Genome-wide Identification Of NsLTP In Gossypium And Their Expression Pattern Analysis

Non-specific Lipid Transfer Protein(nsLTP)is a plant specific protein,playing various functions in plant development.nsLTP could participate in biotic and abiotic stress resistance,stimulate nodule formation in legume roots and promote pollen tube growth via mediating lipid transfer in plant.So far,nsLTP gene family has been systematically investigated in several plants,and numbers of important nsLTP genes have been isolated and functionally verified.Cotton(Gossypium spp.)is an important economic crop;cotton fibre is the world’s most important natural renewable sources for textiles.Although studies have shown that nsLTP genes could participate fiber elongation,little is known about the functions of nsLTP genes in cotton development.Based on the recently published genomic sequence information of Upland cotton G hirsutum TM-1,G raimondii and G arboreum,we identified and analyzed the nsLTP gene family in these three cotton species.Furthermore,by integrating bioinformatics and transcriptional analysis,we isolated the candidate nsLTP genes which were involved in cotton abiotic stress resistance and fiber initiation.1.By bioinformational analysis,153,76 and 83 nsLTP genes were identified in Gossypium hirsutum,Gossypium arboreum and Gossypium ramondii,respectively.According to protein structure and amino acid sequence similarity,the nsLTP genes in Gossypium hirsutum were divided into 14 types,with each type contains the type-specific structure.Ka/Ks analysis revealed that nsLTP gene family is very conservative on evolution.Based on the phylogenetic tree,we can found that group IV and group G containing the GPI-modification site emerged earlier than other groups of nsLTP genes,while group Ⅰ and group Ⅱ emerged later.Gene Ontology(GO)enrichment analysis on G hirsutum nsLTP genes showed that nsLTP genes were mainly involved in lipid transport in plant cell.Promoter analysis showed that abiotic stress and phytohormone responding elements and MYB binding sites were present in nsLTP promoter region,and some nsLTP genes contain the tissue-specific cis-elements.2.The expression pattern of nsLTP genes in cotton development,abiotic stress and hormone treatment was investigated in the G hirsutum transcriptome data.RNA-seq analysis of various G.hirsutum tissues showed that most nsLTP genes are preferentially spatio-temporal expressed.Cluster analysis on nsLTP expression pattern demonstrated that classⅠ,Ⅴ and part of class Ⅱ nsLTP genes are preferentially expressed in root,stem and leaf,might be related with vegetative tissue development;class Ⅲ and class G are highly expressed in developmental cotton fiber;part of class Ⅱ genes are preferentially expressed in pollen;class Ⅳ and Ⅴ are highly expressed in early ovules.Furthermore,RNA-seq data of G.hirsutum leaves showed that some nsLTP genes could be induced by single or multiple abiotic stress conditions and some genes could respond to phytohormone.These results suggested that nsLTP genes could involved in the abiotic stress resistance as downstream signaling molecules or functional effectors.3.Based on the transcriptome data,we selected tempo-spatial expressed and abiotic stress responding nsLTP genes to conduct further studies using qRT-PCR technologies.The results showed that nine nsLTP genes are interesting,of which two genes are tempo-spatial expressed and seven genes are abiotic stress inducible.Additionally,we isolated the fiber initiation related nsLTP genes by employing the cotton mutants and verified their expression pattern by qRT-PCR.We found that GhLtpI.7At and GhLtpIV,2At are differentially expressed between wild type and mutants,might associated with fiber initiation.We also found that 4 nsLTP genes could be significantly induced by drought and salinity,respectively.Besides,three of these nsLTP genes,GhLtpGe.1Dt,GhLtpI.13At and GhLtpIVt2Dt could be induced by multiple hormone treatment.Transcriptome data and qRT-PCR results collectively demonstrated that nsLTP genes play important roles in cotton fiber development and abiotic stress resistance.On the basis of comprehensive identification and analysis on nsLTP genes in this study,we isolated the candidate genes involved in fiber initiation and abiotic stress resistance.This study will provide certain useful information for further application of nsLTP genes in improving cotton fiber qulity and abiotic stress resistance.