Cloning And Functional Analysis Of A Gene Corresponding To Salt-tolerance In Wheat

Salt-tolerance plant has important significance not only for the study of genetic improvement of crops, but also the basic biology of plant as an important component. Therefore, the cloning of salt-tolerance plant gene has caused people′s widespread attention. In recent years, with the rapid development of molecular biology, scientists have found and cloned a number of genes related to salt-tolerance plant, but it is far from the people's expectations. Using our laboratory salt-tolerance wheat varieties RH8706-49 and according to the gene chip information(the expression is rising with the time extention of salt stress). I got two electronic clones through the probe, which contain complete ORF respectively, then I used RT-PCR technology to clone the two genes, and were named TaMIP (Triticum asetivum L. Major intrinsic protein) and TaRSP (Triticum asetivum L. Root specific protein).This experiment focuses on the TaMIP gene, DNAstar software forecasted its biochemical nature,the gene encodes a protein of 300 amino acids, the molecular weight 32270.40 mw, isoelectric point 7.43. NCBI website forecasted its conserved domain, containing two Asn-Pro-Ala (NPA) motifs. Real-time quantitative PCR was used to detect the pattern of the gene TaMIP in wheat.The wheat was treated with NaCl, ABA, PEG and cold. The expression pattern in leaves by NaCl, ABA, PEG and cold stress showed varying degrees of increase, in the root TaMIP in the NaCl, ABA and PEG stress showed varying degrees of increase, and showed suppression in cold stress.Constructed at the same time for the positioning and function of the binary vector TaMIP-GFP and TaMIP, and transformed into Arabidopsis thaliana. The result of subcellular localization showed that the gene location was in the cytoplasm and cell membrane. The study of the transgenic Arabidopsis germination rate, root length, salt-tolerant overall plants showed that the gene in a certain level enhanced the transgenic Arabidopsis salt tolerance. Quantitative PCR detected transgenic Arabidopsis salt stress related gene ADH1,RD29B,KIN2,COR15a and P5CS1,The results showed that compared with the control, P5CS1,ADH1,RD29B upward, especially RD29B up, transgenic plants were about 11 times expression than wild-type; KIN2 and COR15a different degrees of reduction in transgenic Arabidopsis, COR15a was seriously inhibited.Quantitative PCR analysised the genes of the different signaling pathways downstream.In the CDPK pathway FRY1 had no change between wild and transgenic Arabidopsis, while SAD1was slightly rise;In the SOS pathway SOS3 had no change between wild and transgenic Arabidopsis,but SOS2 was downregulated.So speculating TaMIP gene might be downstream genes in salt pathway. Flame atomic absorption detected the content of Na⁺,K⁺,Ca²⁺ in transgenic and wild type Arabidopsis.The content of K⁺,Ca²⁺ were higher in transgenic Arabidopsis,while the content of Na+ was lower.It was accorded with the salt-tolerance mechanism in plant.Through detecting the content of Pro, the result showed the content of Pro was higher in transgenic Arabidopsis than in wild type, It was also accorded with the salt-tolerance mechanism in plant.