Cloning And Functional Analysis Of Ionotropic GABA Receptors In Small Brown Planthopper,Laodelphax Striatellus(Hemiptera:Delphacidae)

lonotropic y-aminobutyric acid receptors(GABARs)are present in vertebrate and invertebrate central nervous systems(CNSs)or neuromuscular junctions,where they mediate rapid inhibitory neurotransmission by allowing the flux of chloride ions into nerve cells.As the important molecular target of cyclodiene and phenylpyrazole insecticides,insect GABARs are always the hot topics in insecticide toxicology.However,it has been reported that there are at least 19 species of insects that have the mutations to these pesticides,and revealed a diversity of subunit isoforms arising from the alternative splicing,which may lead to functional diversity of GABA receptors and develop new specific insecticides for pest management.In this paper,we conducted the experiments on the ionotropic GABA receptors in Laodelphax Striatellus(Fallen)by means of molecular biology technology.1.Cloning and sequence analyses of ionotropic y-aminobutyric acid receptors genes from L.StriatellusTo further understanding of types of GABA receptor subunits,general primers were designed according to the insect sequences of reported and RT-PCR was carried out.The genes of GRD and LCCH3 subunits were successfully cloned to their full length by using the RACE strategy.Both of them had the typical characteristics of GABARs subunits.Further analyses demonstrated that they had high amino acid sequence identity with other insect GABAR subunits previously reported(>90%).Cloning and sequence analysis of the full length Lsrdl cDNA showed three alternative splicing forms in exon 3(designated as 3a,3b and 3c)and two in exon 6(designated as 6a and 6b).We found four variants(designated as Lsrdl-3a6b,Lsrdl-3b6b,Lsrdl-3b6a and Lsrdl-3c6b)in this study.The isoform Lsrdl-3c6b was the most abundant in both strains(50%).2.Expression pattern of GABA receptor genes in L.StriatellusqPCR was performed using cDNA and gene specific primers.We investigated the expression pattern of GABA receptor genes(Lsrdl,Lsgrd and Lslcch3).qPCR results showed transcriptional levels of them differed among developmental stages.For Lsrdl,the highest expression was found in eggs and female adults,followed by fifth-instar nymphs,first-instar nymphs and male adults,and second to fourth-instar nymphs.While for Lsgrd and Lslcch3,the highest expression was found in fifth-instar nymphs.Lsrdl,Lsgrd and Lslcch3 were expressed at the highest level in heads and significantly higher than in thoraxes,abdomens and legs that had similar levels of expression.Additionally,for each genes,no significant difference in the relative transcript levels between the LsFS and LsFR with the A2'N.Different expression patterns may be related to their physiological function.3.AS-PCR technique for detection of the fipronil target resistance in L.StriatellusBased on allele specific PCR(AS-PCR),the specific primers were deigned and the technique for detecting the target-resistance related mutation(A2'N)in RDL subunit of L.Striatellus.With this technique,the target resistance in the susceptible and resistant strains was checked.The results showed that all the individuals in the susceptible strains were susceptible homozygotes.In the resistant strains,most of them were heterozygotes(>91%)and the others were resistant homozygotes.As compared with bioassay,this method had some advantages over the sensitivity and accuracy.4.Function research of RDL GABA receptor by RNAi in L.StriatellusRNAi as one of the most promising techniques to knockdown the expression of corresponding gene has been extensively used for sequence-specific loss of gene function in model insects like Drosophila and also in non-model insects.Relative expression of Lsrdl was reduced by about 27%to 82%compared to the reference(dsegfp injection)at 2-6 days after RNAi injection.Significant down-regulation of Lsrdl occurred on the second day and peaked on the fourth.Survival of the injected individuals during the 6-d period showed no significant difference between dsLsrdl and dsegfp injections(p>0.05)suggesting that Lsrdl knockdown had no adverse effect on survivorship.Fipronil induced mortality was significantly reduced by the knockdown of Lsrdl for both susceptible and resistance strains.Susceptible individuals with Lsrdl knockdown had similar mortality compared to resistance individuals without the knockdown.The mortalities on water treated rice seedlings showed no differences between different injection treatments that rdl knockdown did not affect survivorship.