Cloning And Alternative Splicing Analysis Of A Ryanodine Receptor Gene And A Sodium Chanmel Gene In The Rice Leaffolder,Cnaphalocrocis Medinalis (Guenee)

The rice leaffolder, Cnaphalocrocis medinalis Guenee (Lepidoptera:Pyralidae), is one of the most notorious leaf-feeding pests of rice, Oryza sativa (L.)(Poaceae, Graminales), in subtropical countries. In China, outbreaks of the insect have occurred in recent years in the rice producing regions of southern China and in the middle and lower reaches of the Yangtze River. At present, chemical control is still the primary means in the rice leaffolder management. Developing insecticides that act on novel biochemical targets is important in crop protection due to the ability of insects to rapidly develop insecticide resistance. In this study, the ryanodine receptor gene and sodium chanmel gene were cloned from the rice leaffolder, and the expression profiles of the splice variants of the ryanodine receptor and sodium channel gene in different developmental stages and adult anatomical regions of the rice leaffolder were also studied. The results might assist in understanding the structure-function relationships of insect ryanodine receptor and sodium channels as well as the development of novel insecticides.Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. RyRs are the target sites of two novel classes of synthetic insecticidal chemicals including phthalic acid diamides and anthranilic diamides. Isolation of full-length RyR cDNAs is a critical step toward a structural and functional characterization of insect RyRs and an understanding of the molecular mechanisms underlying the species selective toxicity of diamide insecticides. In this study, a full-length RyR cDNA (named as CmRyR) was isolated from the rice leaffolder. The composite CmRyR contains an ORF of15264bp encoding a574339Da protein of5087amino acid residues, which shares94%and79%overall identity with Bombyx mori and Drosophila melanogaster RyR homologues, respectively. All hallmarks of RyR proteins are conserved in the CmRyR protein. Two consensus Ca2+-binding EF-hands are present in the COOH-terminus of the CmRyR at positions4183-4194and4285-4296, suggesting that this channel, similar to mammalian RyRs, may be regulated by cytosolic Ca2+. The close similarity of the pore regions of CmRyR and mammalian RyRs, including the conserved GGGXGD selectivity filter motif, suggests that CmRyR likely forms functional cation channels with a high single-channel conductance and permeability to Ca2+Multiple sequence alignment illustrates that amino acid residues at positions analogous to the CmRyR residues N4922, N4924, N4935, L4950, L4981, N5013and T5064are unique to lepidopteran RyR homologues, whereas the corresponding residues in non-lepidopteran insect RyRs are highly conserved with other invertebrate and vertebrate RyRs, suggesting that these residues might be involved in difference in channel properties between lepidopteran and non-lepidopteran insect RyRs and in species selective toxicity of diamide insecticides. Furthermore, two alternative splicing sites were identified in CmRyR gene, one of which was located in the central part of the predicted second SPRY domain. Diagnostic PCR assays showed that the usage of the mutually exclusive exons (a/b) exhibited marked developmental and anatomical regulation. Exon a was present in all cDNA clones examined from the egg and adult head cDNA pools, but only in16of the23clones and11of the21clones analyzed from the pupal and adult body cDNA pools, respectively. In contrast, exon b was not detected in the egg and adult head cDNA pools, but was present at low to moderate frequencies (30%and48%) in the pupal and adult body cDNA pools. Developmental regulation of exon inclusion was also observed for exon c. It was present at low frequencies (0%,17%, and10%) in eggs, third instar larvae and fifth instar larvae, respectively, but at moderate to high frequencies (55%and71%) in first instar larvae and pupae. These results implied that alternative splicing might be the major means of generating functional diversity in C. medinalis as well as other insect RyR channels.Voltage-gated sodium channels are the targets of pyrethroid insecticides. Knockdown resistance (kdr) is the major mechanism of pyrethroid resistance in insects. The role of sodium channels in insecticide resistance has prompted the study on insect sodium channels. Indoxacarb is a novel oxadiazine insecticide that acts on the inactivated state of the sodium channel and disrupts nerve-impulse transmission. As an ideal alternative to organophosphate and pyrethroid insecticides, indoxacarb has been used for the control of rice leaffolder in some regions in China. In this study, a cDNA fragment of5773bp in length of sodium channel gene, Cmsc, in the rice leaffolder, was cloned by RT-PCR and RACE. The Cmsc cDNA fragment encodes1921amino acid residues, which shares90%、72%and76%identity with Bombyx mori (Bmsc), Drosophila melanogaster (para) and Blattella germanica (BgNav) para-homologues, respectively. Furthermore, two alternative splicing sites corresponding to exclusive exons1/k and option exon b in para were identified in Cmsc gene. Diagnostic PCR assays showed that the expression levels of splicing variants of Cmsc varied in different development stages and different adult tissues.