Analysis Of Differential Gene Expression Of Cotton Related Drought Stress By Cdna-aflp

Drought is a serious problem all over the world, which is also a major factor that restrict agricultural production in our country. So it is important to improve drought resistance of crop in modern agriculture. Cotton is an important cash crop in China, and even the whole world. Xinjiang belongs to dry area, meanwhile, Xinjiang is the largest cotton production area in China, and cotton needs lots of water, the crisis of water shortage becomes remarkably serious. So drought restricts the improvement of cotton industry severely in Xinjiang. Due to lack of powerful select method and complexity of drought resist trait, for many years, people solve the problem of drought by using traditional breeding methods, but it costs much money and needs a great deal of work, and the efficiency is low. As the development of molecular biology, adversity resistance cultivar breeding through gene engineering project has already become an important aspect in modern agricultural study. In this study, a drought resist cultivar jinmian 13 was used as material, genes relative to drought resistance were screened through cDNA-AFLP and cloned, drought resist mechanism was studied by analyzing gene expression level in different stress times and tissues. This study will provide candidate genes and theoretical basis. The main result is as follows:1. jinmian 13 seedling, control and PEG6000(20% and 30%)treated by 1h,2h,4 h,6h,8h,10h,12h and 24h was cultivated using hydroponic culture method, high quality leave total RNA was obtained.2. cDNA-AFLP was performed on transcriptional products of control and drought treated by using 64 pairs of primers.232 differently expressed TDFs were obtained,102 of them were cloned, sequenced and the sequences were analyzed. The result showed that 49 TDFs have homologous genes on NCBI,8 are repeat sequences, 19 TDFs have homologous genes but lack of biological function,30 TDFs have possible biological function. And still 45 TDFs have no homologous gene or very low homologous genes, they may be genes with unknown function.3. Based on the results of blast identification in NCBI, the possible functions of 30 TDFs involve signal transduction,metabolism,photosynthesis accommodate,stress defense, etc. photosynthesis accommodate proteins involve light-regulated gene(TDF89); signal transduction related genes involve ATP binding protein(TDF3),phosphatidyl glycerol phospholipase C(TDF45 and TDF62),serine/threonine protein kinase (TDF64),F-box domain containing protein(TDF75),receptor protein kinase (TDF93); nucleic acid metabolization related gene is putative reverse transcriptase (TDF34) and retrotransposon protein(TDF7); biosynthesis key enzyme genes involve starch branching enzyme(TDF1 and TDF2); protein synthesis and modulation genes involve RNA helicase-like protein(TDF80),eukaryotic initiation factor 4A(EIF4A) (TDF87); stress defense related genes involve Osmotic stress response protein(TDF 12),rust resistance protein(TDF29),cold stress related gene(TDF31),drought stress related genes(TDF42 and TDF81),pseudo-response reg- ulator (PRR) gene(TDF52),multidrug resistance secretion protein(TDF101); metabolism related genes involve cytosolic acetyl-CoA carboxylase(TDF43),cytochrome P450(TDF5 and TDF44) carbomoylphosphate synthase gene(TDF60),phosphoenolpyruvate carboxykinase (TDF61),phosphotransferase(TDF70),NADH dehydrogenase gene (TDF97),Alcohol dehydrogenase(TDF97); ionic transport related gene involves vacuolar H+-ATPase(TDF33),transcription factor is EREBP(TDF76).4. Three cDNA fragments closely related to drought resistance(TDF42,TDF61,TDF62)were chosen to perform Semi-quantitative RT-PCR to analyze the expression level in cotton leave at different time and in different tissues.The result showed that phosphoenolpyruvate carboxykinase(TDF61)has low transcription level under normal water supply, and increase first and then decrease under drought treatment. It has the highest transcription level at 8h, the highest in leave, stem the second and root the last. TDF42 rarely expresses while under normal water supply, and increases as drought treatment time extended, and has the highest level at 10h, then decreases. It has the highest transcription level in root at 10h, stem the second and leave the last. phospholipase C(TDF62)has low transcription level under normal water supply, and increases as drought treatment time extended, it has the highest transcription level at 12h, then stays mainly stable. So we conclude initially that the three TDFs is related to cotton drought stress.5. The results further proved that it was viable to isolate genes of plants with large genome size via cDNA-AFLP.