Isolation Of Genes Involving In Drought Tolerance And Acquisition Of Transgenic Lines For The Regulation Of In Potato

Water scarcity is an important factor limiting plant growth and development, plants adapt to the lack of water through different mechanisms, such as changing leaf shape, increasing root biomass, so as to ensure the survival, growth and development under drought stress. Plants with low drought-tolerant, such as potato adapt to water shortage stress by inducing the expression of drought-response genes, then changing metabolic pathway. In recent years, it highlights how drought stress induces expression of genes involving in drought tolerance to adapt the stress. Whereas a little is known on idenification of genes for drought resistance on the level of transcriptome. Drought stress specific ESTs were isolated with subtractive cDNA library in our lab.In this paper, two full-length cDNAs were isolated by RT-PCR and RACE based on the drought stress specific ESTs. Biological functions of the cDNAs were predicted by bioinformatic method. Expression of two cDNAs, either in vitro or in vivo, respectively, helps to understand the function of two cDNAs in drought tolerance. The main results are as follows:1. The full cDNA of T3and T8are obtained from potato leaves using the method of RACE. According to the results of BLAST search, we find T3sequence shares100%similarity to that of AK319670in tomato, T8sequence is up to99%similarity to that of DQ207848in potato. Subsequently, the conserved domain of putative T3and T8proteins is respectively analyzed by the conserved domain database in NCBI. So, we preliminary speculate that T3may function as chloroplast thylakoid cavity, and T8may play its role as the ribosomal protein of L6superfamily.2. Recombinant plasmid pET-28c-T3and pET-28c-T3with two full-length cDNA were constructed and transformed into Escherichia coli BL21(DE3), then corresponding recombinant strain were acquired. We measured the growth curve of recombinant strains and found its OD60values at the logarithmic and stable stage are higher than that of control. These results suggested that the expression of T3and T8in vitro were helpful in promoting the growth of the combinant strains and the two recombinant strains had no difference in the different growth stages.3. To further clarify the biological function of cDNA T3and T8, recombinant over and knockout expressing vectors containing T3and T8were constructed, respectively. These constructs were introduced into potato varieties ZiHuaBai by Agrobacterium-mediated transformation method.6PCR positive transgenic lines,4for over and2for knockout expression were obtained from transformation of cDNA T3.The results of this study will lay the foundation for the creation of potato drought tolerant germplasm innovation.