Study On Gene Expression In The Transition From Female Workers To Neotenic Reproductives In Reticulitermes Labralis Based On Transcriptome Sequencing Technique

The workers of the genus Reticulitermes can develop in one of three ways :(i)remain workers;(ii)become sterile soldiers;or(iii)become neotenic reproductives(NR).In R.labralis(Hisa),when colonies are isolated or the reproductives are absent,the female late instar workers became NR after two successive moults and undergo pre-neotenic reproductives(pre-NR).At present,there are few studies on the mechanism of caste differentiation and functional genes of the transition from female workers to NR.In the present study,female late instar workers from the primary colonies of R.labralis,isolated female late instar workers(IW)for three weeks and female neotenic reproductives were used to characterize the transcriptome of R.labralis and to reveal the transformation mechanism from female workers to NR.The main results were show as follows:1.A total of 66.85 Gb clean reads were obtained by transcriptome sequencing.The Q30 of each sample was higher than 90.96%,the GC content was between 43.33% and 44.91%.A total of 112,954 unigenes were assembled.The unigenes were annotated against Nr,Swiss-Prot,KOG,and KEGG databases,17,535 unigenes were successfully annotated in the four databases.In total,40,073 unigenes had significant matches in the Nr database,followed by 29,540 unigenes in the Swiss-Prot database;25,453 and 20,116 unigenes had specific matches in the KOG and KEGG databases,respectively.For the first time,the transcriptome sequencing completed the construction of the database of workers,IW and NR.The results of transprotome not only provided gene database resources for studing on gene function and caste differentiation in Reticulitermes but also provided a molecular basis for revealing the workers caste plasticity.2.Using edge R to screen out differentially expressed genes(DEGs)among workers,IWand NR,the results showed that there were 17,405 DEGs in workers vs IW,30,332 DEGs in IW vs NR and 7,016 DEGs in workers vs NR.The analyzing results not only provided sequence resources for gene function but also provided reference data for selecting of key genes in the transition from female workers to NR.Next,38,070 DEGs were clustered into 8 profiles by STEM,in which a significant profile5 that was consistent with the transition from female workers to NR,combined with GO and KEGG enrichment analysis,Ras and Ca M genes of Ras signaling pathway,Termicin and Transferrin genes specific to termites were selected as key candidate genes.The study on the expression of candidate genes revealed the gene regulation mechanism of the transition from female workers to NR.3.We performed q RT-PCR analysis of five Ras signaling pathway-related genes and seven immune-related genes to test the transcriptome sequencing results.The results of the q RT-PCR and transcriptome results were consistent,indicating that the RNA-Seq analysis were reliable.It provided a valuable gene sequence for biological analysis.4.Ras protein was the expression product of Ras gene,playing an important role in the signal transduction pathway that produced by extracellular signal stimulation.Ras gene was highly expressed in IW that stimulated by environment factors,The expression level of IW was approximately 130-fold(p<0.05)higher than that of workers and 20-fold higher than(P<0.05)that of NRs,respectively.The trend analysis results showed that the Ras gene was enriched in 36 metabolic pathways of profile 5,our finding suggested that the Ras gene played a important role in the transition from female workers to NR.Based on the functional studies of Ras in Drosophila and Bombyx mori,it was speculated that the high expression of Ras during the transition from female workers to NR may promote the secretion of ecdysterone and the growth of workers.Calmodulin(Ca M)initiated DNA synthesis and promoted cells into the mitosis anaphase.IW released the inhibition of sex pheromone,after one week(IW1)the expression level of genes was up-regulated,the expression level of IW was significantly up-regulated and reached the highest level at three weeks(IW).The expression level of IW was approximately 333-fold(p<0.05)higher than that of workers and 47-fold higher than(p<0.05)that of NR,indicating that Ca M promoted cell growth in the transition from female workers to NR.5.Termicin and Transferrin were termite-specific immune genes.The expression level in NR was significantly higher than that in workers and IW,indicating that the NR of R.labralis had strong resistance to pathogenic bacteria.The immune genes verified by q RT-PCR,the expression level of IW was significantly higher than that of workers,which showed that the resistance to pathogenic bacteria was strong during the transition from female workers to NR6.We first study of the differentially expression of gonadotropin-releasing hormone receptor(Gn RHR)gene in the transition from female workers to NR.Workers were used as a control group to analyze Gn RHR differentially expression.The results showed that the expression level in NR significantly higher than that of workers and Pre-NR,the expression level of NR was approximately 12-fold(p<0.05)higher than that of workers and 5-fold higher(P<0.05)than that of Pre-NR,indicating that Gn RHR played an important role in the regulation of gonadal development and oocyte maturation in the transition from female workers to NR.The transcriptome library of worker,IW and NR provided a valuable gene sequence resource for workers caste differwntion in the termite.The study of the genes not only clarified the transformation mechanism from female worker to NR,but also established foundation to construct target gene RNAi system and identify new genes.In addition,it provided molecular biology basis for the development of new drugs for termite control.