Preliminary Functional Analysis Of Four Salt Tolerance Related Genes In Cotton(Gossypium Hirsutum L.)

Cotton is an important cash crop,which is of great significance to the stability and development of China’s national economy.With the decrease of arable land area year by year,the contradiction between grain crops and cotton in competition for arable land has become increasingly prominent,and the demand for cotton planting in saline-alkali land has become more and more urgent.Cotton is a relatively salt-tolerant crop,but the high salinity soil environment will seriously affect the yield and quality of cotton.Therefore,mining key salt-tolerant genes,cultivating new varieties of high salt-tolerant cotton and developing cotton industry in saline-alkali land are effective ways to solve the problems of development and utilization of saline-alkali land and "cotton-grain competition for land".The previous laboratory used 288 upland cotton materials to carry out GWAS(Genome-wide Association study)analysis of genotypes and 10 salt-tolerant phenotypic traits,and obtained a number of SNP loci related to salt tolerance.Candidate genes related to salt tolerance in cotton were excavated based on salt stress transcriptome data.In this study,four candidate genes were selected to identify the expression and location characteristics of candidate genes through bioinformatics analysis,tissue and organ expression,salt-induced expression analysis and subcellular localization.Virus-induced gene silencing(VIGS)vectors of these four genes were further constructed.The salt tolerance function of candidate genes was preliminarily verified by VIGS technology and physiological and biochemical analysis.The main results are as follows:1.Mining of four candidate genes:Based on eight SNP loci associated with GWAS in the early stage of our laboratory,and referring to LD value of Upland Cotton Cultivar population,further mining candidate genes in 500K linkage region upstream and downstream of these loci,combined with upland cotton salt-induced transcriptome database,34 genes were found to be induced by salt stress.Finally,four candidate genes,GhHRGP,GhGASA4,GhBAM2 and GhMURE,were selected for functional verification.The coding products of these four genes are hydroxyproline-rich glycoprotein,GASA protein,leucine receptor-rich protein kinase and acid amino acid ligase.2.Expression and location characteristics of four genes:tissue and organ analysis showed that GhHRGP was predominantly expressed in roots,GhGASA4 was predominantly expressed in pistils and ovules 0 days after anthesis,GhBAM2 was predominantly expressed in roots and stems,and GhMURE was predominantly expressed in leaves.Salt induction analysis showed that these four genes were significantly up-regulated after salt stress.The expression of GhHRGP,GhGASA4 and GhMURE reached the maximum after 12 hours of NaCl treatment,and GhBAM2 reached the maximum after 72 hours of NaCl treatment.Subcellular localization locates GhHRGP on cell membrane,GhGASA4 in nucleus,cell membrane and cytoplasm,and GhMURE in cell membrane and cytoplasm.GhBAM2 could not determine its location.3.Silencing the target gene by VIGS technology to further verify the functional characteristics of four candidate genes under salt stress.The results showed that compared with the control,salt treatment inhibited the growth of plants.After GhHRGP and GhGASA4 were silenced,the leaves of plants withered seriously.After GhBAM2 was silenced,the leaves of plants treated with salt produced macula.It indicated that GhHRGP,GhGASA4 and GhBAM2 were involved in the stress resistance process of plants.Leaf yellowing occurred after GhMURE silencing,which indicated that GhMURE was not only involved in stress tolerance response,but also in plant growth and development.Ten indices,such as plant height,dry matter weight above and below ground,Pro,MDA and H2O2 content,SOD and POD activity,K+/Na+ratio above and below ground,were determined after 15 days of salt treatment and normal watering.The results showed that GhHRGP and GhBAM2 had significant differences before and after salt stress,POD and H2O2 existed,plant height,Pro and SOD activity,K+/Na+ ratio above and below ground compared with CK and TRV:00.The difference is extremely significant.There were significant differences in POD,plant height,Pro,SOD and H2O2 of GhGASA4 before and after salt stress.There were significant differences in GhMURE,aboveground and underground dry matter weight before and after salt stress,and there were significant differences between Pro and SOD.In summary,the four genes play an important role in salt tolerance of cotton,and provide genetic resources for stress tolerance research of cotton.