Effects Of Macrocentrus Cingulum Brischke Parasitism On The Expression Of Calcineurin A Gene In Ostrinia Furnacalis In Biological Control

Macrocentrus cingulum Brischke is an important endoparasitoids,and Asian corn borer Ostrinia furnacalis is akind of key corn pest.The transcriptomic sequencing and real-time PCR were applied to study the expression of immune-related genes after the parasitism of M.cingulum on O.furnacalis larvae for 4 h,24 h,48 h,and contrast to the unparasitized group.The calcineurin A(CaNA)gene,which is associated with the insect innate immune was cloned from the larvae of O.furnacalis,and bioinformaticly analyzed,and the CaNA protein was prokaryoticly expressed in Escherichia coli BL21(DE3).The RNA interference technique was also used to study the function of CaNA gene.The main results were as follows:(1)The NR database was analyzed,and the gene sequences of O.furnacalis transcriptome could map to Bombyxmori,accounting for 18%of the total proportion,and for 11.1%to Danaus plexippus Take GO annotation for the transcriptome gene,the total geneswere mainly divided into biological processes,cell composition and molecular function.After KO annotation,the gene sequences of O.furnacalis transcriptome were classfied according to their participation in KEGG metabolic pathways,and the most genes are involved in signal transduction pathways.The heat map analysis showed that all immune related genes of the transcriptome couldbe clustered in five clades,and the CaNA genes belong to the fourth clade.(2)The expression levels of Of-GRP3,Of-PAP?Of-Serpin1?Of-PPO2?Of-DDC?Of-JHDK.Of-CaNAof O.furnacalis larvae after parasition were analyzed by real-time PCR.The results showed that the up-regulated genes were:?GRP3?PAP,Serpinl,DDC and CaNA.The down-regulated gene were:PP02,JHDK.(3)In the present study,RT-PCR and RACE were applied to cloneCaNA gene from O.furnacalis.The full length cDNA of the CaNA is 1612 bp,including a 1446 bp open reading frame(ORF)?a 166 bp 5-untranslated region(5' UTR),and an unknown 3-untranslated region(3'UTR).ORF is beginning from the 167th nucleotide,the initiation codon is ATG.The ORF encodes a 481-amino acid polypeptide.No signal peptide was detected in N-terminal,but 3 potential S-coupled glycosylation sites and 45 phosphorylation sites were found.The Of-CaNA was predicted with a molecular weight of 54.57 kDa and a pI of 5.81.Bioinformatics analysis showed that the amino acid sequence of Of-CaNA has 99%consistency with Bombyx moriCaNA and Danaus plexippus CaNA.The prokaryotic expression condition of CaNA protein was optimized(28 ?,24 h,200 rpm,IPTG tendency for 1.1 mmol/L),and was confirmed by Western blotting.(4)RNAi was used to study the function of CaNA in O.furnacalis immune defense responses.After effective RNA interference,the expression of CaNA was inhibited in O.furnacalis larvae,and the expression of CaNA gene began to be inhibitedat 60 h postinjection(p<0.05),and continued until 120 h postinjection.