Screening And Function Analysis Of Pheromone Biosynthesis-related Genes In Aelphocoris Suturalis

Pheromones are important mediators of communication for insects in the environments,and play an essential role in survival and reproduction of organisms.Understanding the roles,biosynthesis pathways and evolution of insect chemical communication systems have been an exciting challenge to biologists in recent decades.Currently,the profile of molecular mechanisms of pheromone biosynthesis is becoming more and more clearly in Lepidoptera.And a moderate number of studies have been conducted with the goal of understanding the pheromone biosynthetic pathways in Diptera and Coleoptera.By contrast,little is known about the mechanisms underlying pheromone biosynthesis in Hemiptera.The Adelphocoris suturalis,an important agricultural pest in China,was used in this study.We firstly established an A.suturalis metathoracic scent gland(MTG)transcriptome and screened valid reference genes.Based on this database platform,RNA-Seq technologies were used to identify differential expression genes during MTG development.RT-q PCR and RNA interference(RNAi)methods were used to screen and analyze the pheromone biosynthesis-related genes in A.suturalis.The main conclusions are as follows:1 De novo analysis of the A.suturalis metathoracic scent glands transcriptomeA.suturalis as a non-pattern insect,the lack of genomic information limited the understanding of the molecular bases of the physiological processes.In this study,we sequenced the transcriptome of metathoracic scent glands(MTGs)of A.suturalis via Illumina sequencing platform.A total of 52 million 91-bp-long reads were obtained and assembled into 70,296 unigenes with a mean length of 691 bp.Of these unigenes,a total of 26,744(38%)unigenes showed significant similarity to known proteins in the NCBI database(E-value < 10-5).Out of 26,744 hits,9258 sequences were classified functionally into 25 COG categories,16,473 unigenes were assigned to 242 KEGG pathways.Through bioinformatics analysis,we screened 818 potential pheromone biosynthetic pathway genes.This data provide an invaluable resource for future exploration of molecular mechanisms underlying pheromone biosynthesis in A.suturalis.2 Assessment of suitable reference genes for RT-qPCR analysis in A suturalisIn RT-qPCR analysis of gene-expression,normalization of data against RNA variation by using appropriate reference gene is fundamental.However,appropriate reference genes are not known in case of A.suturalis.We evaluated 12 candidate reference genes(GAPDH,ACT,?ACT,TBP,SDH,?TUB,EF1?,EF1?,EF1 d,RPL32,RPS15 and RPL27)for their expression stability of A.suturalis subjected to various regimes of experimental treatments.Expression stability of the reference genes was analysed by employing five different statistical softwares including ge Norm,Norm Finder,Best Keeper,?Ct and Ref Finder.We recommend three best reference genes EF1?,EF1 d and RPS15 for normalization of RT-q PCR data in A.suturalis.3 Screening of genes involved in pheromone biosynthesis of A.suturalis using the RNA-Seq and RNAi approachesBased on previous A.suturalis metathoracic scent glands(MTG)transcriptome data,four RNA-Seq libraries were constructed from A.suturalis MTG at different developmental stages and sexes,namely,1 day old female MTG(1d FMTG),1 day old male MTG(1d MMTG),8-11 day old female MTG(8-11 d FMTG)and 8-11 day old male(8-11 d MMTG)to investigate the gene expression profiles during MTG development.The RNA-Seq evaluated over 6.9 million clean reads in each MTG sample and revealed3,350 to 12,834 genes that were differentially expressed in these different developmental stages and sexes.Des B,AOX,FAR and some ATF were found to be richly expressed in female MTG during the calling period via RT-q PCR.Then four candidate genes Des B,AOX,FAR and N-ATF10 were selected for pre-experiment to assess whether those genes are involved in A.suturalis pheromone biosynthesis using RNA interference(RNAi)technique.Result showed that only silencing Des B expression potently suppressed the sexual attraction of females to males in the field.We posed this concept as the hypothesis that Des B is related to A.suturalis pheromone biosynthesis.4 Functional characterization of desaturaseB using RNA interferenceDesaturase have been demonstrated to be crucially involved in the pheromone biosynthesis in many insects,which introduce carbon–carbon double bonds at specific positions in fatty acyl substrates.Previous studies have shown that silencing Des B expression potently suppressed the sexual attraction of females to males.We speculated that Des B is related to A.suturalis pheromone biosynthesis.To tested our hypothesis,we isolated and characterized two Desaturases B genes from A.suturalis,and named it asDesaturase B1(Des B1)and Desaturase B2(Des B2).NCBI blast results showed that the only difference between the two Des B sequences is in the 5' untranslated region,namely,they share the same open reading frame.Southern blot analysis revealed that Des B1 and Des B2 are two independent genes.The transcript of Des B2 is more abundant than Des B1 in all of the five tested tissues,especially in metathoracic scent gland(MTG).The Y-Tube olfactometer bioassays and field trapping experiments further demonstrated that down-regulation of Des B expression clearly suppresses the sexual attraction of females.Then we analyzed the MTGs secretions of ds Des B-treated and the ds GFP-treated females respectively via GC-MS.Results indicate that knockdown of Des B significantly increased the content of E4O2 H in MTG,and changed the blend ratios of A.suturalis sex pheromone compositions.5 FAR is required for ovarian development and female fertility in A.suturalisIn many insects(e.g.moths and Hymenoptera),fatty acyl-Co A reductases(FARs)act as the key enzymes required for the production of oxygenated functional groups in the biosynthesis of pheromones.In this study,we isolated a full-length c DNA corresponding to the gene from A.suturalis.The spatial and temporal transcript profiles of FAR revealed that FAR was highly transcribed in the A.suturalis ovary and showed a rising trend of expression with the development of ovary.RNAi results showed that down-regulation of FAR expression by injection of ds RNA led to a significant decrease in the numbers of oocytes,dry weight of ovaries,lifetime fecundity and egg hatchability,ultimately resulting in females producing few viable offspring.Therefore,we demonstrate that FAR plays an essential role in the reproduction of A.suturalis.