Expression Characteristics And Function Analysis Of Cotton Fiber Development Related MRNAs And LncRNAs Under Drought Condition

Cotton is the major source of natural fibers and the most important raw material for the textile industry.The fiber quality of cotton is greatly affected by drought.However,the molecular mechanism of the drought response in cotton fiber cells has not been fully elucidated.By high-throughput RNA-seq technology,researchers can gain a preliminary and comprehensive understanding of complex life activities and efficiently identify target functional genes.In this study,Gossypium hirsutum L.Nongdamian 13(ND13)with superior fiber quality,was used as the research material.Two water conditions were set,including normal irrigation(IR,performed a supplementary irrigation with a capacity of 675 m3/ha just before full bloom)and drought(DR,not carry out the supplementary irrigation).Flowers were tagged on the day of flowering as 0 DPA(day post anthesis).Ovules(0 and 5 DPA)and fibers(10,15,20,25,30 and 35 DPA)were collected to construct RNA library for RNA-seq.By bioinformatics analysis,mRNAs and long noncoding RNAs(lncRNAs)that were differentially expressed during fiber development under the two conditions were identified,respectively.The changes of metabolic pathways related to fiber development in irrigation and drought groups were explored.In addition,differentially and stably expressed mRNAs and lncRNAs under drought condition with irrigation group as control were screened respectively.And candidate genes for cotton drought tolerance trait were mined.This study will provide important theoretical basis and genetic resources for in-depth interpretation of fiber development mechanism,as well as the molecular mechanism of cotton fiber responding to drought stress.The main results were as follows:1.A total of 47 095 mRNAs and 13 051 lncRNAs were identified.Among the 13 051 lncRNAs,11 683 are long intergenic noncoding RNAs(lincRNAs)and 1 368 are long noncoding natural antisense transcripts(lncNATs).The transcript length of lncNATs(mean=1 883 nt)was significantly longer than that of lincRNAs(mean=1 327 nt)and mRNAs(mean=1 307 nt).The majority of lincRNAs and lncNATs contained fewer than 6 exons with an average of 2.34 and 3.06,respectively.While the numbers of exons of mRNAs varied widely and averaged 5.90,which was significantly larger than that of lncRNAs.The overall expression levels and GC contents of lincRNAs and lncNATs were lower than those of mRNAs.2.Through comparison between different time points in each treatment group(including comparisons to 0 DPA and to the previous time point),35 569 mRNAs and 6943 lncRNAs differentially expressed were screened out during fiber development.Among which,32 162 mRNAs and 5 460 lncRNAs,33 027 mRNAs and 6 208 lncRNAs were differentially expressed in irrigated and drought groups,respectively.Drought treatment resulted in a broader response of the transcriptomes of developing cotton fibers.Metabolic pathway enrichment analysis of differentially expressed transcripts showed that some pathways that very significant or significantly enriched in the irrigated group were less or even no longer enrich in the drought group,especially "Pentose phosphate pathway" and"Glycine,serine and threonine metabolism" in "25 vs 20" comparison,"Nitrogen metabolism" in "30 vs 25" comparison,and "Phagosome" and "Ascorbate and aldarate metabolism" in "35 vs 30";Some pathways that were not enriched in irrigation group were significantly or even very significant enriched in drought group,including "Phagosome" in"30 vs 25" and "Zeatin biosynthesis" in "35 vs 30".3.Through comparison between drought group and irrigation group at the same time point,3 427 and 1 021 differentially expressed mRNAs and lncRNAs were screened out,respectively.By analysis of coexpression and genomic colocation,a total of 540(15.76%)differentially expressed mRNAs were predicted to be potentially regulated by differentially expressed lncRNAs and the fiber initiation stage(0 DPA)had a larger proportion of differentially expressed mRNAs(25.44%)that may be regulated by differentially expressed lncRNAs.Pathway enrichment analysis of differentially expressed mRNAs showed that at the fiber initiation stage,five metabolic pathways were significantly enriched,especially "Plant hormone signal transduction" and "Photosynthesis-antenna protein pathway".At the fiber elongation stage(10 and 15 DPA),11 metabolic pathways were enriched