RNAi Knockdown Of Salivary Transcript In Pea Aphid,Acyrthosiphon Pisum

The pea aphid, Acyrthosiphon pisum, is one of important piercing-sucking pests. The saliva plays important roles in aphid’s feeding on host plants and virus transmission. RNA interference (RNAi) has been proven quite effective and used widely in suppression of target gene expression in insects. The published genome of pea aphid provides excellent convenience for the researches of multifacet in aphids.The present study aims to:(Ⅰ) establish a RNAi technique using microinjection system in pea aphid to investigate the function of genes expressed in salivary glands;(Ⅱ) study deeply an unknown protein family that is highly expressed in salivary glands. The main research contents and results are summarized as follows:1) Analysing A.pisum salivary-gland expressed genes43proteines expressed in salivary glands of pea aphid were analysed with bioinformatic tools in NCBI, BCM, ExPASy Proteomics Server, Splign and so on. Eight of the43proteins are function-known proteins and5are being studied by other labs. Genes of twelve proteines are multiple copies, and the homology among copies is between21%to99%; the remaining23proteins are single copy genes.2) RNAi of pea aphidAfter injecting different concentrations (3,4.5,6μg/μL) of dsGFP-RNA to the third instar larvae, no difference was observed in the surviving curve of pea aphid. To ensure the effect of interference, high concentration of dsRNA was used, i.e.6μg/μL, and the injection volume was23nL. After injection of dsRNA, the survival rates of ds-Contig3291, ds-Contig3357, ds-Contig3169and ds-Contig3302aphids were lowered down on the first day after injection compared to ds-GFP aphids while the differential survival rate was observed on the third day after injection for ds-Contig10169aphids.Ct comparison method (2-△△Ct) was used to calculate the relative level of gene expression after dsRNA injection. The interference efficiencies of the five genes expression were①38.69%±10.33and34.44%±6.157for Contig3357on the first day and the third day after injection, respectively;②49.8%±6.14,28.1%±4.93and45.1%±8.44for Contig3291on the first, second and third day, respectively;③67.1%±7.13after two days for Contig3169; ④34.9%±11.8after three days for Contig3302;⑤55.28%±5.644and54.66%±9.806for Contig10169after one day and three days, respectively. These data show that injection of dsRNA into the third instar stage can actually reduce the transcriptional level of target genes.3) Time and spatial expression of a functional unknown protein family of salivay glandsA functional unknown protein family was cloned from salivary-gland EST library of pea aphid. The family consists of13genes that encode14proteins, and four of them are highly expressed in salivary glands. This family is specially espressed in aphids and enriched in cysteines. Fourteen cysteines are conserved,6of which form three CXXC domains. Semi-quantitative reverse transcription PCR showed that there was no developmental stage-specific expression, but tissue-specific. We speculate this protein family probably has the functions similar to oxidoreductases or DNA methyltransferases.The RNAi system for pea aphid set up in this study not only can be used in the functional research of target genes in pea aphid but also provides a new way and a new method to control this pest in the future. The unknown protein family includes several genes highly expressed in salivary glands and these genes have secretory signal peptides, indicating that they are probably important components of the aphid saliva. The control of percing-sucking pests has been challenged in agricultural production. The identification of salivary components can provide new ideas and methods to control piercing-sucking insects. For example, we can interfere their expression and therefore prevent the aphids from feeding on plants or virus tansmission.