1 INTRODUCTION 1. Rationale Research tolerant nature of this plant shows a multi-gene traits. The specific role of each individual gene-related tolerance in plants (P5CS such, RD22 ) has a lot to learn and research analysts. Under the impact of the unfavorable factors of the surroundings, not just the individual genes that are expressed as a system of related gene expression is activated in response to adverse condition that plants encounter. One of the factors that trigger the activity of genes involved in resistance to the transcription factors (transcription factor - TF). NAC transcription factors family (NAC transcription factor) is one of the biggest TF characterized in plant genomes. NAC TF involved in many important physiological processes in the plant body. Many NAC genes involved in response to various pressures from the environment such as drought Many authors have studied NAC TF genes transferred successfully to a number of crops such as rice, wheat, cigarettes In groundnut, the gene AhNAC1, AhNAC2, AhNAC3 related to drought tolerance have been cloned successfully. Moreover, AhNAC2 was used to design a vector carrying a drought tolerance gene and successfully transferred into Arabidopsis. The peanut plant is a short-day crop grown in popularity around the world. In our country, peanuts are grown in all over the regions and suitable for many different ecological zones. The peanut plant is a legume, less drought-tolerant group. Thus, improving the resistance ability will expand the area under cultivation and improve crop yields. Some techniques breeding resistant varieties such as 2 hybrid technology or applications of plant cells has been applied. However, the techniques used to create transgenic varieties resistant to drought has not been studied much. From the above reason, we have carried out the theme: "Researching on isolating and transferring NAC2 gene relating to the tolerance to drought of peanut (Arachis hypogaea L.)"to test the ability of transgenic and advanced directives to drought tolerance of transgenic plants to produce transgenic lines serve in breeding drought-resistant peanuts. Practical application of research to renovate peanut restrictions and break resistant crops on this plant. 2. Objective of the study (1) Isolation of genes encoding transcription factors NAC2 from drought tolerant peanut varieties. (2) Design the structure vector containing the gene encoding transcription factor NAC2. Create and transgenic plants expressing NAC2 protein in tobacco plants. (3) To create transgenic peanut lines. Analysis of transgenic peanut lines. 3. Content of the study (1) Grouping similar level of communication studies assessing drought through drought tolerant callus phase and saplings. (2) Isolation, cloning and sequencing of the gene encoding transcription factor NAC2 related to drought tolerance of peanuts. (3) Design vector containing the gene structure encoding transcription factors NAC2, create A.tumefaciens bacteria carrying the recombinant vector and transferred into the tobacco plant model. (4) Determination of the presence of the transgene on tobacco 3 NAC2, evaluate drought tolerance of the transgenic tobacco lines through changes in proline content. Analysis of recombinant protein expression NAC2 in transgenic tobacco plants. (5) Development of plant regeneration system in transgenic peanut plants and directives (GUS) through callus and somatic embryos. (6) Transfer NAC2 genetic structure in tree nuts, peanuts analysis of transgenic lines. 4. New contributions of the thesis i) Successfully halftone genes encoding transcription factors NAC2 from drought tolerant peanut L12. ii) Design a vector containing the structural gene encoding transcription factors NAC2 and transferred it into tobacco plants, the expression of protein in transgenic plants has demonstrated that structural design has worked well in tobacco . iii) Through regeneration system has been built, tested varieties LVT GUS transgenic and gene transfer structure encoding transcription factors NAC2. GUS expression obtained on peanut somatic embryo and complete. Obtained touch 4 generation T0 lines carrying genes encoding transcription factors NAC2 through initial inspection with PCR. iv) Optimal dissertation and complete the complete process through transgenic callus and somatic embryos in order to serve peanuts to create transgenic peanut purpose improved resistance to water shortages from the lip school. 5. Scientific significanceand practical 5.1. Scientific significance 4 i) The results of the thesis research has provided materials science applications of GM technology in the interests of improving drought tolerance of peanuts. ii) Provide information about NAC transcription factors and genes in peanut NAC2 as well as drought tolerance in peanut research and the role of genes NAC2 in response to the lack of water from the environment. 