Functional Characterization Of Ceramide Synthase Gene GhCS1 Of Cotton

Cotton fiber is not only the main raw material of the textile industry,but also an ideal material for the study of plant cell differentiation,elongation and secondary wall formation.The study on the functions and regulatory mechanism of genes involoved in cotton fiber development has important theoretical and practical value.Sphingolipids membrane components and biological active molecules with a variety of molecular structures play important roles in forming membrane systems,protein transport,signaling pathways,programmed cell death,and response to biotic and abiotic stresses.The previous research of the research group showed that external application of sphingolipid synthesis inhibitor FB1 can significantly inhibit the growth of fibroblasts.Genetic regulation of the expression of the synthetase gene GhGCS1 can affect the development of fibroblasts,which proves that sphingolipid synthesis and metabolism is an essential part in the growth and development of fibroblasts.However,the synthesis pathway of sphingolipids in plants is complicate,and various intermediate products also have corresponding biological functions.Therefore,the function of different synthetase genes in fiber cell development needs to be further studied.Ceramide is an intermediate product of sphingolipid synthesis and of great importance in the sphingolipid synthesis pathway.It is speculated that the ceramide synthase gene plays a critical function in fiber development.To this end,this thesis cloned a ceramide synthase gene GhCS1 from upland cotton,and analyzed their functions in cotton plant and fiber development and related expression regulation mechanisms through genetic transformation of cotton and Arabidopsis.These results provide new evidence to illuminate the regulatory mechanism in fiber growth and development.(1)Cloning and sequence analysis of GhCS1The GhCS1 gene was cloned from upland cotton.Sequence analysis showed that the c DNA sequence of this gene contains a 924 bp ORF,which encodes 307 amino acid residues,with an estimated molecular weight of 36.1 k D and an isoelectric point of 8.19.GhCS1 is a membrane-bound protein with 5 transmembrane domains.The gene is located on the 7th chromosome of the D subgenome.Amino acid sequence alignment showed that GhCS1 contains the conserved domain of TLC,including LAG1,which is necessary for acyl-Co A-dependent ceramide synthesis.In terms of phylogenetic relationship,Cotton GhCS1 is closely related to CSs of hibiscus,durian and cocoa.(2)Expression analysis and subcellular localization of GhCS1Real-time fluorescence quantitative RT-PCR results showed that GhCS1 was predominantly expressed in fibers and roots during the rapid elongation period,indicating that GhCS1 participated in the rapid elongation of cotton fibers.The plant expression vector containing the GhCS1::e YFP fusion gene controlled by the Ca MV 35 S constitutive promoter was constructed and then transiently expressed in tobacco leaves.The subcellular localization observation results showed that the GhCS1 protein was localized in the cell membrane and endoplasmic reticulum.(3)The effect of ectopic expression of GhCS1 gene on the growth and development of Arabidopsis thalianaThe Arabidopsis mutant Atloh1 has a smaller leaf and shorter inflorescence than the wild type.Atloh2 has a phenotype such as larger leaf area and sharp leaf shape.GhCS1 gene was overexpressed in Arabidopsis At LOH1 mutant Atloh1 and At LOH2 mutant Atloh2,and stable genetic transformants were obtained.It was found that GhCS1 could not restore the phenotype of Atloh1,but can restore the Atloh2 phenotype to a certain extent,from which indicated that GhCS1 can complement the function of At LOH2.The overexpression vector was transferred to wild-type Arabidopsis,and it was found that the leaf seat area of the transgenic Arabidopsis was reduced and the bolting time was delayed,which was consistent with the phenotype of overexpression of At LOH2.The wild-type Arabidopsis,mutant Atloh1,Atloh2 and heterologous overexpressing GhCS1 Arabidopsis were treated with FB1,and it was found that Arabidopsis thaliana expressing GhCS1 were less sensitive to FB1.(4)The effect of regulating the expression of GhCS1 gene on the growth and development of cotton plants and fibersThe plant over-expressing GhCS1 grows dwarf,compact,leaves and petals are smaller,petals are curled and cannot open normally,as well as anthers and pollen are abnormal.Sepals become smaller and cannot be separated,flower buds are few and fall off 2 to 3 days after flowering,unable to set fruit,and highly sterile.On the surface of transgenic ovules overexpressing the GhCS1 gene,fibrocytes start later than the wild type,and the ovules are smaller than the wild type,and the development tends to stagnate after 1 DPA.At 2-DPA stage,the surface of the ovule began to collapse and shrink,and more reactive oxygen species accumulated in the ovule.The results of in vitro culture of ovules showed that the fibers of OE-GhCS1 were shorter than the wild type and the ovules were always smaller than the wild type during the initial,elongation,and secondary wall synthesis stages of cotton.At the same time,the sensitivity of OE-GhCS1 fiber growth to FB1 also decreased.(5)Analysis of sphingolipidome and transcriptome in transgenic ovulesIn the transgenic ovules(containing fibers)overexpressing the GhCS1 gene,the content of sphingosine and some phytoceramides decreased,and the content of some ceramides and glucosylceramides increased.It shows that increasing the expression of GhCS1 gene promotes the synthesis of ceramide and complex sphingolipids.In transgenic ovules(containing fibers)overexpressing the GhCS1 gene,the GhCS1 gene was upregulated by 5.21 times.In addition,some other genes in the sphingolipid synthesis pathway also changed significantly,such as α,1,4-glycosyltransferase and 3-ketoacylCo A synthase.The expression of multiple homologous genes of these synthases was upregulated several times,indicating that up-regulation of GhCS1 gene expression can promote the production of glucosylceramide and very long chain fatty acids,and GhCS1 is a useful gene in the sphingolipid synthesis pathway.(6)Cloning and functional analysis of GhCS1 promoterIn this study,the regulatory sequences of 642 bp,1113 bp,1468 bp,1980 bp,and2380 bp upstream of GhCS1 were cloned.In addition to core transcription elements and a large number of light response elements,these regulatory sequences also contained abscisic acid response elements and anaerobic induction.Response elements,stress response elements,defense and stress response elements,and salicylic acid response elements and many other specific response elements.The cloned promoter was fused with the GUS gene and transformed into Arabidopsis thaliana.After obtaining stable genetic transformants,GUS staining analysis was performed.The results showed that the promoter was mainly expressed in the anthers and petals of flowers,and the expression intensity of different 5’-deleted promoter fragments is different.The staining of the 1113 bp to 1468 bp fragment gradually deepens,and there are more enhancer elements between the two fragments.After the 1113 bp promoter fragment was treated with FB1,the taproot began to color,indicating that FB1 did have a certain effect on the upstream promoter fragment of GhCS1.