Study On Gene Silencing And RNAi Efficiency By Dsrna Feeding In Amphitetranychus Viennensis

Hawthorn spider mite(Amphitetranychus viennensis)is the most devastating pest in apple industry in China.Amphitetranychus viennensis is widely distributed in the main apple producing areas and has caused significant economic losses.The management of A.viennensis has primarily relied on the synthetic chemicals.The repetitive and extensive use of acaricides beyond rules and regulations has led to the development of resistance,which,in return,compromises the efficacy of acaricides and subsequently negatively affects the apple production in China.As an emerging biotechnology,RNA interference(RNAi)technology has been gradually applied in the field of pest control.However,in order to facilitate the adoption of RNAi into the existing integrated pest management strategies of A.viennensis,the first step is to obtain the target genes of and detect the efficiency of RNAi.In this study,the transcriptome sequencing of A.viennensis was carried out,and a total of 10 GB of Clean Data and 34,631 Unigenes were obtained,of which 20,398 were functional annotations.The data quality was evaluated by four indexes: single Base mass,Base content distribution,GC content distribution and Sequence Base quality,indicating that the sequencing results were good.Through in-depth analysis of transcriptome database,the high-throughput potential target genes were obtained.After the gene sequence of hawthorn mite was obtained,the fragment V-ATPase A gene of A.viennensis was amplified by PCR.This gene was used as a target to verify the feasibility of RNAi method for A.viennensis.In this study,we tried three RNAi methods: petiole absorption method,leaf absorption method,and drying leaf absorption method.The results show that the drying leaf absorption method is the optimal method.We analyzed the stability of nine HKGs identified from A.viennensis transcriptome under RNAi condition.These candidates,including: 18 S ribosomal RNA(18S),28 S ribosomal RNA(28S),elongation factor 1-α(EF1A),actin 3 gene(Actin3),ATPase B subunit(V-ATPase B),α-tubulin gene(α-Tubulin),ribosomal protein gene(RPL13),40 S ribosomal protein S9(RPS9),and glyceraldehyde 3-phosphate dehydrogenase Gene(GAPDH).Based on Ref Finder,a comprehensive analytical software,the top three most stable reference(GAPDH,V-ATPase B and RPS9)genes under RNAi condition were recommended.In order to study the RNAi efficiency of other genes in A.viennensis,three important genes for the growth and development of A.viennensis were selected: CREB binding protein(Av CBP),Farnesoic acid O-methyl transferase(Av Fa Met)and an ATP-dependent RNA helicase(Av Belle).The expression levels of these three genes under different developmental stages of A.viennensis were studied by RT-q PCR.Av CBP and Av Belle had the highest expression levels during the egg stage.The expression of Av Fa Met increased from egg to protonymph stage,and the highest levels were found under deutonymph and adult stages.Starting from deutonymph,RNAi was continuously performed on these three genes,and the RNAi efficiency was studied.The results show that Av Fa Met and Av Belle can be inhibited well,but there was no significant difference on Av CBP.This indicates that the RNAi efficiency of different target genes varies greatly.In summary,this study conducted transcriptome sequencing on A.viennensis,explored the RNAi methods,screened out the most suitable reference genes under RNAi conditions,and studied the RNAi efficiency of different genes in A.viennensis,which lays foundation for the RNAi based gene function research and the development of pest control technology.