày Figure 3.6 Fluctuations of similar sugar lost L18, L23 and the limited selection in terms of physiological Table 3.11 The correlation between the activity of α-amylase and sugar content of communication like L18, L23 and the selection stage seed germination the Regression equation No Varieties selection R44 1,13x + 0,72 0,9881 R46 1,61x + 0,57 0,8972 R48 1,04x + 0,77 0,8379 RM46 1,83x + 0,34 0,9811 RM47 0,54x + 1,04 0,9945 RM48 2,66x + 0,66 0,9999 RM49 1,04x + 0,68 0,9862 L18 1,07x +0,70 0,8466 L23 2,26x + 0,19 0,9983 and Correlation Assessing drought tolerance at seedling stage of the selection fifth generation by determining the rate of withered tree, the tree recovered in the first days time limit and determine the relative drought indices for RM48 line with the highest drought index 13 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn (10,089.20%), then to the R46 and L23 cultivar At least the current R44, RM49, and like the original L18 In the seventh line of communication like the original selection and L18 have lines selected drought index is relatively high, including two lines derived from callus resistant blower (R46; R48) and three lines derived from tissue scar affected by irradiation combined with a blower (RM46; RM47; RM48) 3.2.2.3 Assessing the differences between the genomes of the communication techniques selected by RAPD The results of analysis with 25 primers RAPD reaction randomly collected a total of 1254 segments, estimate the size of segments ranging from 0,2 kb to 4,2 kb Total segment polymorphisms as 255 segments, accounting for 20.33% of the segment to be cloned The results compare differences in rates at the molecular level between the genomes of the selected pair and like the original show, gene system error rate from 1,521% to 9,143% The level is the biggest difference dong dong RM48 and R48 (rate difference of 9,143%) Figure 3.10 describes the relationship of the seven original species and the selection at the molecular level with 25 random primers in RAPD reactions L18 RM46 R44 R46 RM47 RM49 Figure 3.10 Diagram describes the relationship of seven lines with similar selection at the molecular level the original L18 with 25 random primers The results in Figure 3.10 shows the selected communication line and the same original L18 are classified into two groups with genetic distance is 12% Group I: There is only one line of RM48, which is derived from the scar tissue affected by a combination of radiation and blow dry, with genetic distance from the line and just rest in branch is 22% Group II: consists of the R48, RM49, RM47, R46, R44, RM46, and like the original L18 Genetic distance of the original species selection and L18 is 9.5% (1-.905 = 095) Group II is divided into two branches, first branch is the original species (L18); second branch, is divided into several small branches of the remaining selection, R44 and R46 along two lines originating from the scar tissue under blowing dry, which differs from the original species with a low 1.521% and 1.522% 14 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn Coefficient of genetic diversity (HRAPD) of seven selected communication line within the 25 primers used at the molecular level were identified: HRAPD = 11.09% Have identified five molecular indicators RAPD featured in two communication lines with primers selected randomly appear RM48 line features four sizes with four primers In particular, specific primers OPA07 size appears at 750bp (RM48/OPA07-750bp) primer OPA08 size characteristics appear at 500bp (RM48/OPA08-500bp) primers specific OPB05 size at 900bp (RM48 / OPB05-900bp) specific primers UPC348 size appears at 200bp (RM48/UPC348-200bp) OPH08 prey appears a characteristic 250bp in size with the RM47 (RM47/OPH08-200bp) Gather the results analysis of the characteristics of agricultural biology seed quality resistant and molecular biology characteristics of selective line derived from callusis blow dry and callusis irradiated combined with blow dry breeds L18 is presented in ficture 3.11 and table 3.16 Table 3.16 Some characteristics of selective lines and variety L18 Characteristic RM48 RM47 R46 L18 Plant Regenerate from calli treatment with irradiated+ dry blow Plant Regenerate from calli treatment with irradiated+ dry blow Plant Regenerate from calli treatment with dry blow Imported from China Plant height (cm) 35,46 ± 1,02 41,63 ± 0,54 45,77 ± 0,79 30,62 ± 0,84 Weight of 100 pods (g) 108,27 ± 6,98 103,43 ± 4,46 145,12 ± 5,05 116,59 ± 2,84 Weight of 100 seed (g) 46,53 ± 2,89 41,11 ± 2,50 56,27 ± 1,32 47,81 ± 0,55 Protein Content (% KLK) 27,72 ± 1,96 38,97 ± 4,25 32,08 ± 2,54 34,47 ± 2,85 38,67 ± 0,88 36,89 ± 0,89 34,67 ± 0,77 36,00 ± 2,67 10089,20 5272,80 7662,38 4725,09 Source Lipid content (% KLK) Drough Index Specific Primer RAPD RM48/OPA07-750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348-200bp; RM47/OPH08-250bp; 15 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn RM48 RM48 RM47 RM47 R46 R46 L18 L18 Figure 3.11 Images of fruits and seeds communication lines selectively