Cloning And Alternative Splicing Analysis Of Ryanodine Receptor Gene In Dialeurodes Citri And Toxoptera Citricida

Ryanodine receptor is a distinct class of ligand-gated channels which controlling the release of Ca2+ from intracellular stores. It exists in sarcoplasmic reticulum of muscles and endoplasmic reticulum of neurons and other cells, which could play an important role in balance of Ca2+. There are three different isoforms of RyR in mammals(RyR1, RyR2, RyR3). While insects have only one RyR gene, which shares under 50% sequence identity with either of the three mammalian RyRs. It makes developed for efficient and safety insecticide to be possible. Dialeurodes citri and Toxoptera citricidus are two citrus pests which belong to Hemiptera. During these years, due to monoculturing single cultivar in citrus and overuse of insecticide, many natural enemies were killed. It cause imbalance of citrus ecosystem, and insecticide resistance is increasingly prominent. Therefore, it’s popular to study the chlorantraniliprole which acted at ryanodine receptor. Our study focused on the molecular cloning, characteristics and alternative splicing of D.citri and T.citricidus. Aimed at provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. The main results as follows: 1. Susceptibility of D.citri and T.citricidus to five class insecticidesThe susceptibility of D.citri and T.citricidus, collected from the citrus orchard of SouthWest University, Chongqing, to five(avermectins, chlorantraniloprole, imidacloprid, chlorpyrifos and cypermethrin) different types of insecticide were determined through a modified impregnated filter and slide dip method. The toxicities of these five insecticides to D.citri. from high to low is avermectins, imidacloprid, chlorantraniloprole, chlorpyrifos and cypermethrin. As for T.citricidus, it’s most sensitive to imidacloprid, followed by chlorantraniloprole, chlorpyrifos, cypermethrin and avermectins. 2. Molecular cloning and characteristics of ryanodine receptor gene in D.citri and T.citricidusBased on the transcriptome data, the ORF cDNA sequence of RyR gene was cloned from D.citri and T.citricidus using RT-PCR techniques. DcRyR(KP733848) contained the complete open reading frame(ORF) with 15,381 bp and encoded a peptide with 5,126 amino acid residues, with a predicted molecular weight of 579.45 kDa and a pI of 5.40. On the other hand, TciRyR(KP733849) contained a 15,315 bp ORF encoding a deduced peptide with 5,104 amino acid residues. The calculated molecular weight and p I of TciRyR were approximately 580.08 kDa and 5.49, respectively.Multiple protein alignments shows that the similarity between DcRyR、TciRyR and other insect RyRs from 70% to 90%.The highest similarity(95.1%) was observed between DcRyR and B. tabaci RyR(BtRyR) at the amino acid level. TciRyR was highly similar to RyRs from M. persicae(98.2%) and A. pisum(98.1%) at the protein level, respectively. The similarity between DcRyR and TciRyR with the three RyRs isoforms of H.sapiens ranged from 43.0% to 46.0%.Several conserved structural features crucial for RyRs’ functions were also identified in DcRyR and TciRyR. These include one MIR(mannosyltransferase, IP3 R, and RyR) domain, two RIH(RyR and IP3 R homology domains) domains, three SPRY(spore lysis A and RyR) domains, and four RyR repeat domains. In addition, the RIH-associated domain was also conserved in DcRyR and TciRyR. The C-terminal of DcRyR and TciRyR contained six predicted transmembrane helices(TM1-TM6), which corresponded to six highly hydrophobic regions. In addition, both DcRyR and TciRyR also had the well conserved sequence motif, GXRXGGGXGD, which constitutes part of the pore-forming segments of the Ca2+ release channels. Moreover, one pore helix and two consensus Ca2+-binding EF-hand motifs were also found in DcRyR and TciRyR. These regions exhibited high sequence identity to other insect RyRs. In a word, both DcRyR and TciRyR had all the hallmark features of ryanodine receptors, suggesting their high structural and functional conservation with known RyRs. 3. mRNA expression profiles of DcRyR and TciRyRThe qRT-PCR was performed to investigate the mRNA levels of DcRyR and TciRyR. The expression pattern revealed that DcRyR and TciRyR were expressed in all of the tested developmental stages and body parts. DcRyR mRNA expressed in the adult head and adult thorax were significant higher than in abdomen. The developmental expression profiles showed the highest and lowest transcription levels of DcRyR mRNA in egg and larva, respectively. The mRNA expression levels of TciRyR in adults were significant higher than those in nymphs, especially in adults head. There was no significant difference between thorax and abdomen. 4. Alternative splicing analysis of DcRyR and TciRyRAligning multiple cDNA clones revealed the presence of several alternative splicing variants in DcRyR and TciRyR, leading to the discovery of four variable regions in DcRy R(AS1a、AS1b/c、AS1d) and one in TciRyR(AS2). Diagnostic PCR was performed to detect the presence of each putative alternative exon in the individual cDNA clone. AS1.a(+) was detected at higher frequencies in adult thorax, while AS1d(+) was high expressed in nymphs. As mutually exclusive exons, AS1 b was predominantly detected in all body parts and all developmental stages of D.citri. By the same manner, AS2 was identified in T.citricidus, subsequently. The genome sequence flanking AS2 was cloned and sequenced, which indicated that this alternative exon was derived from intron retention, inclusion of which could lead to the production of truncated proteins. Variants retaining the alternative exon in nymph, adult, adult head, adult thorax, and adult abdomen were detected at lower frequencies compared with those with the alternative exon spliced out.Taken together, we estimated the susceptibility of D.citri and T.citricidus, collected from the citrus orchard of SouthWest University, to five different types insecticides. Cloned the ORF sequence of DcRyR and TciRyR based on their transcriptome data, respectively. qPCR analysis revealed the expression profiles during different development stages and different body parts. Furthermore, alternative splicing exons was identified four in DcRyR and one in TciRyR. Diagnostic PCR was settled to analysis the relative frequencies. Our results enriched our current knowledge about insect RyRs, lay the foundation for understanding the structural and functional properties of DcRyR and TciRyR. As well as estimating the potential of the new insecticides, which is selectively acting on the target pests.