Cloning And Function Analysis Of GhEF1A8 Promoter And Development Of Glyphosate-tolerant Transgenic Cotton

Cotton(Gossypium SPP.) belongs to angiosperms dicotyledonous, malvaceae hibiscus subfamily, as an important economic crop, cotton was found in China as early as the 9th century, now China has become one of the world’s largest cotton planted country, the planting area ranged among the domestic 25 provinces and autonomous regions, and created a lot of economic benefits and social value. In recent years, the large-scale promotion and application of transgenic herbicide-resistant transgenic cotton has change the traditional cotton cultivation pattern, greatly increased the yield and quality of cotton product. Currently GM cotton research in China has made great progress, but the development of transgenic herbicide-resistant cotton started relatively late, and yet there are no efficient and stable herbicide-resistant transgenic cotton varieties were bred, therefore this study stand at the perspective of plant gene engineering, by cloning the organization efficient expression promoter and optimization of glyphosate resistance genes, carried on a new exploration toward glyphosate-resistant genetically modified cotton research.The expression characteristics of nine members of the Gh EF1 A gene family were analyzed, and then with the genome walking(TAIL- PCR) technology,Gh EF1A8 gene promoter region was obtained. After analysis of substructure and its potential cis element in regulatory regions by bioinformatics tools, we constructs the promoter full length, 5 ’UTR absent and a series of promoter 5’ end truncated plant expression vector. Transgenic tobacco GUS histochemical staining and fluorescence enzyme activity determination showed tissue expression pattern of the promoter and its molecular mechanism of regulation.at the same time new EPSP synthase gene and N- acetyl transferase gr79 and gat gene codon modification and structure optimization was carried out, combining Gh EF1A8 promoter and cotton chloroplast leader peptide(Gh CTP) a series of single and double gene plant expression vector were constructed, through the molecular biology analysis and the resistance ability against glyphosate, characterization of the of transgenic tobacco and transgenic cotton, the bivalent glyphosate resistant transgenic cotton were obtained. Based on the above research, the main results are as follows:1. The nine cotton Gh EF1 Agene family members(Gh EF1A1- Gh EF1A9) have different expression characteristics, which Gh EF1A8 gene has the highest expression level in leaf, wich as high as eight times of the Ghubi expression level, far higher than other family members. In addition Gh EF1A8 gene in cotton stem tissue also can efficiently express, made above Ghubi gene expression quantity 5 times.2. The upstream Gh EF1A8 gene promoter region were isolated for the first time,1679-bp sequence fragments, which contains 143- bp Gh EF1A8 gene coding sequence. Using Plant CARE, PLACE and Scan WM-P bioinformatics tools to analyze the promoter substructure and predict potential cis element, result showed that not only the TATA box, CAAT box, GC-rich basic regulatory elements, Gh EF1A8 promoter and also contains a variety of stress response and transcriptional regulatory elements, such as motif, GT- 1 W- box, LTR, and A/T- rich, etc. In addition, the cloning ofsequence fragment contains a length of 242- bp 5 ’UTR region, this area contain a non-translation exon, a plant intron and a 5’ UTR Py- rich stretch components.3. Based on bioinformatics analysis results, we further construts Gh EF1A8 full length promoter, 5 ’UTR absent and a series of promoter 5’ end truncated plant expression vector and carry on the study of transformation of tobacco. GUS histochemical staining and fluorescence analysis shows that, Gh EF1A8 promoter express mainly on the up-ground plant tissue of the tobacco, expression level in leaf and stem tissues were 2 times and 4 times higher than of Ca MV promoter, espectively.5 ’UTR can enhance Gh EF1A8 promoter activity in almost all tissues and organs, especially in tobacco leaf and the stem tissues, GUS activity increased by 400% and 600%, respectively. In addition, the 5 ’end sequences analysis results show that the missing- 1081 /- 869 and 869-869 /- sequence area is important control area of promoter, A/T-rich elements also can rise the promoter activity.4. EPSP synthase gene and N- acetyl transferase geneg79 andgatwere codon modification and structure optimization, combined with the Gh EF1A8 promoter and cotton chloroplast leader peptide(Gh CTP) we building a series of single and double gene plant expression vector and the study of transformation of tobacco and cotton proving that transformed tobacco and cotton with theg79 andgat gene obtained certain glyphosate resistance, and a preliminary comparison of the gene of glyphosate resistance difference was carried out.5. Bivalent glyphosate resistant transgenic cotton T0 and T1 generation plant was further analyzed, Southern blot, Western blot and glyphosate resistance experiment results show thatg79 andgat genes have stable integration in the cotton genome, but there are differences between the gm strains gene copy, gr79 genes can be correct translation for the target protein in cotton. In cotton T0 the T1 generation reproduction process, two exogenous genes and glyphosate resistant plant basically stable.