earnings at fifth generation, just like touch touch L18 and L23 16 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn 3.3 ISOLATION AND SEQUENCING GENES CYSTATIN FROM PEANUTS 3.3.1 Amplified DNA genomes of cystatin genes from peanuts After the extraction of total DNA glass cleaner and headlight on the appropriate concentrations we have conducted gene cloned from DNA by PCR cystatin genomes of seven lines of seed selection L18 and same L23 Human genes are check result on 1% agarose gel with marker (Figure 3.12) L23 RM49 RM48 RM47 RM46 R48 R46 R44 L18 M Figure 3.11 Photos electrophoresis products of the same human cystatin gene L18, L23 and line research communicationh PCR reaction product obtained in Figure 3.11 shows, only one DNA band, approximately 500bp in size, consistent with theoretical calculations on the size of cystatin genes that Yan et al (2004) when This gene was isolated from mRNA Therefore, we believe that it is the result of PCR with primer pairs-AraF Cys / CysArar Cystatin gene was cloned from the DNA like us L18, L23 and selective communication line near 500 bp in size However, to affirm correctly that cystatin genes, we performed separate line, identifying the sequence comparison with cystatin gene sequence was published We perform a separate line and determine the order of cystatin genes dong R46, RM48 (two lines have the highest tolerance), similar to the original L18 (drought-resistant cream) and similar to L23 (with tolerance good) for comparison purposes cystatin gene sequence of lines and varieties 3.3.2 Results of cloning and sequencing of genes cystatin Result of splitting the colonies carrying cystatin genes were tested by clonyPCR reaction Defining the nucleotide sequence obtained from the plasmid of the sample is treated with DNAstar software shows that cystatin genes of the same communication L18, L23, R46 and RM48 lines are 461 nucleotides in size 17 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn Comparison of sequences with nucleotide sequences isolated from cDNA of cystatin genes in plant gene bank communication on international codes AY722693 is the sequence used to design primers and sequence bearing codes EU723567, we get that the cystatin genes encoding proteins that we isolated, including two exons and one intron The first exon of the gene segment of 102 nucleotides starting from position to 102 and the second exon 195 nucleotides, from position 267 to 461 zone in the middle of a 164 nucleotide intron from position 103 to 266 3.3.3 Results comparing the gene sequence and protein cystatin 3.3.3.1 Comparison of nucleotide sequences of the cystatin gene Results comparison of nucleotide sequences of the same tribe of cystatin genes L23 with two lines touch L18 R46 RM48 is created by the technology of plant cell tissue cultured has the wrong In particular the sequence of nucleotides and the R46 L18 varieties derived from callussuffered dehydration is the same entirely The wrong nucleotide position RM48 has 19 different than the same root (L18) Cystatin-like tribe's genome Nucleotide L18 has 14 different compared to the same position wrong L23 Cystatin genes of leguminous green relatively conservative there is no difference in terms of genes of drought well and cystatin-like drought worse Results identified the nucleotide difference cystatin genes peanuts (table 3.17) shows the difference of cystatin genes appear in both the Club and the intron exon In exon just the wrong positions L18 has L23 and 12 other wrong locations other than the RM48 In exon just the wrong positions L18 has four different varieties of L23 and had a wrong locations other than with the RM48 In addition cystatin genes and RM 48 breeds there are 11 L23 position discrepancy lies in the intron As such is derived from the same L18 two lines touch the wrong selection can vary in the sequence of nucleotides of cystatin genes and the difference between the same expression is likely to be poor (L18) with the same term is likely to be a better term (L23) Research results demonstrate effective processing gamma rays associated with blow dry raises cystatin gene mutation frequency in comparison with callusis only liable to blow dry 3.3.2.2 Comparison of nucleotide sequences in the coding region Because cystatin gene isolated from DNA containing the intron of communication does not exist in the process of translation, we proceed to remove the pairing of the intron and exon, the result is the 297 nucleotide long encoding cystatin Compared with the protein sequence of the gene segment encoding cystatin of plant sizes, respectively published in the international gene banks 18 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn Results evaluated through analysis of level differences, similarity level (Table 3.18) found four genes encoding the order disrupted communication co cystatin levels similar to cystatin the order of other crops published in gene bank international, from 45.9% to 100% Nucleotide sequence similarity of peanut-Definition from 98.0% to 100% Especially, not have any