Cloning, Expression And Transformation Of TaSUT1, A Responsive Gene In Triticum Aestivum To Drought Stress

Water shortage had become a global crisis, and drought was the main limitation factors of the world’s food crop production. Clarifying the mechanism of drought resistance of plant, cloning drought-related gene, cultivating varieties of drought resistance crop applicated with modern molecular biology techniques and the means of combining traditional breeding, to implement water-saving cultivation, not only is the production of sustainable development, and the key to ensure food supply, but also to alleviate the situation of water crisis.In this research we obtained the TaSUTl gene by homology cloning technology from seedlings at two-leaf stage of Shijiazhuang No.8 drought-resistant Triticum aestivum Linn; Then constructed the plant expression vector of subcellular localization successful, infecting the tobacco through Agrobacterium tumefaciens EH 105, confocal laser microscopy showing where was the expressed protein of TaSUT1 located. Semi-quantity RT-PCR and Real-time quantitative PCR analysis detected the TaSUT1 gene expressing in roots, stems and leaves in wheat under water stress condition; The plant over-expression and RNAi vector of TaSUT1 were constructed successfully, SUT gene that examined and analysised was transformed into wheat callus by Agrobacterium tumefaciens infecting and the particle bombardment.The main results are as follows:1. The full-length cDNA of SUT gene named TaSUTl was cloned from wheat treated with 20% PEG6000 stress. The sequence analysis showed that the cDNA was 1935 bp, which including the 1566 bp of ORF, encoding 522 amino acid, molecular weight of 55.072 kDa, and isoelectric point of 8.102. The analysis on the deduced amino acid sequence showed that the expressive protein of TaSUT1 gene belong to Major Facilitator Super-family (MFS), which has a typical structure of 12 membrane spanning helices.2. Localization in the tobacco cells indicated that TaSUT1 expressive protein located in the cell membrane.3. Semi-quantity RT-PCR and Real-time quantitative PCR analysis showed that the TaSUT1 gene could expressed in roots, stems and leaves in wheat of drought resistance treated with water stress; the expression of TaSUT1 gene could increased when treated with drought; The expression of TaSUT1 gene could gradually increased when treated with Glucose and drought. The expression of TaSUT1 in wheat of no drought resistancegene decreased when treated with drought and Glucose. So we suggested the TaSUT1 gene could play an important role in stress response.4. We constructed three over-expression vectors, TaSUT1-pCAMBIA1300, TaSUT1-pUBI::cas and TaSUT1-pMWB003.TaSUT1-pCAMBIA1300 transformed into wheat callus by transfection of Agrobacterium tumefaciens. We did not get resistent plant by Hyg-resistent scanning and got two resistent plants without Hyg, but no positive plant by the molecule test of the PCR amplification. The result indicated that Hyg inhibited the wheat callus differentiation and did not suitable regeneration system of effective screening. TaSUT1-pUBI::cas and TaSUT1-pMWB003 transformed into the callus of wheat immature embryos with the particle bombardment, We totally got 216 resistent plants by PPT-resistent scanning and three positive plants, positive rate is 1.4%.5. We constructed three RNAi vectors, R1, R2, R3-pMWB003, transformed which into the callus of wheat immature embryos with the particle bombardment respectively, and finally got 216 resistent plants by PPT-resistent scanning and seven positive plants, positive rate is 2.5%. Under the condition of the same transformation, R1-pMWB003 vector obtained 97 regenerated plants, R2-pMWB003 vector obtained 153 regenerated plants, R3-pMWB003 vector obtained 30regenerated plants. This indicated that the transformation efficiency differ with different interference segments of TaSUT1 gene.