Screen Of Salt Tolerant Genes And Functional Analysis Of Protein Tyrosine Phosphatase In Cotton

Cotton (Gossypium hirsutum L.) is the most important fiber and oilseed crop in the world. At present, salt stress is the main disaster in production. With the molecular biotechnology development, plant gene engineering research on salt tolerance got a lot of achievements. Biological resistance to salt is a quantitative trait controlled by multiple genes, and the physiological and biochemical processes are the results of genes interactions and co-regulation, so exploiting cotton salt-related genes and exploring cotton salt response pathway are the important goals. This study set up cotton VIGS (Virus Induce Gene Silencing, VIGS) system under hydroponic condition. Multiple cotton salt-related genes were screened by VIGS-cDNA library. A cotton protein tyrosine phosphatase GhPTPl gene was cloned successfully, uncovered GhPTP1 played a negative regulatory role in salt response pathway. GhPTP1 interacting protein was screened by yeast-two-hybrid system. The main results were as follows:Cotton VIGS system under hydroponic condition was set up. Silencing efficiency was induced by syringe-infiltrated time at 3 to 5 days after seeding emergence and optimum growth temperature at 24℃ under hydroponic condition, but syringe-infiltrated concentrations could not affect VIGS silence efficiency. Moreover, pTRV-GFP as null fragment could alleviate the adverse effect of insertion fragment for plant growth. Silencing phenotype could be visible earlier in hydroponics culture than soil culture, and the experimental period was significantly shortened under hydroponics condition. In addition, GhCLA1 could be silenced in all tested varieties (lines) under hydroponics condition. Cotton plants silenced GhCTRl were severely dwarfed.VIGS-cDNA library was screened by VIGS system, obtained several cotton salt-tolerant and-sensitive phenotype. Sequencing results showed that the candidate genes mainly encode photosynthesis related protein, phosphatase, ACR proteins and other function-unknow proteins. This study cloned GhPTP1 gene. The full length of GhPTP1 including family characteristic sequence (V/I)HCXXGXXR(S/T) was 849 bp, encoding 283 amino acids. Multiple sequence alignments showed that GhPTP1 and plum, orange, grape, cocoa, tobacco proteins are highly similar. Phylogenetic analysis indicated GhPTP1 and AtDSP8, AtDSP10 belonged to the same clade. Tissue specific expression and subcellular localization results showed that high expression of GhPTP1 gene were observed in cotton root and leaf, and GhPTP1 located on the plasm-membrance. Results of qRT-PCR showed that NaCl up-regulated GhPTP1 gene expression in different tissues of cotton. GhPTP1 gene can be also up-regulated by ABA and PEG, but down-regulated by H2O2, which hinted that GhPTP1 may participate in signal pathways of plants under stress conditions. Cotton seedlings of GhPTP1 silenced showed obvious salt-resistant phenotype under salt stress, dry weight was higher than that of the control, and the expression of GhNHX1, GhSOSl and GhSOS2 were higher than the controls. In Arabidopsis, over-expression GhPTP1 plants were sensitive to salt, cotyledons greening and root length were lower than the WT, and plant Na+ accumulation was higher than the WT. The results showed that GhPTP1 as a negative regulatory in salt response signaling pathway. At the same time, GhPTP1 gene silencing reduced plants sensitivity to osmotic stress, relative water content of leaves of VIGS-GhPTP1 were significantly increased; and water lost in cotton leaves were significantly decreased. The over-expression GhPTP1 Arabidopsis showed obvious drought stress phenotyse. The results showed that GhPTPl mainly involved in osmotic and Na+ stress pathway induced by NaCl, and the content of Na+ was affected by the involvement of GhPTPl in SOS pathway.More than thirty positive clones were screened by yeast-two-hybrid system interacting with GhPTP1. These clones included several transcription factors, binding proteins, ion transport proteins and so on. Interaction between GhPTPl and GhANN5 was identified by Co-IP and transient expression in tobacco, and the interaction between GhPTPl and GhANN5 can be induced by salt stress.