differences on any of the same nucleotide selectively communicate with the L18 and R46 of the same nucleotide sequence Arachis hypogaea L on the international gene bank (code AY722693) Establishing the genetic relationships of the gene encoding cystatin of 10 sequences, the relationship diagram of the gene encoding cystatin be classified into two groups: Group I sub-divided into three large groups and small groups of women includes the same communications and other crops, including communications lines and similar distribution in the same group Group and the same communication lines (R46, RM48, L18, L23, AY722693) is closely related to cystatin with green beans (number AM712476), then the seed of rice cystatin-number S49967, barley and riceY12068 -AB125973 Group II is the longest distance to the remaining group and only one species is kiwi fruit (AY390352), error rate is different than the peanut plant cystatin was 41.5% Table 3.18 High level similarities and differences in the coding sequences of genes Similarity level 1.L18 cultivar R46 line RM48 line L23 cultivar Arachis hypogaea (code AY722693) Oryza sativar (code: S49967) Actinidia deliciosa (code AY390352) O sativar japonica (code AB125973) Vigna radiata (code: AM721476) differences level 10 Wheat (code Y12068) 3.3.2.3 Comparison of amino acid sequence of the protein cystatin Nucleotide sequence of the cystatin gene encoding the same communication L23, L18 and two communication lines R46, RM48 was translated by software code to create molecular DNAstar of 98 amino acid protein, with a code beginning with AUG 19 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn provisions of methionine (M) and the code ending UAA Results comparing the order of research with four specialized software BioEdit represented by Figure 3.16 Comparison of amino acid sequence of the communication research, we found that all four amino acid sequence contained two regions of the group is conservative and QVVAG LARFAV The specific locations of the two segments are conservative: L22A23R24F25A26V27 and Q49V50V51A52G53 Compared with the classification of Margis et al cystatin gene (2008) and Martinez et al (2008), we found cystatin genes of the same communication and research under the category of phytocystatin Figure 3.16 Comparison of amino acid sequences of the four sample Combined with the results of genetic analysis, research results show that the location of glutamine 34 (Q34) and asparagine 35 (N35) is a pair of amino acids encoded by the first three exon junction of the cystatin Position corresponding to position it as arginine 34 (R34) and asparagine 35 (N35) of cystatin RM48 line Glutamine and arginine at position 34 is two different amino acid structure characteristics of the original R, in which glutamine is the amide of glutamic acid; different from the amino acid arginine amino acid control group This difference is related to molecular structure and activity of cystatin in the inhibition of cysteine proteinase activity, is suggested for further study of cystatin in groundnut Besides differences in amino acid position 34, resulting in amino acid sequence comparison of the same communication L23, L18 and R46 lines, RM48 we found more variation in the position amino acid sequence, bringing the total number of amino acid changes in the cystatin touch location is Specific differences shown in position 29, 30, 31, 32, 33, 34 and 36 of the line and the same study (Figure 3.16) 20 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn If transmission of the same cystatin L18 compare la base, we found that, similar to peanut L23 has a different position is wrong amino acid position 36, alanine replaces glycine The biggest difference of cystatin cystatin of the same communication line L18 is RM48 (7 other wrong position) Analyze each other related amino acid sequences of lines, the same analysis with different level of drought indicates that, similar to L23 has good drought tolerance, the error is different from the sequence of drought-cream group in position tri 36, cystatin RM48 line now also have differences However, not find every relevant variation of the amino acid at position 29, 30, 31, 32, 33, 34 RM48 line with the order studied The results of analysis of cystatin amino acid components like lost L23, L18 and R46 selected second communication line, RM48 showed significant differences on the amino acid and the same communication lines However, the statistical results the total number of amino acid hydrophilic, hydrophobic or group of amino acids essential for people who not receive the difference between lines and selective breeding The amount of hydrophobic amino acid occupies a relatively large proportion of cystatin communication component (45.92%) allows predicting the residence of the cystatinrelated drought tolerance of peanut plants Clear but will distributed hydrophobic protein on the cell membrane or system offers, the preferred water-soluble protein primary translation Therefore, with functional cysteine proteinase inhibitor activity can be