Cloning And Expression Analysis Of Salt-tolerance Related Genes (GhPTAC,GhGnT) On Gossypium Hirsutum L.

Land salinization has become a focus of the world and it is imminent to solve. The researchers considered palnting salt-tolerant crop on mild or moderate saline-alkali soil as a simple, quick, economic and effective improvement measures. The cotton became an important study material as main economic crop and salt resistance crop. It provided a good approach for improving cotton salt-resistant ability and cultivating salt-resistant breed that studying cotton salt-resistant mechanism with molecular biology methods. The salt-resistant material Zhong 9806 and salt-sensitive material Zhong S9612 were studied as research material. Two genes were excavated by gene chips and SSH librarys, cloned by RACE and RT-PCR, named GhPTAC and GhGnT separately. Salt-resistant related expression analysis studied for the two genes by Real-time PCR. More in-depth researches for GhGnT gene were studied by GFP sub-cellular localization and yeast expression technology. Specific research results are as follows:1. Isolated full length cDNA of two salt-tolerant genesThe full length cDNA sequences of GhPTAC and GhGnT were obtained by RACE technology combined with RT-PCR. The full length of GhPTAC is 1564bp, including 5'-UTR 41bp, complete ORF 1038bp and 3'-UTR 485bp, which encodes a polypeptide of 345 amino acids. The full length of GhGnT is 2140bp, including 5'-UTR 587bp, complete ORF 1353bp, 3'-UTR 200bp, and PolyA 27bp, which encodes a polypeptide of 450 amino acids.2 Real-time PCR analysesThe Real-time PCR results in cotton seed germination periods showed: The GhPTAC gene expression raising follows the increasing of salt stress level for Zhong S9612, and descending for Zhong 9806 in finite salt stress extent. The GhGnT gene expression raised at first, and then descended follows the increasing of salt stress level for both the experiment materials.The Real-time PCR results in cotton seedling stage showed: The GhPTAC gene expression raised induced by salt stress in both experiment materials, and it was higher in Zhong 9806 compared with Zhong S9612. The GhGnT gene expression rised induced by salt stress in both experiment materials. It was earlier to be induced by salt stress in Zhong S9612 compared with Zhong 9806, but the expression was lower than in Zhong 9806.The Real-time PCR detection found it is closely correlated for the genes expression changes and the salt stress.3 Sub-cellular localization for GhGnT gene encoded proteinPlant expression vector PBI121-GhGnT-GFP was built and transformated into onions endepidermis cell by particle gun. The result showed that the GhGnT protein was located at cell nucleus.4 Expression and analysis for GhGnT gene in yeastYeast expression vector PYES2-GhGnT was built and transformated into yeast strains AXT3 which is salt-sensitive by LiAC method. The yeast strains transformed GhGnT gene growed faster than CK, so we supposed that the high expression of GhGnT can improve the salt-resistant ability.