Cui Li, Yang Daibin, Yan Xiaojing, Rui Changhui, Wang Zhenying, Yuan Huizhu
Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
PLoS One. 2013 Oct 1;8(10):e75825. doi: 10.1371/journal.pone.0075825. eCollection 2013.
Ryanodine receptor (RyR) Ca(2+) release channel is the target of diamide insecticides, which show selective insecticidal activity against lepidopterous insects. To study the molecular mechanisms underlying the species-specific action of diamide insecticides, we have cloned and characterized the entire cDNA sequence of RyR from Ostrinia furnacalis (named as OfRyR). The OfRyR mRNA has an Open Reading Frame of 15324 bp nucleotides and encodes a 5108 amino acid polypeptide that displays 79-97% identity with other insects RyR proteins and shows the greatest identity with Cnaphalocrocis medinalis RyR (97%). Quantitative real-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highest level in adult. The relative expression level of OfRyR in first, third and fifth-instar larva were 1.28, 1.19 and 1.99 times of that in egg. Moreover, two alternative splicing sites were identified in the OfRyR gene. One pair of mutually exclusive exons (a/b) were present in the central part of the predicted SPRY domain, and an optional exon (c) was located between the third and fourth RyR domains. Diagnostic PCR demonstrated that exons a and b existed in all developmental stages of OfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of these exons showed a significant difference between different developmental stages. These results provided the crucial basis for the functional expression of OfRyR and for the discovery of compound with potentially selective insect activtity.
兰尼碱受体（RyR）钙释放通道是双酰胺类杀虫剂的作用靶点，这类杀虫剂对鳞翅目昆虫表现出选择性杀虫活性。为了研究双酰胺类杀虫剂物种特异性作用的分子机制，我们克隆并鉴定了亚洲玉米螟（Ostrinia furnacalis）RyR的完整cDNA序列（命名为OfRyR）。OfRyR mRNA具有15324个碱基对的开放阅读框，编码一个5108个氨基酸的多肽，该多肽与其他昆虫的RyR蛋白具有79 - 97%的同一性，与稻纵卷叶螟RyR的同一性最高（97%）。实时定量PCR显示，OfRyR在卵中的表达水平最低，在成虫中的表达水平最高。OfRyR在一龄、三龄和五龄幼虫中的相对表达水平分别是卵中的1.28、1.19和1.99倍。此外，在OfRyR基因中鉴定出两个可变剪接位点。在预测的SPRY结构域中部存在一对互斥外显子（a/b），一个选择性外显子（c）位于第三个和第四个RyR结构域之间。诊断性PCR表明，外显子a和b存在于OfRyR cDNA的所有发育阶段，但在卵cDNA中未检测到外显子c。这些外显子的使用频率在不同发育阶段之间存在显著差异。这些结果为OfRyR的功能表达以及发现具有潜在选择性昆虫活性的化合物提供了关键依据。
