Screening Of Salt Tolerance Genes And Preliminary Functional Analysis Of Ghspx1 Gene In Xinjiang Cotton Varieties

Xinjiang is one of the most serious salinization areas in China,and one third of cultivated land is composed of saline alkali soil.Soil salinization is the most important environmental factor restricting agricultural production,especially cotton production in Xinjiang.Cotton is a kind of economic crop with strong salt tolerance,which plays an important role in the development of agricultural economy in Xinjiang.It is one of the main ways to improve cotton yield to study the mechanism of salt tolerance and screen out the functional genes related to salt tolerance.In this study,through transcriptome sequencing technology,we compared and analyzed the differential genes before and after salt stress in cotton,screened out the regulation genes related to salt tolerance in cotton,cloned the full sequence of Ghspx1(cot-ad 50458 in the transcriptome),and constructed the overexpression vector and transformed into Arabidopsis thaliana to further verify the salt tolerance related function of the gene.This study provided a theoretical basis for further understanding the molecular regulation mechanism of Ghspx1 gene in salt stress response of cotton.The main results of this paper are as follows:1.The results showed that the germination rate,chlorophyll content,cat and SOD activities of Xinluzhong 69,Guoshenmian206-5,Xinluzhong 72 and Xinluzhong 42 were higher than other cotton varieties under different salt stress.’Xinluzhong 69’ is the best material for transcriptome sequencing of salt tolerant cotton.2.In order to reveal the molecular mechanism of salt tolerance in cotton and screen the genes related to salt stress response in cotton,"Xinluzhong 69" was selected as the research object,and the transcriptome analysis of its roots was carried out under salt stress.The results showed that 891 genes were up-regulated and 666 genes were down regulated.Go enrichment analysis showed that more differentially expressed genes were mainly concentrated in molecular function,cell components and biological processes.KEGG analysis showed that the differentially expressedgenes were mainly involved in lignin biosynthesis and plant MAPK signaling pathway.3.According to the ORF sequence of cot-ad 50458 in transcriptome database,primers were designed to amplify and clone the gene sequence using cotton root c DNA as template.Cot ad 50458 ORF is 792 BP in length,encodes 263 amino acids,MW is 30.3559 k Da,PI is 6.80,there are 15 phosphorylation sites.Phylogenetic tree analysis showed that cot-ad 50458 was highly homologous with SPX of Gossypium raimondii.Cot-ad 50458 contained a typical conserved domain of SPX.Cot-ad 50458 was named Ghspx1 according to this structural feature.The recombinant plasmid p MD18-T-Ghspx1 was constructed.According to the ORF sequence of Ghspx1 gene,primers with Xba I and Eco R I were designed.The Ghspx1 fragment was amplified with the recombinant plasmid p MD18-T-Ghspx1 as template.The PCR product was digested and ligated with plant expression vector p BI121,and then transformed into E.coli DH5α The recombinant plasmid of p BI121-Ghspx1 was extracted from competent cells and identified by double enzyme digestion.4.In order to further verify the function of Ghspx1 gene,the constructed expression vector p BI121-Ghspx1 was transformed into Arabidopsis thaliana by Agrobacterium mediated floral pathway.The results showed that: the root development of T1 generation of transgenic Arabidopsis L1 was shorter than that of wild-type Arabidopsis under salt stress;the germination rate of T1 generation seeds of transgenic Arabidopsis L1 was lower than that of wild-type Arabidopsis seeds under salt stress,which indicated that Ghspx1 gene could improve the sensitivity of transgenic Arabidopsis to salt stress.