Functional Analysis Of The Fiber Development Related Genes GhCLASP2 And GhAlaRP In Cotton

Object:Cotton is the most important source of the natural fiber and the textile industry materials in the world.Cotton fiber is differentiated and developed from the ovule epidermal cells.Many genes are involved in the regulation of cotton fiber development.Microtubule is the main components of plant cytoskeleton,which also plays an important role in the cotton fiber elongation and the secondary wall thickening.Cytoplasmic linker-associated protein(CLASP Protein)is a microtubule associated protein that specifically binds with microtubules and participates in regulation of microtubules structure and function.Previous studies have shown that CLASP is involved in the root and the hypocotyl cell elongation,cell signal transduction and leaf trichome branching.Cotton fiber is the trichome of cotton seeds.Due to the structural and genetic similarities,the development of cotton fiber cells may have similar regulatory mechanisms with the development of Arabidopsis trichomes.GhAlaRP(Ala-Rich-Protein)is a cotton fiber-specific expressed gene.The expression level of GhAlaRP is the highest in the 6 DPA cotton fiber tissues;we speculate that this gene may relate to the elongation of cotton fiber development.In view of the hypothesis that GhCLASP and GhAlaRP gene may play regulatory effects in fiber development.In this study,we cloned GhCLASP and GhAlaRP genes from the cotton fiber,analyzing their regulatory functions and molecular mechanism during cotton fiber development,laid the foundation for use genetic engineering to improve the quality of cotton fiber.Method:GhCLASP2 and GhAlaRP gene sequences were analyzed by the bioinformatics software.The subcellular localization vectors,the overexpression vectors and the RNA interference(RNAi)vectors of GhCLASP2 and GhAlaRP genes were constructed,and injected with the tobacco(N.benthamiana),Arabidopsis(Col-0 and clasp-1)and cotton(YZ-1),respectively.The transgenic plants were identified and the phenotypes were observed.The functions of GhCLASP2 and GhAlaRP were analyzed in the transgenic plants.Protein network that regulates cotton fiber development in interaction with GhAlaRP was identified by the yeast two-hybrid system and the bimolecular fluorescence complementation.The CRISPR/Cas9-GhAlaRP genome editing vector was constructed,and the cotton(YZ-1)was injected,the transgenic cotton was identified to provide a mutant material for studying the function of GhAlaRP.Results and Conclusion:(1)GhCLASP2 gene was high similarity with AtCLASP sequence and GhCLASP2 was cloned from cotton fiber.Bioinformatics analysis shows GhCLASP2 protein have two HEAT repeat domains and two CLASP-N domains.GhCLASP2 belongs to the CLASP family I subfamily.qRT-PCR analysis shows that GhCLASP2 was predominantly expressed in cotton fibers,especially in the secondary cell wall thickening stage(27 DPA and 30 DPA)during cotton fiber development.The result indicates that GhCLASP2 is involved in the regulation of cotton fiber development,which may be related to the formation of cotton fiber quality.The results of subcellular localization in tobacco(N.benthamiana)showed that GhCLASP2 was located on the microtubules of cells.Ectopic expression of GhCLASP2 in Arabidopsis can increase the branching number of leaf trichomes and rescue the phenotypes(sunch as the trichomes,the hypocotyls,the roots and so on)of clasp-1.Compared with the control cotton of YZ-1,the cotton fibers strength of the overexpression of GhCLASP2 transgenic lines was increased.This result may be related to the change expression levels of tubulin(TUA and TUB),cellulose synthase(CESA),and expansin(EXP)genes.Suppression of GhCLASP2 caused shorter internodes and semi-dwarfism,abnormal flower stigma,aborted anthers without pollen grains,and sterility.These phenotypes are similar with those observed in the Arabidopsis clasp-1 mutant.These results suggest that GhCLASP2 and AtCLASP may have similar functions to regulate cotton growth and development by regulating the microtubules.(2)Sequence analysis showed that GhAlaRP is a protein containing two transmembrane domains.Phylogenetic tree revealed that GhAlaRP have the closest evolutionary relationship with TCAlaRP.Subcellular localization indicated that GhAlaRP was localized on the plasma membrane,the nucleus and the endoplasmic reticulum,which indicate that GhAlaRP may participate in the transport of substances in cotton fiber cells.The results of the yeast two-hybrid system and BIFC show that GhAlaRP could co-express and interact with GhAnnexin(Gh_D11G2184)and GhEXPA(Gh_A10G2323)in cells.Transgenic cotton fiber which inhibits GhAlaRP expression becomes shorter in length,and the expression levels of GhAnnexin and GhEXPA were decreased.The above results indicate that GhAlaRP is involved in the regulation of cotton fiber elongation development;suppression of the expression of this gene has a significant decrease in fiber length.GhAlaRP interacts with GhAnnexin and GhEXPA proteins to form a regulatory network to regulate the cotton fiber development,and there may be other proteins involved in this process.In addition,the cotton alarp mutants were obtained by using the CRISPR/Cas9 genome editing technology.The fiber length of the cotton alarp mutant was also significantly shorter than that of the control cotton YZ-1;this result is consistent with the phenotype of the GhAlaRP-RNAi transgentic cotton fiber.These results further indicate that GhAlaRP is involved in regulating the elongation of cotton fiber development.