Cloning And RNA Interference Of Two Intracellular Ca²⁺Channel Genes In The Red Flour Beetle,Tribolium Castanuem

The red flour beetle, Tribolium castaneum (Coleoptera:Tenebrionidae), is a serious global pest of agriculture grain, and is of the most efficient systemic RNA interference response in insects. Ryanodine Receptors (RyRs) and Inositol1,4,5-Trisphosphate Receptors (IP3R) are homotetrameric Ca2+channels regulating Ca2+release from intracellular stores. RyRs are the targets of novel pesticides including flubendiamide and chlorantraniloprole, and IP3Rs are the important potential targets to develop new selective insecticides. However, the research about function and structure of RyRs and IP3Rs is relatively limited at home and abroad, and there is few report about their roles in the development process of insect. In this paper, we cloned the RyR and IP3R gene, and analyzed their expression profiles in different developmental stages of T. castanuem. Furthermore, the function of RyR and IP3R gene inT. castanuem were studied using RNAi technology. The results will assist in understanding the structure-function relationships of insect intracellular Ca2+channels as well as the development of novel insecticides.Full-length RyR and IP3R cDNAs were cloned from T. castanuem, and named as TcRyR and TcIP3R, respectively. The composite TcRyR contains an ORF of15285bp encoding a577.09KDa protein of5094amino acid residues, which shares81.5%、82.2%and78%overall identity with Nilaparvata lugens, Bombyx mori and Drosophila melanogaster RyR homologues, respectively. The sequence motif, GXRXGGGXGD, which constitutes part of the pore-forming segments of the Ca2+release channels, was highly conserved in TcRyR (4943-4952), and residues corresponding to I4897, R4913, and D4917of rabbit RyR1, which play an important role in activity and conductance of theCa2+release channel, were also conserved in TcRyR (I4950, R49665and D4970), suggesting that TcRyR likely forms functional cation channels with a high single-channel conductance and permeability to Ca2+. Two consensus Ca2+-binding EF-hand motifs were also predicted in TcRyR at positions4181-4208and4216-4243, suggesting that this channel, similar to mammalian RyRs, may be regulated by cytosolic Ca2+. The composite TcIP3R contains an ORF of8172bp encoding a308.68KDa protein of2724amino acid residues, which shares69.4%and70.0%overall identity with D. melanogaster and Aedes aegypti IP3R homologues, respectively. The eleven amino acid residues within the IP3-binding core domain of mouse IP3R1(265R,266T,267T,268G,269R,504R,508K,511R,567Y,568R及569K), which are responsible for the correct recognition of IP3, are conserved in TcIP3R (267R,268T,269T,270G,271R,496R,500K,503R,560Y,561R,562K). Analysis of conserved structural domains showed that the NH2-terminal region of both TcRyR and TcIP3R contain suppressor-domain-like domain (SD), MIR (Mannosyltransferase, IP3R and RyR) domain, two RIH (RyR and IP3R Homology) domain, and RIH-associated domain. Like mammalian RyRs, three copies of a repeat termed an SPRY (SPla and RyR) domain and four copies of a repeat termed a RyR domain were also predicted. Six transmembrane helices (TM1to TM6) were also predicted in the COOH-terminal region of TcRyR and TCIP3R. Genomic structures of TcRyR and TcIP3R were predicted by comparison of the composite cDNA sequences with the genomic sequences retrieved from contigs in the whole genome shotgun release for T. castaneum. The TcRyR comprises55exons, while TcIP3R was split into26exons. The5’donor and3’acceptor site sequences in both TcRyR and TcIP3R are in agreement with the GT/AG consensus sequence, except5’donor sequence (GC) for intron7in TcRyR. Additionally, we found an alternative splicing site in TcRyR and TcIP3R genes, respectively.The mRNA levels of TcRyR and TcIP3R in different developmental stages of T. castanuem were analyzed using qRT-PCR. The results showed that the mRNA levels of TcRyR is the highest in1-day old female adults, while there is no significant difference between egg, larvae and pupa stages.. The highest and lowest mRNA expression levels of TcIP3R were observed in the1-day larves and3-day old eggs, respectively. Furthermore, the RNAi experiments showed that, when the mRNA expression levels of TcRyR were suppressed in late larval stage, the adult eclosed normally, but the hind wings of65.94%individuals could not fold properly, and all individual adults could not crawl, and died after two weeks. This result suggested that TcRyR palys an important regulatory role in hind wings folding and action ability. When the mRNA expression levels of TcIP3R were suppressed in late larval stage,64.67%larvae were unable to cast their molts completely and could not undergo normal larval-pupal metamorphosis, and thus died entrapped in their larval cuticles during pupal stage. While the rest larvae could develop into pupae, the pupae could not undergo normal pupal-adult metamorphosis. This result suggested that TcIP3R plays an important regulatory role in the metamorphosis of T. castanuem.