5.2. Practical significance Results of evaluation of drought tolerance in peanut callus level and young plants are used as a basis to assess and apply measures to improve the drought tolerance of peanuts. Initial results obtained on transgenic peanuts is a foundation for evaluationing at the next level and serving continuous research on improving drought tolerance of peanuts. 6. The structure of the thesis The dissertation consists of 103 pages (including references), is divided into sections: Introduction consists of 2 pages; Chapter 1: Overview document, page 40, Chapter 2: Materials and Methods, page 15, Chapter 3: Results and Discussion, page 47; Conclusions and suggestions: 1; published works related to the thesis: 1; references: 14 pages with 134 references in Vietnamese and English. The thesis has 25 tables, 36 figures. Chapter 1. LITERATURE REVIEW The reference of theisis summarize 17 Vietnamese documents and 117 foreign ones with related content, including: (1) Limit and limit the impact of the plant, (2) The transcription factor related to drought tolerance in plants, (3) Improving drought tolerance of plants 5 by gene transfer techniques. With the data collected, analyzed results confirmed, optimism is short-term agricultural crops grown in popularity around the world. General trend of climate change makes unusual drought in many areas, this is the main reason for the reduction in crop yields includind peanuts. The understanding of drought tolerance mechanisms will help to understand the resistance characteristics of peanut plants and provide a basis for studies on the ability to respond to water shortages from the environment. Mechanisms of plant tolerance to drought is complex, involving not only morphological anatomy but also related to the biochemical changes in cells, regulating activity of the genes involved in resistance limit of vegetation. The success of gene transfer techniques has been published in many plant species and characteristics related to drought tolerance of plants has improved . Currently, there are two trends of research for genes involved in plant tolerance to conditions of abiotic ecological disadvantage. The first one is to find genes that are directly related to drought tolerance, cold tolerance, waterlogging or alkaline Secondly resistant genetic studies towards the control elements such as transcription factor ( TF ) in oder to regulating overall better plant when conditions are unfavorable. They NAC TF is one of them featured in the largest plant genome. NAC transcription factors functionally related to many different processes in the plant body, such as development, aging, response to biological and non- biological stress. Many studies mainly focused on the cloning and identification of genes related to drought like NAC TF: ANAC019, 6 ANAC055 and ANAC072 from Arabidopsis, BnNAC from cabbage, AhNAC1, AhNAC2, AhNAC3, AhWSI 153, AhWSI 308 from peanuts, GmNAC2, GmNAC3 and GmNAC4 in soybean, ONAC045, SNAC1 in rice, TaNAC2, TaNAC4, TaNAC8 in wheat Once isolated, the gene promoter is designed with strong, specific (CaMV35S promoter, promoter rd29A, ) and converted into a number of plant species by genetic engineering techniques. Several published studies have obtained successful transgenic plants have the ability to enhance the tolerance to water shortage conditions of the environment such as: A.thaliana, tobacco, rice The results obtained demonstrate the increase resistance better with adverse conditions of environment as high salt concentration, low temperature, high temperature, drought, thereby improving growth and development. This is the basis for our understanding and analysis of gene NAC2 on peanuts. Chapter 2. Materials and methods 2.1. Plant Materials Thesis used 10 varieties of seeds have different drought tolerance by the Center for Research and Development beans - Institute of Food Crops and Plants provide food. 2.2. Chemicals and equipment The chemical which are purity and common origining from reputable companies such as Taq-polymerase, PCR buffer, primer specificity of goods Invitrogen, EDTA, SDS, Tris Vector cloning, gel and separated only plasmid kit , Types of Fermentas restriction enzymes. The growth stimulant Sigma 2.3 . Research Methodology 7 2.3.1. Method physiological, biochemical Rapid assessment methods drought tolerance by dry blowing callus under Le Tran Binh et al (1998). Rapid assessment drought tolerance at seedling stage under Le Tran Binh et al (1998 ). Quantification of proline by the method of Bates et al (1973) 2.3.2. The method used in vitro Create tobacco lines and communications in vitro culture techniques. Tobacco variety C91 is supplied from a plant cell technology with the seedlings have developed 3-4 true leaves are used to conduct experiments for transgenic serving: Regenerated shoots and transgenic, create a complete tree. Methods of communication through tissue regeneration and scarring somatic embryos was conducted based on several methods of published authors. Callus was in the dark for 10 days, 7 weeks in somatic embryogenesis, plant regeneration and create a complete tree. Methods of gene transfer through callus is based on research conducted by a number of authors. 