very optimistic participants cystatin protect trees bearing cells will be effective in conditions of extreme water shortage CONCLUSIONS AND RECOMMENDATIONS Scar tissue processor communications bandwidth of 10 similar techniques blower continuously for hours, hours, hours and 11 hours were determined to be the same optimistic L18 tolerant low water At the threshold selected nine hours of irradiation with gamma rays 2krad reduced rate of recycling plants and the phenotype appears less in lost crops Regeneration communication line 198 is derived from scar tissue and scar tissue under dehydration under irradiation with gamma rays to cause dehydration, analyzed through five generations have selected three communication lines (RM48, R46, RM47) can high drought tolerance from drought-tolerant varieties less optimistic L18 Three lines are not selective about the advantages Vietnamese drought tolerance, but 21 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn also changes in the phenotype: The RM48 is the highest lipid content (38.22% KLK), a genetic distance compared to the same line and Original is 22% RM47 Series has the highest protein content (38.97% KLK) R46 Series volume 100 results (145.12 g), 100 grains (56.27 g) was highest Two lines R46 RM47 and genetic distance for L18 is 9.5% of original breeds Dong RM48 refreshing sound promising new varieties Have determined the coefficient of genetic diversity of the selected communication line is HRAPD = 11.09% There are five unique RAPD indicators identified in the two communication lines: lines appear RM48 indicator featuring four new sizes: RM48/OPA07-750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348200bp), the RM47 features OPH08 primer 200bp in size (RM47/OPH08-200bp) Amplification and separation of the cystatin gene from DNA into the genome of a selected communication line and the same root Cystatin gene of peanut has 461 nucleotides, with two exons and one intron, the first group of phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%) The protein encoded by the gene has 98 amino acid, including 45.92% hydrophobic amino acid, amino acid 54.08% and 28.57% hydrophobic amino acid essential to the total amino acid protein of cystatin Compare the order of the rows of peanut protein cystatin select and breed seeds have been detected in seven amino acid changes, including co dong RM48 biggest change This has affirmed the process of scar tissue irradiated by gamma rays have done major changes in the peanut genome structure leads to a change of phenotypic larger than just the scar tissue processed by the dry wind RECOMMENDATION Continue to monitor, analyze and communicate the advantages refreshing Vietnamese is R46, RM47, RM48 to introduce assaying Design vector carrying cystatin gene and moved on peanuts and other crops as well as to study the effects of cystatin drought resistant and some other characteristics in plants 22 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn PUBLICATIONS Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau (2009), Selection for dehydration tolerance in vitro of some peanut cultivars, Science and technology journal of Agriculture & Rural development, 7: 14-19 Vu Thi Thu Thuy, Nguyen Vu Thanh Thanh, Chu Hoang Mau, Nguyen Thi Tam (2009), Analysis of cystatin gene sequenceing of L18 peanut cultivar (Arachis hypogaea L.), Báo cáo Hội nghị Sinh học toàn quốc: 397-400 Vu Thi Thu Thuy, Dinh Tien Dung, Nguyen Thi Tam, Chu Hoang Mau (2010), The results of selection of some peanut lines from losing water scar tisue from L18 cultivar, Journal of science and technology, Thai Nguyen University, 72(10):122-126 Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau, Nguyen Vu Thanh Thanh (2011), Characteristics of nucleotide sequence of cystatin gene of some peanut lines derived from irradiated and dehydrated, Journal of Biology, 33(1): 86-95 Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau, Nguyen Vu Thanh Thanh (2011), The selection of the variational lines which resistant to dehydration and radiation of peanut crop (Arachis hypogaea L), Journal of Biotechnology: 9(3): 1-8 Submistion 04 cystatin gene sequence on NCBI (1) Vu,T.T.T., Nguyen,T.V.T., Chu,M.H and Nguyen,T.T (2010), Arachis hypogaea cystatin gene 1, exons 1-2, EMBL, GenBank, Accession FN811133 (2) Vu,T.T.T., Chu,M.H., Nguyen,T.T.V and Nguyen,T.T (2010), Arachis hypogaea cystatin gene for cystein proteinase inhibitor, cultivar L23, EMBL, GenBank, Accession FR691053 (3) Vu,T.T.T., Nguyen,T.T., Chu,M.H and Nguyen,T.T.V (2010), Arachis hypogaea cystatin gene for cystein proteinase inhibitor, cultivar L18, EMBL, GenBank, Accession FR745399 (4) Vu,T.T.T., Nguyen,T.T., Chu,M.H and Nguyen,T.T.V (2011), Arachis hypogaea partial cystatin gene for cystein proteinase inhibitor, cultivar L18, EMBL, GenBank, Accession FR828803 23 Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tai lieu chia se tai: wWw.SinhHoc.edu.vn The work was completed at the Department of Genetics & Biology Modern Biology-Agricultural Engineering, University of Pedagogy-Thai Nguyen University Science Instructor: Prof Dr Chu Hoang Mau Prof Dr Nguyen Thi Tam Objection Objection Objection The thesis will be protected before the Board level thesis dots Meeting at the University of Pedagogy-Thai Nguyen University At last hours, days May 2011 Thesis can be found at: - National Library - Learning Resource Center Thai Nguyen University - Library Pedagogical University, Thai Nguyen 24 Tai lieu chia se tai: wWw.SinhHoc.edu.vn [...]