The Study On The Transcriptome In Early Development Of Cotton Fibers And Expansin Gene Family

Cotton is a natural fiber crop widely planted in the world,which is a natural resource with the highest cellulose purity.The mechanism of fiber development has been a focus field for scientists.Cotton fiber is derived from ovule epidermal cells,which can develop into lint(long fibers)and fuzz(short fibers).Lints are the material source of textile industry,so it is of great significance to reveal the mechanism of lint development for creating new germplasm with excellent fiber quality.Transcriptome sequencing technology has been widely used in the mining of genes related to cotton fiber development,however the current materials used for fiber transcriptome sequencing can not exclude the interference of fuzz development.Therefore,the transcriptome of the early fiber development were studied between n2 with fuzzless-linted and Xuzhou142 with fuzzless-lintless(XZ142FLM,Control)in this study.The fuzz fibers were neither developed on n2 nor XZ142FLM ovules,thus the interference of fuzz fiber development could be effectively eliminated.Transcriptome analysis showed that the expansin genes were up-regulated on the 0 DPA(Days Post Anthesis)and then their gene family was further studied.The main results were summarized as follows:1.The RNA samples of ovules of n2 and XZ142FLM were sequenced at 0,1,2,3,4 and 5 Days Post Anthesis(DPA)by transcriptome.At first,the differential expression genes of n2 at other time points were compared with the n2₀ DPA,then the genes of n2 and XZ142FLM were compared at each same time point.Take the intersection of the corresponding time after the two comparisons,the differential gene set was obtained for specifically up-regulated genes of n2 material.The results of the differential gene set at each time point are as follows:245,337,1123,1583,1411 and 2181 genes were up-regulated on day 0,1,2,3,4 and 5 after flowering,respectively.2.GO functional enrichment and metabolic pathway analyses were carried out for up-regulated differential genes at 0,1,3 and 5 DPA.The main results of enrichment pathway and fiber specific gene expression in n2 with fuzzless-linted are as follows:(1)the most abundant pathways were cell wall relaxation and metabolism at 0 DPA stage,including two important up-regulated expansin genes,Ghir D10g012330 and Ghir A10G015240;(2)The most abundant pathways were VLCFA synthesis and lipid metabolism at 1 DPA stage,including 4 VLCFA synthesis and 4 lipid transporter genes,which were up-regulated from 1DPA;(3)During the 3 DPA phase,cytoskeletal proteins,hormone metabolism and calcium signaling pathways were significantly enriched,in which 3 actin and three tubulin related genes were up-regulated from 3 DPA;(4)During 5 DPA,carboxylic acid,amino acid and organic nitrogen metabolism pathways were significantly enriched,which included Ghir_D04g003610 and Ghir_D01g021670 encoding fatty acid amide hydrolase and aldolase.The dynamic process model of cotton fibers early development starting from cell wall relaxation was proposed,and the timing characteristics of certain key genes of fiber elongation was verified by qPCR.3.The expansin gene families of Gossypium hirsutum,G.arboreum and G.raimondii were identified at the genome-wide scale,including 93,49 and 45 members respectively.Cotton expansins contain two typical conserved domains,DPBB-1 and Pollen_allerg₁(CBM63),while the majority of them contain a signal peptide.Upland cotton is the main source of cotton fiber,so the expansin family of upland cotton was analyzed in detail in this study.The chromosomal location of GhEXP genes was identified in G.hirsutum.The results showed a total of 93 expansin genes distributed on 24 chromosomes,excluding Ghir_A02 and Ghir_D06.A phylogenetic tree analysis showed that the expansins were divided into four major subfamilies,named EXPA,EXPB,EXLA and EXLB,and 15 subgroups.The analysis of cis-acting elements in the promoter region showed that there were many cis-elements in the promoter regions of the 93 GhEXP genes,including hormone regulatory elements development related elements,environmental stress elements and light responsive elements,and so on.The analysis of the characteristics of gene family members laid a foundation for explaining the function of fiber-specific expansin.4.The results of RT-qPCR analysis and gene expression profile analysis showed that there were different expression patterns of expansin genes in different stages of fiber development.Fourteen key genes of fiber development in upland cotton were identified,including 3 genes of initiation stage,9 genes of elongation stage,and 2 genes of transition stage from elongation stage to secondary wall thickening stage.5.The heterologous expression of GhEXPA8g in Arabidopsis thaliana showed that GhEXPA8g could significantly promote the trichome elongation of Arabidopsis thaliana.The dual luciferase assay showed that the transcription factor GhACE3 was the upstream activator of GhEXPA8g.These results provided candidate genes for studying the mechanism of expansin in fiber development.In conclusion,a number of up-regulated genes were obtained by transcriptome analysis in the early development of long fiber,and an orderly dynamic process of fiber development in ovule epidermal cells from cell wall relaxation,fatty acid synthesis,to a variety of hormone signals transduction and cytoskeleton reconstruction was proved.14 dominant expansin genes were identified by study of expansin gene family in fiber development.Among them,GhEXPA8g,regulated by GhACE3,could significantly promote the trichome elongation in Arabidopsis thaliana.This research analyzed the key gene expression and regulation pattern of cotton fiber development,and provided a theoretical basis and candidate genes for genetic improvement of cotton fiber quality traits.