Study On Cloning LEA Gene With The Function Of Standing Against To Water Stress And Sequence Analysis

It was well known that water deficiency had become a critical ecological problem, and it was worse in China. To resolve this problem, the effective way was to study the mechanism of drought resistance, clone relative genes and cultivate new species. Acquiring the novel drought resistance genes with knowledge property right became very important to our country. The research demonstrated that plant could adapt water stress through signal transduction, osmotic adjustment, desiccation protection and metabolic changes. Of these above, the researchers paid much more focus on desiccation protection. The reason was as followed: When more severe stress desiccation occurs, accumulation of desiccation protectants such as LEA(Late Embryogenesis Abundant) protein and sugars avoided damage of biomacromolecules, especially membrane system in some extent. Special construction of LEA made itself as a perfect desiccation protectant, and LEA could take part in osmotic regulation as a regulatory protein and molecule chaperone, therefore it was paid attention again. The hypothesis that LEA protein played an important role under water or salt-stress conditions was further proved by the fact that increased tolerance was acquired by transgenetic plant when Deping Xu transformed the HVA1 (it belongs to group 3 LEA)gene to rice.The purpose of this thesis was to clone two different group 3 LEA genes. On one hand, we designed and synthesized a pair of degenerate primers according to the foreign reports. A 500bp DNA fragment was obtained with PCR technique from the DNA of highly drought-resistant china leymus. After purification, the DNA fragment was cloned into PMD-18T vector directly.The result of identification by PCR method and restriction digestion indicated that the recombinant plasmid had contained a 500bp DNA fragment, which demonstrated that we had cloned a part of DNA encoding HVA1 successfully. Compared with the published materials abroad, the identity of DNA sequence and deduced amino acid sequence reported here was above 90%. This result hinted that the gene was highly conservation evolution. Meanwhile, the discovery of novel gene might be a basis of function research and breeding resistant plant. On the other hand , we searched the sequence of rice (group 3 LEA} from gene bank anddesigned specific primes. The interest fragment with 776bp was obtained by RT-PCR and cloned by T-overhang vectors. The next step was to clone this gene into PYES2, a expression system of yeast, which would offer further functional detecting between different species. Molecular biology software Primer5.0, DNAsis, DNAclub, GENEDOC were used during the experiment and data analysis, which had been a good assistant. The application of biology information was one of highlight in this thesis, and it might be a new tendency of cooperating the biology technology with information technology in future development of biology. In addition, this thesis discussed experimental method for the purpose of good design, as well as attained better experiment results.In conclusion, in the thesis we reported the working of cloning two different group 3 LEA genes and analyzed their sequence particularly, which would be a basis of breeding novel species.