Silencing Of C.suppressalis GABAergic Neural Pathway Genes By RNA Interference

Rice stem borer(Chilo Suppressalis(Walker))is a very terrible pest of rice and other graminaceous crop,and is widely distributed in many countries in Asia,Europe,Oceania and Africa,including China.GABA is the most important inhibitory neurotransmitter in the central nervous system(CNS).The production,transportation,performing function and decomposition processes of GABA are all accomplished through GABAergic pathway,which is an important branch of the tricarboxylic acid cycle(TAC).Thereinto,GABA receptor,as a molecular target of various insecticides,has been widely studied in the development of agricultural insect control and pest resistance.Based on the genomic and transcriptome databases,the full-length cDNA sequences of GRD and RDL2 subunits of GABA receptor were obtained by molecular cloning,and the ORFs of other related genes in the GABAergic pathway were identified as well.RT-qPCR was used to detect the relative mRNA expression of GABA receptor subunits in different organs and developmental stages of C.suppressalis.At the same time,RNAi was used to investigate the effects of GABAergic pathway genes towards the growth and development of C.suppressalis and sensitivity to different insecticides.1.Identification and bio-informatic analysis of GABAergic neural pathway genes from C.suppressalisThe full-length cDNA sequences of GABA receptor GRD and RDL2 subunits,and the ORF sequences of GAD1,GAD2,VGAT,GAT,GAB AT genes in GABAergic pathway were obtained following the C.suppressalis genomic and transcriptional databases,and molecular cloning technique.Bioinformatics analysis revealed that the ORFs of GRD,RDL2,GAD1,GAD2,VGAT,GAT and GABAT contained 1722,1491,1623,1530,1617,1824 and 1482 bp(including stop codon),and encoded 573,496,540,509,538,607,and 493 amino acid residues,respectively.Among them,GRD was detailed and the result showed the putative GRD has a protein molecular mass of 65.12 kDa with an isoelectric point of 7.16,and is a neutral protein.Three strong hydrophobicity regions were formed at the N-terminus,C-terminus,and middle part around the 300th amino acid residue of the GRD.Five transmembrane domain were predicted.The C suppressalis GRD showed 76.20%、33.57%、38.00%、38.05%、35.17%and 36.51%homology with those of B.mori,D.melanogaster,A.mellifera,T.castaneum,L.striatellus and N.vitripennis,respectively.2.Spatial and temporal expression of C.suppressalis GABA receptor mRNAThe relative mRNA expression of C suppressalis GABA receptor(RDL1,RDL2,GRD and LCCH3)genes in different organs and developmental stages was detected and analyzed by RT-qPCR.The results showed that the relative expression of GABA receptor mRNA were high in ventral nerve cords and head,of which the former is the highest,and was relatively low in other parts.In addition,the expression pattern of GABA receptor mRNA was associated with developmental stages with the expression level gradual decrease,and the expression level was the lowest in the 6th instar larva and relatively high in the adult.3.Silencing study on C.suppressalis GABAergic neural pathway genes by RNAiThe dsRNA of C.suppressalis GABAergic neural pathway genes(GAD1,GAD2,VGAT,RDL1,RDL2,GRD,LCCH3,GAT,and GABAT)were generated in vitro.After injecting dsGAD1,the development of C.suppressalis was affected,and expressed manifested stunting and death.The relative mRNA expression of RDL1 and RDL2 were significantly decreased 43.0%and 39.1%,respectively at 48 h after RNAi.After injecting dsRDL1 or dsRDL2 and feeding by artificial food with sub-lethal doses of fluralaner,the mortality of treated insects significantly reduced while compared with the control group;however,no change was observed with other genes’ dsRNA treatment.Meanwhile,no change was observed when the C.suppressalis treated with chlorobenzamide containing food after RNAi.The dsRNA of C.suppressalis GABA receptor(RDL1,RDL2,GRD,and LCCH3)genes from Escherichia coli strain were expressed in vitro and RNAi was conducted by feeding 1st-instar larvae with artificial food containing expressed dsRNA.The treated C.suppressalis exhibited developmental delay and death,and RT-qPCR results showed a significant decrease of relative mRNA expression level after RNAi.