Gene Function And Transcriptome Analysis Of Hyphantria Cunea Based On RNA Interference

Hyphantria Cunea is an important forestry quarantine pest in China.It frequently breakouts in Northeast,North and Central China,which poses a serious threat to forest health and ecological safety.RNA interference,an important molecular biology tool for studying functional genes,has been certified to silence important developmental genes which significantly affect the normal growth and development in many insects.Moreover,it's a potential tool to control pests through molecular biology.RNA interference is more difficult to achieved in Lepidoptera and rarely used in H.cunea.In this study we explore the application of RNAi in H.cunea.Two developmental key genes were selected for testing,and SID-1 gene which is a key gene of systemic RNAi was cloned and analyzed.Finally,transcriptome sequencing technology was used to analyze the effects of different RNAi treatments on gene expression of H.cunea larvae,and found some genes related to RNAi process.The main results are as follows:?1?RNAi interference on the chitinase gene HcChi of H.cuneaThe expression levels of HcChi in fourth and fifth instar larvae of H.cunea were detected by qRT-PCR to exploring the role of chitinase in the molting process.It was showed that the expression levels of HcChi were maintained at a lower level during the instars while it was increased 13 to 17 times during molting process.Injection of 5th instar larvae with different dsChi doses,2.5?g could reduce the gene expression up to 66.8%,while 0.5?g lead to the pupation and eclosion stage deformity with a pupation rate of 31.03%.The expression of HcChi in 4th instar larvae could be reduced by different injection time.It also caused malformation during molting process and lead to a pupation rate of 53.8%⁷7.8%.The chitinase dsRNA expression system was constructed using E.coli HT115 strain.2^nd and 3^rd instar larvae were started to feed on dsRNA expressed by HT115,and caused the weight decrease of 40.7%and10.7%,respectively.But the pupation and mortality rates were not affected and no abnormal phenotype emerged.Compared with injection,fed on dsRNA expressed by bacteria had little effect on larval growth,but had a certain cumulative effect.?2?Clone and RNAi analysis of two acetylcholinesterase gene HcAceAcetylcholinesterase is a key enzyme in nervous system,and is a target of organophosphorus pesticides.In this study,the full-length cDNA of two acetylcholinesterase genes was cloned from the larvae by RT-PCR.The length of coding region of HcAce1 was2085bp,which predicted coding 694 amino acids.The coding length of HcAce2 is 1917bp,which predicted coding 638 amino acids.Both HcAce have typical characteristics of acetylcholinesterase.Phylogenetic analysis showed the conservation of acetylcholinesterase in insects.The relative expression of HcAce1 and HcAce2 decreased by 22.1%and 66.6%,and the activity of AChE enzyme decreased by 43.3%and 76.2%,while the weight of larvae decreased by 25.4%and 31.9%compared with the control due to dsRNA injection,respectively.Silencing HcAce1 also resulted in 89.7%mortality of larvae,indicated that AChE1 play a leading role of AChE in H.cunea.The HcAce1 and HcAce2 dsRNA expression system was constructed using E.coli HT115 strain,which provide the basis for following test.?3?Clone and functional verification of HcSID-1 geneFull-length cDNA of SID-1 was cloned from H.cunea larvae by RT-PCR and RACE.The ORF of HcSID-1 is 2613bp and encodes 870 amino acids.It is predicted that the gene has typical SID protein domains such as a long extracellular N terminal and 11 transmembrane domains.Phylogenetic analysis revealed the homology between HcSID-1 and other SID genes.Temporal and spatial expression patterns analysis showed that the gene was expressed in all tissues and all development stages of H.cunea,with the highest expression level was found in midgut.The expression of HcSID-1 could be reduced to 53.5%by RNAi.The downregulation of HcSID-1could reduce the interference efficiency of dsChi up to 7.32 times,suggesting that HcSID-1 may be involved in the systematic RNAi process in H.cunea.?4?Transcriptome sequencing analysis of H.cunea after different RNAi treatmentIn this study,five injection RNAi treatments were set as ddH₂O,dsGFP,dsSID-1,dsChi,and dsSID-1+dsChi,and the control group was naturally grown larvae.Transcriptome sequencing analysis was performed after 12 hours treatment.A total of 132.55 Gb Clean Data was obtained,with an average of 6.69 Gb per sample.After assembling,103647 Unigene was obtained,with an average length of 699.99bp.18051 annotated Unigene were obtained which accounting for 17.4%of the total after database comparison.A large number of differentially expressed genes were obtained after analysis of differentially expressed genes among different RNAi treatments.It was found that chitinase is involved in the pathway of amino sugar and nucleotide sugar metabolism by KEGG analysis.In this pathway,several downstream genes affected by chitinase RNAi.29 genes related to RNAi mechanism were obtained by UniGene search.It included core components such as Dicer,Argonaute,RISC complex components and dsRNA transport related genes.These genes were involved in multiple physiological pathways,and the expression of multiple genes in these pathways could be regulated by RNAi.RNA interference and other molecular biological methods are new strategies for ecological prevention and control pests.In this study,we explored theRNAi effects on H.cunea targeting to two important growth genes.We also cloned a key gene involved in dsRNA transport,which provide the basis for the application of RNAi in H.cunea.We explored the regulation of related genes after RNAi using transcriptional sequencing technology.We also obtained some pathway genes related to target genes and some important genes involved in the RNAi mechanism.The above results will provide theoretical and data support to intensive study RNAi in H.cunea and control of H.cunea using RNAi.