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

摘要/Abstract

摘要： 【目的】二酰胺类杀虫剂的作用靶标鱼尼丁受体（ryanodine receptor, RyR）是目前所知的最大的离子通道蛋白，该受体可控制细胞内Ca²⁺的释放，对细胞内Ca²⁺的稳定起着关键作用。克隆获得桃小食心虫鱼尼丁受体基因（CsRyR）全长序列，进一步解析该基因在桃小食心虫(Carposina sasakii)各发育阶段的表达模式。【方法】通过同源序列比对的方法，利用cDNA末端快速扩增技术（RACE）获得该基因的全长cDNA序列；应用生物信息学软件对该基因的开放阅读框、编码的氨基酸序列、功能结构域等信息进行预测分析，并基于最大似然法构建该基因与其他昆虫相关基因序列的系统发育树，明确其系统进化关系。分别提取桃小食心虫各发育阶段RNA，以GAPDH为内参基因，应用RT-qPCR技术，解析CsRyR在桃小食心虫各发育阶段（卵、幼虫、蛹和成虫）的表达模式。【结果】桃小食心虫CsRyR的cDNA全序列长度为15 766 bp，开放阅读框15 405 bp，编码5 134个氨基酸。氨基酸序列比对结果显示，CsRyR与脊椎动物RyRs的一致度分别为45%—47%；与秀丽隐杆线虫（Caenorhabditis elegans）RyR的一致度也为46%。在昆虫RyRs中，与鳞翅目一致度为91%—94%，与同翅目、双翅目昆虫一致度均为79%。系统发育树显示该基因编码的蛋白质与鳞翅目夜蛾科和螟蛾科害虫RyRs亲缘关系最近。结构域分析结果显示，CsRyR具有RyR的典型结构域，如位于C-末端的6个跨膜结构域（AA 4 467-5 029）、释放Ca²⁺的通道形成基序GVRAGGGIGD、Ca²⁺结合位点EF-hand和3个ATP结合位点GXGXXG等。二酰胺类杀虫剂可能的作用位点AA 183-290（BmRyR），AA 4 610-4 655（DmRyR）和4 946G（PxRyR）在CsRyR中无特殊性。此外，CsRyR中存在AA 2 490- 2 496 TQAPRPG和5 131-5 134 SQAK两个基序，在其他物种中均没有发现。RT-qPCR分析结果表明，CsRyR在蛹期表达量最高，分别是1日龄卵、6日龄卵、初孵幼虫、老熟幼虫和成虫的25.19、7.73、6.48、4.74和3.58倍。【结论】克隆了CsRyR基因全长cDNA序列，证明其表达具有发育阶段特异性。

关键词: 桃小食心虫, 鱼尼丁受体, 基因克隆, 发育阶段, 表达模式

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

孙丽娜，张怀江，闫文涛，马春森，仇贵生. 桃小食心虫鱼尼丁受体基因克隆及表达模式分析[J]. 中国农业科学, 2015, 48(10): 1971-1981.

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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