Cloning Of Drought-tolerance Genes In Shixiya Ⅰ (Gossypium Arboreum L.)

Drought is one of most important factors which effect the expression of good traits of plants in adverse circumstance. In China, the majority of regions where cotton were grown are short of water, so, it is necessary to establish a simple, fast and easy operated method to evaluate the drought tolerance of cotton because it is extremely urgent in cotton breeding.This experiment was conducted separately with 8 upload cottons, 2 Pima cottons and 3 Asian cottons as the material to establish the PEG evaluation the fast cotton seedling drought resistance evaluation system. The 3～6-leaf stage seedlings with 17%(W/V) PEG6000 treatment, after 12, the results suggested that osmotic adjustments of PEG could be used to evaluate simply the drought tolerance of cotton, though the cotton after PEG-treated differed slightly in physiology with cotton after drought-treated. The method with osmotic adjustments of PEG is simple, fast and easy operated, could be used to evaluate the drought tolerance of cotton in principle, and will establish foundations for the study on cotton drought-tolerance molecular biology.In drought resistance level test, the evaluation result of Asian cotton Shixiya I is in the drought-resistent level (survival rate > 80%). In 1～5 hours by inducing the drought resistance gene the expression with 17%(W/V) PEG6000 treatment,we separates three drought resistant genes' conserved fragment, which is P5CS,cytosolic Cu/Zn-SOD and chloloplast Cu/Zn-SOD by RT-PCR. Furthermore, we got full-length sequence of two genes by RACE and submitted to GeneBank, named Gacytsod and Gachlsod.In order to study the expressive quantity in drought press, after the blast and analysis to the sequences of nucleic acid and protein, 3～6 leaves stage seedling of Shixiya I with 17%(W/V) PEG6000 treatment for 0, 1, 2, 3, 4, 5 h were gathered.The cDNA of them were obtained and were used for semi-quantitative RT-PCR, inner referenced primers were designed from the UBQ7 partial sequence (198bp). The results revealed that: expressive quantity of P5CS raised constantly; expressive quantity of Gachlsod kept steady with almost none changes; but Gacytsod went down at first, and then, after 2 hours stepped up by hours. The dynamic diversification of Gachlsod's expressive quantity is accordant to the activity changes of SOD enzyme after the same 17%(W/V) PEG6000 treatment。The plant transformation vectors of the Gacytsod gene were built——pBISOD and pBIRDSOD,which were promoted by 35s and rd29A promoter. The results have been testified by two enzyme cutting and PCR amplification. The right clones were transferred in to Agrobacterium tumefaciens LBA4404 for the succedent plant translation to validate the function of Gacytsod gene...