Genetic And Biochemical Analyses Of The GhFSN1 Transgenic Cotton (Gossypium Hirsutum) Progenies

Cotton in China and the world's agricultural economy are in an important position,but the quality and yield of traditional cotton has been difficult to meet the needs of modern society.In order to solve this problom,the application of transgenic technology in cotton is becoming more and more widely,therefore,the basic research on the function of cotton gene,it is very necessary therefore.In this study,a NAC family of gene,named GhFSN1,was cloned in cotton fiber.It has high homology with genes NST1-3 which are involved in the regulation of secondary wall synthesis in arabidopsis.In this paper,the gene was further explored,and the following results were obtained:1.GhFSNl overexpression of transgenic cotton phenotypic analysisIn order to further understand the regulation function of GhFSN1 gene in cotton fiber development,we used GhFSN1 transgenic cotton as material to analyze the genetic phenotype of T2,T3 and T4 transgenic cotton lines.First of all,through the positive identification and expression analysis,we screened out the transgenic cotton lines which have a significant difference in expression lever compared with the wild type.The phenotype of T2 transgenic cotton was observed,we found that GhFSN1 RNAi plants had no significant difference with the wild type,regardless of plants height,leaf morphology,pod size,cotton fiber length,seed size,or cotton fiber thickness,suggesting that there is a function redundant gene with GhFSN1 in cotton.However the GhFSN1 overexpression of plants height became significantly shorter,the leaves appeared upturned,seeds smaller,cotton fiber length was shorter,and the fiber thickness increased,suggesting that GhFSN1 plays an important role in the development of cotton fiber.2?GhFSN1 transcriptome analysisTo uncover the differentially expressed genes(DEGs)between GhFSN1 overexpression transgenic lines and wild type,we performed transcriptomic analysis to find genes regulated by GhFSN1 in 18 DPA fibers.Of the 2857 differentially expressed genes,1486 genes were upregulated and 1371 were downregulated in the GhFSN1 overexpression fibers The genes upregulated in GhFSN1 overexpression fibers were mainly enriched in biosynthysis of secondary metabolites,phenylalanine metabolism,flavonoid biosynthysis,amino sugar and nucleotide sugar metabolism,and phenypropanoid biosynthesis processes.By contrast,downregulated genes were involved in the biosynthysis of secondary metabolites,cutin and suberine and wax biosynthesis,biosynthysis of unsaturated fatty acid and pectin,fatty acid metabolism,and fatty acid elongation process.Further examination of the identities of these DEGs revealed that they include not only transcription factors(TFs),but also a number of genes related to cellulose biosynthesis,xylan biosynthesis,lignin biosynthesis,pectin catabolism,fatty acid biosynthesis,and cytoskeletal organization.These gene expression levels were verified using real-time PCR,and the results were consistent with the RNA-Seq analysis.We hypothesized that GhFSN1 gene could promote the synthesis of secondary wall of cotton fiber through different regulatory pathways.3.The interaction between GhFSNl protein and GhE2 protein was verified by pull-downPreviously,the interaction between GhFSNl and GhE2 protein was confirmed by yeast two-hybrid technique,suggesting that GhFSNl protein degrades through ubiquitination pathway,This experiment would like to further verify this result through the pull down technology,Thus,the pMAL-GhE2 vector and the pGEX-4T-1-GhFSNl vector were constructed.Then,the MBP-GhE2 protein and the GST-GhFSNl protein were purified respectively.The result of pull down test showed that GhFSNl protein can interact with GhE2 protein.