2.3.3 . Methods of molecular biology Study information about genes and primers for cloning genes: - Operation: i) collected nucleotide sequences from NCBI GenBank; ii) design primers; iii) Quality control of PCR primers. - Total DNA extracted by the method of Gawell et al. - Total RNA extraction by Trizon. cDNA was synthesized according RevertAidTMH Minus First Strand cDNA Synthesis Kit. 8 - Isolation of genes by PCR, RT - PCR. Gene cloned by the method of Sambrook et al (2001). - Gene sequencing on automated sequence analysis by the method of Sanger. - GM design structure: the reaction cut, spliced to create vector, creating the recombinant vector and transformed into cells A.tumefacien by means of electrical impulses . - Analysis of gene expression by GUS staining, cell staining method of Jefferson et al (1987). - Evaluate the presence of proteins by gene transfer technique created by Western hybrid method of Sambrook et al (2001). 2.3.4. Method of calculation and data processing The data are processed in biostatistics methods on a computer with Excel. Analysis of gene and DNAstar BioEdit software. 2.4. Research locations Technology Office, Plant cells, key Laboratory of Gene Technology - Institute for Biotechnology. Chapter 3. RESULTS AND DISCUSSION 3.1. Evaluation of drought tolerance breeding research 3.1.1. Rating resistant to dry blown at callus phase Callus dry blowing is used as a technical measure to assess the impact of the loss of water to drought tolerance, heat tolerance of plant cells. By dry blowing the callus of rice, peanut, many studies have selected the lines of tissue is capable to resist to high temperatures, dry phenomenon when dehydration after being treated in culture conditions. 3.1.1.1. Ability to create scar tissue of the same study 9 Ability to create scar tissue is the first parameter reflects the adaptability of the model to study the environment to use for future experiments in evaluating drought-tolerant varieties of peanuts. We use the original sample source embryonic axis peanuts on environmentally supplements stimulate the growth of 2.4 D. The study results showed that all the varieties studied were capable of creating callus. The highest ability of formation callus was observed in the same L12 and L16 (100%) and the lowest is the same LVT (69.56%). Sources scar tissue collected will be used in the experimental evaluation of drought tolerant varieties at this stage. 3.1.1.2. Water retention of the scar tissue used To determine the degree of dehydration of scar tissue we conducted blown mass of scar tissue and scar tissue separation with different ratios (1/2, 1/4, 1/8) with sterile air box of implantation. Conduct blown 3h, 6h, 9 h continuously to determine scar tissue dehydration. Experimental results show that the dehydration of the peanut scar tissue after dry blowing processed by different changes in each breed. The fastest rate of water loss observed after the first 3 hours. The amount of scar tissue water decreased rapidly in all varieties. Speed dehydration after 3 hours of the whole scar tissue is less than the separated callus. Among varieties, LVT had the highest degree of dehydration after being dry blown for 3 hours and the lowest was L12. The Water continued to decline after next 6 hours of blowing dry in compared to the weight of initially fresh callus. 3.1.1.3. Survival rates of the scar tissue used 10 Water retention of scar tissue after dry blowing affects the survival rate of them. From the result of scar tissue dehydration showed a survival rate of scar tissue is inversely proportional to the period time of dry blowing. L12 had the highest survival rate as the biggest scar tissue in most of the processing threshold (100%, 100%, 89,99%, corresponding to blow dry time is 3, 6, 9). The survival rate of TB25 is the lowest, at 9pm only 40,63 % threshold. High survival rate observed in the scar tissue blocks separated 1/2. Low resilience observed in callus mass splitting fourth and eighth. Survival was lowest at 12,50 % just after 9 hours blown TB25 (split 1/4 ) and 12,00 % in just after 9 hours blown LVT (split 1/8). 3.1.2. Evaluate drought tolerance at seedling stage The percentage of wilted plants, plant recovery rate after artificial dought The results obtained after processing the term artificial seedling stage by stopping irrigation in different times during the experiment showed no wilting plant ratio decreases with the time limit cause. Plant recovery ratio decreases with time causes the limit. At a time limit of 5 days to recover most of the same whole, only 4 like L08, L15, LVT, TB25 rate has decreased. Resilience lowest observed in the same TB25 (55,24 %). After 7 days there is just cause term resilience L12 highest (73,34%) and lowest LVT (18,18%). Very low recovery rate after 9 days cause term, the withering of the dominant tree recovery, little green back. Lowest recovery rate observed in the same TB25 (2,94%). Like the L12 has a higher resilience both (30,38%). [...]