Study On The Mechanism Of Adenylate Cyclase Activator Improving The Salt Tolerance Of Cotton Seeds

Cotton is the main natural fiber crop in the world and one of the important oil crops.It plays an important role in the national economy and agricultural production of cotton-producing countries around the world.As the global climate continues to deteriorate,the area of land available for agriculture continues to shrink,the contradiction between population and resources is becoming increasingly prominent,and the land competition between food crops and cotton and other economic crops is becoming more and more serious.By cultivating drought-tolerant and saline-alkali-tolerant cash crops and planting cash crops on potentially valuable land such as drought and saline-alkali land,it can not only ease the conflict between food crops and cotton and other cash crops to ensure food security,but also expand the agricultural land area.Abiotic stresses affect the entire cycle of plant growth and development.The ability to resist stress during seed germination determines the emergence rate of the plant and is the basis for the formation of plant yield.By studying the mechanism of response to stress during seed germination of cotton and other plants under stress conditions such as drought,salt and alkali stress,it is important to further explore the key genes that regulate seed resistance to stress stress,which is of great significance for drought-tolerant and salt-tolerant cotton molecular breeding techniques.cAMP is the second messenger in plant cells,which plays an important role in the resistance of plants to stress.Forskolin can significantly increase the cAMP content in plant cells by activating cyclic adenylase activity,further activate cAMP-dependent stress-related genes,and enhance the plant’s ability to resist stress.In this study,H₂O,120μM Forskolin,25μM cAMP,0.4%NaCl,0.4%NaCl+120μM Forskolin,0.4%NaCl+25μM cAMP were used to treat the salt-tolerant’P30’,’Henan No.4’and non-salt-tolerant Alkali’Yangtze A3’upland cotton seed.Analyze the differences in germination potential,germination rate and hypocotyl development during the germination of cotton seeds;determine the oxidase SOD and CAT activities in cotton seeds,determine the content of plant endogenous hormones IAA,SA,JA,GA,and determine the osmotic adjustment substance The content of lysine was measured in the second messenger cAMP content and other physiological and biochemical indicators.At the same time,the high-throughput RNA-Seq sequencing technology was used to analyze the differential genes between different treatments.Combining physiological and biochemical indexes and bioinformatics algorithm analysis,a group of cotton salt related genes were identified during the germination of cotton seeds and the VIGS technology was used to carry out preliminary functional verification of the related genes.The main research results obtained are as follows:1.Cotton seed germination experiments showed that three kinds of cotton seeds were treated with six solutions of H₂O,120μM Forskolin,25μM cAMP,0.4%NaCl,0.4%NaCl+120μM Forskolin,0.4%NaCl+25μM cAMP.Forskolin treatment compared with H₂O treatment Germination potential and germination rate increased by an average of 9.63%and 2.22%,respectively.Compared with NaCl treatment,the germination potential and germination rate of seeds were increased by an average of12.97%and 9.26%,respectively.elongation..2.The determination of biochemical indicators during the germination process of cotton seeds showed that H₂O,120μM Forskolin,25μM cAMP,0.4%NaCl,0.4%NaCl+120μM Forskolin,0.4%NaCl+25μM cAMP solution were soaked in the three cotton seeds for 4h to complete the uptake process In the petri dish,continue to process with the corresponding solution of wet filter paper until 16h,and measure the biochemical index.Compared with H₂O treatment,Forskolin treatment had an average SOD enzyme activity of 88.70 U/g and CAT enzyme activity decreased by an average of 247.57 U/g;GA3,cAMP,SA and IAA contents increased by an average of 0.218nmol/g and 0.067 nmol/g,respectively.18.39μg/g and 6.7×10^^-3nmol/g;ABA,JA and proline content decreased by an average of 170.38ng/g,2.68pmol/g and 59.04ng/g,respectively.Compared with NaCl