Identification Of CRK Gene Family In Upland Cotton And Functional Analysis Of GhCRK25

The cysteine-rich receptor kinase(CRK)is a kind of plant receptor kinase,and plays important roles in plant growth and development,hormone signal transduction and Stress tolerance.So far,few cotton CRK genes have been reported.The cotton genome database was searched using the BLASTp program to identify the CRK gene family of cotton;The mapping of the chromosomal localization,gene structure and protein domains of the CRK gene family of upland cotton was made using TBtools;Download the RNA-Seq data from the NCBI database,the calculation of TPM value using transcriptome quantitative tool Kallisto,draw the heat map of CRK gene expression in upland cotton through online tools Morpheus..Silencing GhCRK25 with VIGS technology was used to study the role of GhCRK25 in resistance to Vd991 in upland cotton.Transferring GhCRK25 into Arabidopsis thaliana to study its role in resisting Vd991;lays the foundation for in-depth study and utilization of CRK family genes in upland cotton.The main findings are as follows:1.There are 70 CRK genes in the genome of upland cotton distributed on 14 chromosomes,52 genes(74.3%of the total)are intensively distributed in clusters on A6/D6,A9/D9 and A10/D10 chromosomes,and show highly collinear relationships between A/D chromosomes.These CRK genes encode proteins containing 302-901 amino acids,58 proteins(82.9%)has a transmembrane domain,mainly located in the chloroplasts,plasmaleuna and extracellular.Phosphorylation site prediction results showed that Upland Cotton and Arabidopsis CRK share 5 consensus phosphorylation sites.The promoter regions of 65 cotton CRK genes(account for 92.9%)contain at least one Stress hormone response element,and 69(98.6%)genes' contain at least one biotic or abiotic Stress response element.Based on RNA-Seq data analysis,CRK gene in upland cotton showed different types of tissue expression patterms;salt,drought,cold,heat Stress and inoculation with Verticillium dahliae lead to some of CRK genes to alter their expression levels,and thus they can be divided into three types of Stress response patterns.2.GhCRK25 has 7 exons and 6 introns.The ORF length is 1965 bp,encoding 654 amino acids,the relative molecular mass is 73.37 kD,and the theoretical isoelectric point is 7.63.The promoter region of GhCRK25 contains multiple phytohormone responsive elements,as well as Stress and defense response elements.GhCRK25 is predominantly expressed in roots,stems,leaves,and ovules,but hardly expressed in fibers.Treatments of cotton seedlings with ABA,GA3,SA,PEG-6000,NaCl and Vd991 all stimulated the up-regulated expression of GhCRK25.Silencing GhCRK25 reduced the resistance of upland cotton to Vd991.Overexpression of GhCRK25 enhanced the resistance of transgenic Arabidopsis thaliana to Vd991.The upland cotton CRK fanily genes are tandemly clustered in the genome;they play an important role in resisting various biotic stress and abiotic stress in upland cotton.The GhCRK25 gene is associated with Verticillium wilt resistance in cotton.