... substrate X - Gluc (Figure 3.23 A) After 4 weeks on the environment created SIM1 played does not contain selected , the buds were transferred to a medium containing BAP concentrations SIM2 2mg/l The sample rate of survival after 2 weeks on selective environment contains statistics of 146 players These buds will survive the switch to the root environment and continued selective The peanut plant was fully... SUMMARY Thai Nguyen – 2013 26 The work was completed at Department of Genetics and Modern Biology, Department of Biology-KTNN, College of Education, Thai Nguyen University Supervisors: Prof Dr Le Tran Binh Reviewer 1: Reviewer 2: Reviewer 3: This thesis will be defended in front of the Thesis Assessment Board of Thai Nguyen University in room No………………………… Time: …… …………Date 2013 Thesis can be found... sensors form a mass of scar tissue about the size of 3x3x3 mm, greenish yellow, viscous or non-white foam After 7 weeks the only animal to light yellow callus volume, viscosity not capable of creating new clusters of somatic embryos Results regeneration from somatic embryo clusters showed that the addition of BAP 2mg/l on SIM environment, sample rate and number of shoot buds produce average gain is highest... raw materials put out peanuts in vitro natural After two weeks we moved the tree to the ground 3.4.2 Transfering of GUS indicator through A.tumefaciens bacteria The efficiency of gene transfer process not only depends on the regeneration system that depends on many factors, including genetic characteristics of the breed Some successful studies in transgenic peanut callus via embryo has been announced... cells A.tumefaciens efficient Transfer structural genes encoding transcription factors in tobacco NAC2 through A.tumefaciens bacteria CV58 The transgenic tobacco lines after a certain period of time surviving in the wild has been tested by our PCR with specific primers NAC2XbaI/NAC2SacI The results showed that, in 9 line after random screening tests would give a positive reaction, the wells appear electrophoresis... drought brought NAC2 transcription factors in peanut callus via micro LVT bacterial A tumefaciens follow the procedure was optimized The process of gene transfer was conducted on 4 batches with a total of 935 samples are used for gene delivery, have created 595 23 embryos form soma, buds formed in 1416, during the 189 selected antibiotics on plant survival and crop prices in the grid Figure 3.26 Image transfer... regeneration process and the GUS gene transfer via somatic embryo tissue and callus in peanuts GUS genes are expressed in somatic embryo stage and a complete peanut plant 6 Created 4 transgenic lines at the generation of T0: 24, 49, 52, 89 from less drought-tolerant varieties LVT Recommend - Further analysis of transgenic drought-tolerant communication lines serving peanut breeding oriented improve drought... Proline content of the peanut processing before and after drought treated Proline content was viewed as an important indicator in evaluate drought tolerance We conducted a content analysis of proline in seedling stage in time: pre-term and post-term Proline as signal response of plants to extreme elements of the environment Experimental results showed that proline content increased after term than the term... of a system for regeneration and transformation in peanut (Arachis hypogaea L.) using somatic embryo, Journal of Biology, vol 34 (3), pp 370-376 3 Nguyen Thi Thu Nga, Le Van Son, Le Tran Binh, Banh Thi Mai Anh (2013), “Cloning and characterization of the Nac – like gene NAC2 in Arachis hypogaea L.“, Journal of Biology, vol 35 (2), pp 234-242 4 Nguyen Thi Thu Nga, Le Van Son, Le Tran Binh (2013), “Vector... with A tumefaciens, B Transgenic somatic embryos after 7 weeks; C Buds from transgenic somatic embryos on selective medium; D Transgenic shoots on selective medium; E Transgenic peanut plant rooting environment; F Transgenic peanut plants on land prices can: Husk: sand To check the presence of the transgene, after about 1 month the tree, the leaves of the tree line form T0 is conducted to collect a . results of the thesis research has provided materials science applications of GM technology in the interests of improving drought tolerance of peanuts. ii) Provide information about NAC transcription. level and serving continuous research on improving drought tolerance of peanuts. 6. The structure of the thesis The dissertation consists of 103 pages (including references), is divided into. pages with 134 references in Vietnamese and English. The thesis has 25 tables, 36 figures. Chapter 1. LITERATURE REVIEW The reference of theisis summarize 17 Vietnamese documents and 117