significantly,mainly "Plant hormone signal transduction" and"Phenylpropanoid biosynthesis".At the secondary cell wall biosynthesis stage(25,30 and 35 DPA),16 metabolism pathways including "Lipid metabolism","Energy metabolism","Amino acid metabolism" and "Phenylpropanoid biosynthesis" involved in the cell wall development and 1 pathway related to ATP-binding cassette(ABC)transporters were significantly enriched.Only three pathways related to genetic information processing were enriched at fiber development transition 1 stage(5 DPA).GO enrichment analysis of target differentially expressed mRNAs of differentially expressed lncRNAs showed significant enrichment in“Cell wall organization or biogenesis" and "Macromolecule metabolic process",indicating that these differentially expressed lncRNAs mainly regulate cell wall development.4 pairs of regulatory relationships between differentially expressed mRNAs and lncRNAs mediated by microRNAs(miRNAs)responding to drought were predicted by analysis of putative common miRNA binding sites.4.By comparison between drought and irrigation groups at the same time point,4 247 mRNAs and 27 lncRNAs stably expressed among the two groups were screened out.Based on this,a weight gene co-expression network was constructed,which could be divided into four modules and named ME1,ME2,ME3 and ME4.Genes in each module were predominantly expressed at the early phase of fiber development(0 and 5 DPA),elongation phase(5-15 DPA),secondary wall biosynthesis phase(20-35 DPA)and both the elongation and secondary wall biosynthesis phases(10 DPA to 35 DPA),respectively.Functional enrichment analysis showed that the genes in module ME1 were mainly enriched in the biological processes related to fiber initiation or transition from initiation to elongation,including "Trichome morphogenesis","Flower development","Regulation of ethylene-activated signaling pathway",and "Jasmonic acid mediated signaling pathway".Genes in module ME2 were mainly enriched in biological processes related to protein ubiquitination and the regulation of reactive oxygen species metabolism necessary for fiber cell elongation.Genes in module ME3 continued to be enriched in biological processes related to protein ubiquitination,as well as in "Ethylene response" and "Sugar mediated signaling pathway".Some genes in module ME4 were significantly enriched in protein ubiquitination related biological processes similar to that in module ME2 and ME3,and the other genes were mainly enriched in "Multidimensional cell growth".The 30 core genes with the highest connectivity in each module were selected as candidate key genes to maintain normal cotton fiber development under drought condition.5.According to the transcriptome data,it was found that the important genes in the inositol oxygenase metabolism pathway,the inositol oxygenase gene(MIOX)and phosphoinositol synthase gene(MIPS),were differently expressed between the two treatment groups and during fiber development.Functional analysis of MIOX,MIPS and another two genes in the pathway,myo-inositol phosphate phosphatase(IMP)and glucuronic acid kinase/glucuronokinase(GlcAK)were taken in model plant Arabidopsis thaliana.The results showed that compared with the wild type,the leaf trichomes of MIOX and GlcAK overexpressing A.thaliama were extremely longer as well as the hypocotyls from MIOX,MIPS,IMP and GlcAK overexpressing Arabidops is,suggesting that these four genes play an important role in cell elongation and are potential genes related to fiber elongation development.Additionally,the wilting degree of Arabidopsis plants overexpressing MIOX was lighter than that of wild type after drought stress,implying that MIOX was indeed related to drought stress tolerance.In conclusion,differentially expressed protein coding genes and differentially expressed lncRNAs,stably expressed protein coding genes and stably expressed lncRNAs were identified in cotton fibers under irrigation and drought conditions.A fiber development expression profile was constructed and pathways related to fiber development and drought stress response were mined.The potential ways of some lncRNAs regulating mRNAs were preliminarily speculated,which provided valuable information for further studying their molecular functions.Overexpression of fiber development related gene MIOX,MIPS,IMP and GlcAK promoted cell elongation in model plant A.thaliana;overexpression of MIOX improved the drought tolerance of transgenic A.thaliana.