... activity of cystatin in the inhibition of cysteine proteinase activity, is suggested for further study of cystatin in groundnut Besides differences in amino acid position 34, resulting in amino acid sequence comparison of the same communication L23, L18 and R46 2 lines, RM48 we found more variation in the position 6 amino acid sequence, bringing the total number of amino acid changes in the cystatin touch... of the cystatin Position corresponding to position it as arginine 34 (R34) and asparagine 35 (N35) of cystatin RM48 line Glutamine and arginine at position 34 is two different amino acid structure characteristics of the original R, in which glutamine is the amide of glutamic acid; different from the amino acid arginine amino acid control group This difference is related to molecular structure and activity... dehydrated and callusinfluenced irradiation combined with handle causing dehydration we selected 3 lines has outstanding features, including: (1) The R46 has a mass of 100 fruit and seeds; (2) Lines of RM47 protein content higher; (3) Line of RM48 lipid content higher 3.2.2.2 Results evaluated tolerance of the selective in the fifth generation Drought tolerance of plants in general and in particular peanut. .. highest tolerance) , similar to the original L18 (drought- resistant cream) and similar to L23 (with tolerance good) for comparison purposes cystatin gene sequence of lines and varieties 3.3.2 Results of cloning and sequencing of genes cystatin Result of splitting the colonies carrying cystatin genes were tested by clonyPCR reaction Defining the nucleotide sequence obtained from the plasmid of the sample... Cystatin gene was cloned from the DNA like us L18, L23 and 7 selective communication line near 500 bp in size However, to affirm correctly that cystatin genes, we performed separate line, identifying the sequence comparison with cystatin gene sequence was published We perform a separate line and determine the order of 2 cystatin genes dong R46, RM48 (two lines have the highest tolerance) , similar to. .. OPH08 primer 200bp in size (RM47/OPH08-200bp) 5 Amplification and separation of the cystatin gene from DNA into the genome of a selected communication line and the same root Cystatin gene of peanut has 461 nucleotides, with two exons and one intron, the first group of phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%) The protein encoded by the... (L18) Cystatin- like tribe's genome Nucleotide L18 has 14 different compared to the same position wrong L23 Cystatin genes of leguminous green relatively conservative there is no difference in terms of genes of drought well and cystatin- like drought worse Results identified the nucleotide difference cystatin genes peanuts (table 3.17) shows the difference of cystatin genes appear in both the Club and. .. changes in the peanut genome structure leads to a change of phenotypic larger than just the scar tissue processed by the dry wind RECOMMENDATION 1 Continue to monitor, analyze and communicate the advantages refreshing Vietnamese 3 is R46, RM47, RM48 to introduce assaying 2 Design vector carrying cystatin gene and moved on peanuts and other crops as well as to study the effects of cystatin drought resistant... fruit (42.9%) The protein encoded by the gene has 98 amino acid, including 45.92% hydrophobic amino acid, amino acid 54.08% and 28.57% hydrophobic amino acid essential to the total amino acid protein of cystatin 6 Compare the order of the rows of peanut protein cystatin select and breed seeds have been detected in seven amino acid changes, including co dong RM48 biggest change This has affirmed the... EU723567, we get that the cystatin genes encoding proteins that we isolated, including two exons and one intron The first exon of the gene segment of 102 nucleotides starting from position 1 to 102 and the second exon 195 nucleotides, from position 267 to 461 zone in the middle of a 164 nucleotide intron from position 103 to 266 3.3.3 Results comparing the gene sequence and protein cystatin 3.3.3.1 Comparison ... RM48/UPC348-200bp; RM47/OPH08-200bp Discovery of peanut cystatin genes of group I phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%) The gene contains... Provide information on the cystatin and cystatin genes in peanut Handling of scar tissue by radiation combined with water is blown causing agent appeared cystatin gene mutations as evidenced by differences... soybean and green beans, P5CS gene in soybean, cystatin genes in green bean plants, DREB genes in Arabidopsis, etc Cystatin gene (Cys) of vegetation was first published on rice by Abe et al (1987),