Isolation And Functional Verification Of Salt Stress Response Genes In Upland Cotton (Gossypium Hirsutum L.)

Cotton is the pioneer crop used in salt and alkali land. To reveal the stress mechanism and to increase the tolerance of cotton are very important to genetic improvement in Upland Cotton. Based on the previous research on the SSH libraries of Upland cotton roots under salt stress, we have selected five candidate genes, including GhSnRK2.6, GhCIPK6, GhCPK5, GhC2H2 and GhWRKY41. We cloned the full length sequences of these genes, constructed the plant expression vectors, and transformed into wild-type Arabidopsis and cotton breeding lines. We constructed transient expression vectors, to analyze subcellular localization. We have investigated and analyzed the salt-tolerance of transgenic materials, analyzed these candidate genes functions, and provided the material source for the breeding of cotton salt tolerance.(1) We have isolated the full length of gene GhSnRK2.6, whose GenBank accession number is JN872373. The full length of ORF (opening reading frame) was 1,086 bp, coding a 361 amino acid polypeptide. The gene included 9 exons and 8 introns. Through constructing the transient expression vector p35S-GhSnRK2.6:GFP, and transforming into onion epidermal cells with particle bombardment, the results was showed that Subcellular localization of GhSnRK2.6 protein was in the nucleus. We constructed the plant expression vector, p2301M-GhSnRK2.6, transformed into wild-type Arabidopsis and cotton breeding line to analyzethe salt tolerance of transgenic materials. The results showed that the salt tolerance of transgenic Arabidopsis was improved at the stage of both seedling and mature stages. The transgenic cotton exhibited significant higher germination rate, and the salt tolerance was also higher than the receptor line. Moreover, the yield and fiber quality of transgenic cotton were better than receptor cotton under salt stress.(2) We have constructed the transient expression vector, p35S-GhCIPK6:GFP, and the results of transient expression assays showed that subcellular localization of GhCIPK6 protein kinase was in the cytoplasm. BiFC assay showed GhCIPK6 interacted with GhCBLl and GhCBL8. GhCBL1-GhCIPK6 complex was located in the nucleus, and the GhCBL8 and GhCIPK6 co-expressed in the nucleus and membrane. Then we transformed the recombinant vector into the cotton breeding line using pollen-tube-pathway method. Then we analyzed the salt tolerance of transgenic cotton lines. Under salt stress, GhCIPK6involved in multiple pathways to regulate the balance of cellular K+, maintain the cell membrane stability, and increase salt tolerance of transgenic cotton.(3) We constructed transient expression vectorto analyse the subcellular localization. The result showed that the transcription factor GhC2H2 was in the nucleus. Then we transformed the recombinant vectorpBI-GhC2H2into the cotton breeding line using pollen-tube-pathway method. Through analyzing the salt tolerance of transgenic cotton lines, we found that GhC2H2 over-expression transgenic cotton exhibited higher lint percentage, boll weight and fiber length than receptor, and increased salt tolerance.(4) We analysed the subcellular localization of GhCPK5 protein kinase using transient expression assays, and the results showed that the protein was localised in the nucleus. Then we transformed the recombinant vector pBI-GhCPK5 into the cotton breeding line using pollen-tube-pathway method. Then we analyzedthe salt tolerance of transgenic cotton lines, and found that the fiber quality of transgenic lines was higher than receptor. The boll weight, fiber length and fiber strength of transgenic lines increased, and micronaire decresed.(5) We analysed the subcellular localization of GhWRKY41 through constructing the ransient expression vector, which showed the transcription factor was in the nucleus. We transformed the recombinant vector p2301M-GhWRKY41 into the cotton breeding line using pollen-tube-pathway method. Over-expression of GhWRKY41 in transgenic cotton could improve germination rate under salt, cold and drought stresses. Except the fiber quality, the transgenic cotton lines could maintain boll weight and lint percentage under salt treatment.