Proteomics And Transcriptomics Analysis Of Fiber Differentiation And Initiation

Cotton is an important global economic crop that is widely grown for production of textile fiber materials,and more than 90% of cotton's value originates from the seed fibers.The cotton fiber qulity and quantity is largely dependent on the time of fiber differentiation and initiation.The epidermal cells differentiate before or on the day of anthesis,and then develop into lint fiber.The number and time of prefiber cells differentiation lead to the cotton fiber yielding.Great progress has been made in illuminating cotton fiber initiation mechanisms,most studies have been focused on cytological,morphological,physiological and biochemical,genetic aspects.Cotton fiber molecular mechanisms largely focus on gene cloning,exprssion and functional analysis.The preliminary research reflects the molecular mechanism of fiber initiation by multiple gene regulation network system,and the molecular mechanism research remains to be further studied.Using of high-throughput technology,from the transcriptomics and proteomics level,helps to reveal the key metabolic pathways and the key genes on cotton fiber initiation.In this study,the isotope labeling relative and absolute quantitative technology(iTRAQ)combined with liquid chromatography tandem mass spectrometry(MS)and RNA-seq technology were used to carry out the cotton ovule proteome,phosphorylation proteome and transcriptome analysis between the Xuzhou 142 fl mutant and its parental WT.The specific results were as follows:1.Our approach was sensitive enough to identified 5 045 proteins with a false discovery rate of less than 1%.A set of 202 proteins was differentially expressed between WT and fl.121 of these proteins were up-regulated,81 were down-regulated in WT.The abundance of proteins mainly involved in fatty acid metabolism,flavonoid biosynthesis,amino sugar and nucleotide sugar metabolism,hormone signal transduction.In these fiber-initiation related proteins,most of Arabidopsis orthologs proteins were related to the Arabidopsis epidermal hair and root hairs development,suggesting the both have a similar regulation mechanism.Together,out data provide insight into regulatory processes underlying fiber initiation and provide a community resource for using biological technology tools to enhance fiber initiation and improve fiber properties.2.To better understand the molecular mechanisms involving phosphorylated proteins(phosphoproteins)and signaling networks during cotton fiber differentiation and initiation,we investigated the cotton ovule phosphoproteome using a liquid chromatography-tandem mass spectrometry(LC-MS/MS)enriched by titanium dioxide(TiO2)affinity chromatography.A total of 1592 phosphorylation sites(1423 serine,151 threonine,18 tyrosine)from 619 phosphoproteins were identified.Among the identified phosphosites,492 were conserved across the P3 DB public database.we found 8 motifs unique to cotton,belonged to casein kinase 2 motif,transforming growth factor-?(TGFB)receptor kinase motif,protein kinase B motif,extracellular-signal-regulated kinases motif.These results suggest that these kinases motif may be the major kinase group involved in phosphorylation of the identified phosphoproteins during cotton fiber initiation.Among the detected phosphopeptides,69 were differentially expressed between the fl mutant and its WT in ovules at-3 and 0 DPA.A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction,protein modification,carbohydrate metabolic processes,and cell cycle and cell proliferation,suggesting that phosphorylation is a critical post-translational modification involved in regulation of cotton initiation.We also find some important fiber-initiation-related phosphorylated proteins,such as,GTPase and UDP-glucose 6-dehydrogenase.Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation,and also offers a helpful dataset for elucidation of signaling networks in fiber development of G.hirsutum.3.Paired-end cDNA libraries were constructed and then sequenced using the Illumina Genome Analyzer(HiSeqTM2000 Sequencing System).About 578 million high-quality reads representing over 57 Gb of sequences were obtained by deep sequencing.A novel hybrid strategy combining reference-based and de novo analysis was then applied.The reads were assembled into 166 963 unique transcripts(average length 1 051 bp).Among the 166 963 assembled sequences,109 518 had a significant match to at least one entry in these databases.In the assembled transcriptome,15 196 simple sequence repeats(SSRs)were detected in 14 026 unigenes;primers were designed from 11 179 of them.193 755 single nucleotide polymorphisms(SNPs)were identified(142 800 from the cotton AADD genome and 50 955 from the de novo strategy).Of these,11 488 SSRs and 181 981 SNPs were identified for the first time.Twenty-five SNPs and 75 SSRs were selected for validation by high-resolution melting and PCR,respectively.8 503 DEGs were identified between the fl mutant and its WT in ovules at-3 and 0 days post-anthesis.2 470 DEGs with sequence varation including SNPs or SSRs were mapped on chromosomes.This study is the first comprehensive transcriptome analysis of cotton fiber-initiation-related molecular markers using reference-based alignment and de novo assembly.The SNPs,SSRs,and DEGs represent good resources for genetic,breeding and whole genome association studies in cotton.