treatment,NaCl+Forskolin treatment increased SOD and CAT enzyme activities by an average of 553.33U/g and 192.77U/g,respectively;GA3,cAMP and SA contents increased by an average of 0.546nmol/g,0.053nmol/g and 3.42μg/g;ABA,JA,IAA and proline content decreased by an average of 182.11ng/g,4.56pmol/g,0.154 nmol/g and 48.60ng/g,respectively.3.Difference analysis of the transcriptome of samples from different treatments:soak the three cotton seeds in H₂O,0.4%NaCl,120μM Forskolin+0.4%NaCl solution for 4h to complete the swelling process,move to a petri dish,and continue the treatment with the corresponding solution of wet filter paper until 16h,RNA-Seq sequencing,a total of 190,352,219 clean reads were obtained.Screening with p-Value≤0.005 yielded 163,636 genes with significant differential expression（DEGs）.There were 4097 differentially expressed genes in the three treatments of the’P30’material,22260 differentially expressed genes in the three treatments of the’Henan No.4’material,and 25606 differentially expressed genes in the three treatments of the’Yangtze A3’material.The differential gene expression analysis of the same treatment between different materials showed that:under H₂O treatment,there were a total of42822 differentially expressed genes in the three materials;under NaCl treatment,there were a total of 48752 differentially expressed genes in the three materials;under NaCl+Forskolin treatment,the three materials were different A total of 20071 genes were expressed.Combining all the differential expression data shows that the number of differential genes between different treatments of the same material is small,and the number of differential genes in the same treatment of different materials is more,which may be due to the different genetic background and salt tolerance between different materials.4.Enrichment analysis of transcriptome data:GO→KEGG→Inter Pro→Domain analysis of all the differential genes,and the Pathway results obtained by statistical analysis and annotation show that under NaCl treatment,’P30’,’Henan No.4’,and’Yangtze A3’Pathway is mainly enriched in:fatty acid biosynthesis,secondary metabolite biosynthesis,C5 isoprenoid biosynthesis,glyoxylic acid and dicarboxylic acid metabolism,Pathway is mainly enriched in NaCl+Forskolin treatment:C10-C20isoprene biosynthesis,biosynthesis of secondary metabolites,pyruvate metabolism,glutathione metabolism,citrate cycle（TCA cycle）,carbon metabolism,glyoxylic acid cycle,pentose phosphate pathway.5.Functional validation of the salt-tolerant candidate gene VIGS:Since gene silencing induced by VIGS occurs mainly at the seedling stage,the ability of the candidate gene to respond to salt-alkali stress at the seedling stage of cotton needs to be verified.The seedlings of salt-tolerant and salt-sensitive materials were treated with H₂O,0.4%NaCl,120μM Forskolin+0.4%NaCl for 16h,and the expression of candidate genes was analyzed by qRT-PCR.Select the gene with the same expression method in the same treatment as the seed germination stage to construct the VIGS vector,and use the Agrobacterium-mediated method to transform cotton seedlings.After the seedlings grow to 5-6 true leaves,the transformed seedlings are treated with1%NaCl medium for three days to preliminarily identify gene function,and qRT-PCR is used to detect gene expression.The results showed that Gh WRKY33,and other genes were significantly reduced in salt tolerance;after Gh CYP82A3 gene silencing,plant salt tolerance was significantly improved.The research in this paper shows that Forskolin activates adenylate cyclase activity and increases cAMP content in the body.CAMP participates in cotton salt tolerance-related pathways.Increasing intracellular cAMP content can significantly increase the germination efficiency of cotton seeds in saline soil.At the same time,transcriptome and VIGS verification showed that Gh WRKY33,Gh CYP82A3 and other genes were regulated by cAMP,which was related to the salt tolerance of cotton seeds during germination.In addition,the genes obtained in this paper can provide genetic resources for the genetic improvement of cotton salt tolerance,and the research strategy of this paper can provide new ideas for the study of the tolerance mechanism of other crops..