桃小食心虫鱼尼丁受体基因克隆及表达模式分析

doi:10.3864/j.issn.0578-1752.2015.10.010

Abstract

Abstract: 【Objective】Ryanodine receptor (RyR), the target of diamide insecticides, is the largest known iron channel protein. The receptor is the key to the stabilization of Ca²⁺ by regulating the release of Ca²⁺ in cell. The objectives of this study are to isolate the cDNA of ryanodine receptor from the peach fruit moth Carponsina sasakii (CsRyR), and to analyze the expression of RyR mRNA at different developmental stages of this pest. 【Method】 According to the sequences of RyR gene of other species obtained from the NCBI database, degenerate and specific primers were designed for reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR to clone the full-length sequences of CsRyR. The open reading frame (ORF), animo acid residues, the conserved structure domains, phylogenetic tree and other characteristics were analyzed using the bio-software. And the relative expression levels of RyR mRNA at different developmental stages (egg, larvae, pupe and adult) were investigated using quantitative real-time PCR (RT-qPCR), GAPDH was used as the reference. 【Result】 The full-length cDNA sequence of CsRyR was isolated from the peach fruit moth using RT-PCR and RACE-PCR. The CsRyR mRNA has an open reading frame (ORF) of 15 405 bp nucleotides and encodes 5 134 amino acid residues. CsRyR displays 45%-47% identity with vertebrate RyR isoforms, and 46% identity with RyR from Caenorhabditis elegans (CeRyR). And CsRyR shares 91%-94% identity with that of Lepidoptera insects and 79% identity with those of Homoptera and Diptera insects. Meanwhile, the phylogenetic tree analysis showed that CsRyR has the closest genetic relationship with insects of Noctuidae and Pyralididae.CsRyR shares common structural features with known RyRs. Six transmembrane domains (AA 4 467-5 029) are at the COOH-terminal. The sequence motif, GVRAGGGIGD, which constitutes part of the pore-forming segments of the Ca²⁺ release channels. The sites of RyR binding with Ca²⁺and ATP (EF-hand and GXGXXG (3 motifes)) exist. Furthermore, three putative sites potentially binding with diamides were AA 183-290 (BmRyR), AA 4 610-4 655 (DmRyR) and 4 946 G (PxRyR) showed no differences in the CsRyR. And the relative expression abundance of RyR mRNA from the pupae of C. sasakii was the highest, which were 25.19, 7.73, 6.48, 4.74 and 3.58 fold compared to 1-day-old eggs, 6-day-old eggs, infant larvae, mature larvae and adults, respectively. 【Conclusion】 The full length of RyR cDNA was cloned from C. sasakii, and the mRNA expression has significant differences at different developmental stages.

Key words: Carposina sasakii, ryanodine receptor, gene clone, developmental stage, expression profiling

SUN Li-na, ZHANG Huai-jiang, YAN Wen-tao, MA Chun-sen, QIU Gui-sheng. Molecular Cloning and Expression Profiling of a Ryanodine Receptor Gene in the Peach Fruit Moth (Carposina sasakii)[J].Scientia Agricultura Sinica, 2015, 48(10): 1971-1981.

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Molecular Cloning and Expression Profiling of a Ryanodine Receptor Gene in the Peach Fruit Moth (Carposina sasakii)

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