The Function Of GhFDRT2 In The Development Of Cotton Ovule And Fiber

Cotton is an important economic crop in China,and cotton fiber is an important raw material for textile industry,which plays an important role in Chinese national economy.Cotton fiber is a single cell fiber formed by polar elongation and secondary wall thickening of the epidermal cells of the ovule.Cotton fiber cell is one of the longest single cells in plants,and is a good material for studying the growth and development of plant cells.Trihelix transcription factors play important regulatory roles in plant growth and development,but their function on cotton fiber elongation and secondary cell wall(SCW)synthesis have been rarely reported.Previously,we have demonstrated that exogenous applyment of fumonisin 1(FB1),an inhibitor for sphingolipid synthesis could strongly inhibit the growth of fiber cells by chemical biology methods.To screen the sphingolipid related genes in regulating cotton fiber development,we analyzed the transcriptome data of FB1 treated ovules(with fibers),and the results showed that there were 615 significant changed genes in the inhibited samples.These genes may plays important roles in the fiber growth.In this study,we cloned a trihelix transcription factor gene Gh FDRT2(FB1 down-regulated Trihelix transcription factor 2)that significantly inhibited by FB1,by means of molecular biology and genetics,we characterizied the gene function and explored its action mechanism in the growth and development of fiber and ovule.Our results provide some new data for further analysis of the regulatory mechanism of the fiber cell growth and development.1.Cloning and sequence characterization of Gh FDRT2The cloned c DNA sequence of Gh FDRT2 gene was 1162 bp and contained 19 bp 5 ’-UTR,1050 bp ORF and 81 bp 3’ –UTR,which encodes a trihelix transcription factor with 349 amino acid,an estimated molecular weight of 39.2 k D and an isoelectric point of 9.601.This gene is located on chromosome 4 of the D subgenome of Upland cotton sequence characterization showed that the N-terminal of Gh FDRT2 contained a highly conserved trihelix binding domain.The trihelix transcription factor members were divided into SIP1,GT-2,GT-γ,SH4 and GT-1sub-family.Phylogenetic analysis showed that Gh FDRT2 belongs to SIP1 sub-family and was closely related to two Arabidopsis genes,At3G54390-1 and At3G54390-2,These results indicate that Gh FDRT2 gene is a SIP1 sub-family trihelix transcription factor.2.Spatiotemporal expression of Gh FDRT2RT-q PCR results showed that the expression levels of Gh FDRT2 were significantly different in different tissues and organs.The expression of Gh FDRT2 was low in petals but high in hypocotyls,stems and fibers.At various developmental stages,Gh FDRT2 expressed at lower level at the early fiber elongation stage(5 DPA),and then was gradually increased with the development of cotton fiber and reached a peak at 15 DPA.These results indicate that Gh FDRT2 preferentially expressed in cotton fiber elongation and transition stage.Therefore,Gh FDRT2 may play roles in the growth and development of cotton fiber cell.3.Subcellular localization and transcriptional activation of Gh FDRT2We constructed plant expression vector in which the Gh FDRT2:: e GFP fusion gene was controlled under Ca MV 35 S promoter to detect the subcellular localization of Gh FDRT2.Using the tobacco transient expression system.We verified that Gh FDRT2 was mainly localized in the nucleus,the results showed that the full length of Gh FDRT2 did not have self-activating activity.4.Effects of ectopic expression of Gh FDRT2 in ArabidopsisThe ectopic expression of Gh FDRT2 gene led to the enlargement of the rosette leaf and earlier flowering in Arabidopsis.Staining the stem with phloroglucinol showed that the heterologous overexpression of Gh FDRT2 gene led to the decrease of lignification of the interfascicular cell wall,indicating that the heterologous overexpression of Gh FDRT2 gene promote plant growth and inhibited the deposition of secondary cell wall(SCW).5.Effects of suppressing Gh FDRT2 expression on growth and development of cotton plant and fiberCompared with the wild type,the antisense Gh FDRT2 transgenic plants displayed higherplant,more compact in plant architecture,shorter internode of lateral branches,and smaller flowers,leaves and balls.These results indicated that down-regulation of Gh FDRT2 gene expression had a certain effect on the growth of cotton plants.The changes of transgenic fiber growth were observed using the in vitro cotton ovule culture system.The results showed that the elongation of transgenic fiber wassignificantly inhibited.The length of mature fiber of transgenic cotton was significantly also shorter than that of wild type.The SCW of transgenic mature fibers was significantly thicker than that of wild type.6、Screening and validation of target genes of Gh FDRT2The results of yeast one hybrid showed that Gh FDRT2 directly bind to the promoter of Gh TLC which of the sphingolipid-related gene.At the same time,RT-q PCR result show that the expression of Gh TLLC gene is significantly down-regulated in antisense inhibited Gh FDRT2 transgenic cotton fibers.The Gh TLC gene is predominance expressed in cotton fiber rapid elongation,and down-regulation of Gh FDRT2 gene expression inhibited fiber growth.That is consistent with our mature fiber phenotype.Overall,we confirmed that Gh TLC is a downstream target gene of Gh FDRT2.