【作者】 王剑；

【导师】 王建军；

【作者基本信息】 扬州大学 ， 农药学， 2013， 硕士

【摘要】 褐飞虱Nilaparvata lugens (Stal)属同翅目、飞虱科(Homoptera:Delphacidae),是我国和许多亚洲国家重要的水稻害虫,通过直接取食和传播水稻草状矮化病毒(Rice grassy stunt virus, RGSV)和水稻齿叶矮缩病毒(Rice ragged stunt virus, RRSV)而危害水稻。棉铃虫Helicoverpa armigrea (Hubner)属鳞翅目、夜蛾科(Lepidoptera:Noctuidae),是亚洲、大洋洲、非洲及欧洲危害棉花及其他作物的主要害虫之一。目前对褐飞虱和棉铃虫的主要防治方法还是依靠化学手段,由于药剂使用不当等原因使得这两种害虫对多种杀虫剂产生了抗性,因此,研发具有新型作用机制的杀虫剂至关重要。鱼尼丁受体(ryanodine receptors, RyRs)是一种控制Ca2+从细胞内部释放到细胞质中的配基门控钙离子通道,最近发现的邻苯二甲酰胺类和邻甲酰胺基苯甲酰胺类新型杀虫剂使得鱼尼丁受体的研究再次受到关注。本文克隆了褐飞虱和棉铃虫的鱼尼丁受体基因,分析了鱼尼丁受体基因在褐飞虱和棉铃虫不同发育阶段的表达模式,研究结果对进一步提高对昆虫鱼尼丁受体结构和功能的认识以及新型高效杀虫剂的创制具有重要的理论意义和实践价值。通过反转录多聚酶链式反应技术(RT-PCR)和cDNA末端快速扩增技术(RACE)克隆了褐飞虱和棉铃虫RyR全长cDNA,命名为NlRyR和HaRyR. NlRyR的开放阅读框长15423bp,编码5140个氨基酸残基。HaRyR开放阅读框长15429bp,编码5142个氨基酸残基。NlRyR和稻纵卷叶螟Cnaphalocrocis medinalis、小菜蛾Plutella xylostella、果蝇Drosophila melanogaster的RyR的氨基酸序列一致性分别为81%、79%和78%。HaRyR和稻纵卷叶螟、小菜蛾、果蝇RyR的氨基酸序列一致性分别为94%、92%和78%。作为Ca2+释放通道的孔道形成区域(pore-forming segments)一部分的GXRXGGGXGD基序在NlRyR(4989-4998)和HaRyR (4994-5003)中高度保守,表明NIRyR和HaRyR可形成功能性Ca2+通道。分别位于NlRyR和HaRyR蛋白C末端4199-4226和4234-4261处与4218-4245和4253-4280处的两个EF-hands表明NlRyR和HaRyR和哺乳动物RyRs相似,可以调节细胞内Ca2+释放。保守性结构域分析发现,NlRyR和HaRyR都含有20个结构域,其中包括6个跨膜结构域。通过荧光定量PCR研究了NlRyR和HaRyR在不同发育阶段的mRNA表达水平。结果发现,NlRyR和HaRyR在不同发育阶段的mRNA表达水平存在较大差异。NlRyR的mRNA表达水平在卵和幼虫期没有显著差异,但从1龄到4龄逐渐下降,4龄到5龄明显上升。长翅雌虫NlRyR的mRNA表达水平显著高于短翅雌虫和长翅及短翅雄虫。HaRyR在棉铃虫卵中的表达量最低,在3龄幼虫、雌蛹、雄蛹、雌成虫的表达量没有显著差异,但雄成虫的表达量显著高于其它发育阶段,同时HaRyR在棉铃虫头部的表达量也显著高于胸部和腹部。此外,在NlRyR中发现了3个选择性剪接位点,包括2个选择性外显子和一对互斥外显子,在HaRyR中发现了一个选择性剪接位点。对NlRyR互斥外显子的特异性PCR检测发现,在褐飞虱的卵、1龄和3龄若虫及长翅雄虫和短翅雌虫所有cDNA克隆中都检测到外显子A,外显子B在5龄幼虫、长翅雌虫和短翅雄虫中的表达频率分别为5%、23.8%和4.7%。更多还原

【Abstract】 The brown planthopper (BPH), Nilaparvata lugens (Stal)(Homoptera:Delphacidae), is a serious pest of rice crops throughout Asia. This pest damages the rice plant both directly by feeding and indirectly by vectoring rice virus diseases, such as rice grassy stunt virus (RGSV) and rice ragged stunt virus (RRSV).The cotton bollworm(CBW), Helicoverpa armigera Hiibner (Lepidoptera:Noctuidae), is one of the serious pests of cotton and other crops throughout Asia, Oceania, Africa and Europe. The use of chemical insecticides has been the primary strategy for controlling N. lugens and H. armigera. As a consequence of extensive exposure to insecticides, both N. lugens and H. armigera have developed resistance to a wide range of insecticides. Novel chemicals that act on novel biochemical targets with no cross-resistance to the current insecticides are thus needed to cope with N. lugens and H. armigera resistance. In the present study, we isolated full-length RyR cDNAs from N. lugens. and H. armigera (named NlRyR and HaRyR). Ryanodine receptors (RyRs) are a distinct class of intracellular calcium (Ca2+) release channels that regulate the entry of Ca2+into the cytosol from the intracellular organelles including the sarcoplasmic reticulum of muscles and the endoplasmic reticulum of neurons and many other cell types. Recent discovery of two novel classes of synthetic insecticidal chemicals targeting RyRs, phthalic acid diamides and anthranilic diamides, has prompted the studies on insect RyRs. In this study, the RyR genes were isolated from the N. lugens and H. armigera, and the expression profiles of the RyR genes in different developmental stages were also studied. The results might assist in understanding the structure-function relationships of insect ryanodine receptor as well as the development of novel insecticides.Full-length RyR cDNAs were cloned from N. lugens and H. armigera using RT-PCR and RACE, and named as NlRyR and HaRyR, respectively.The composite NlRyR contains an ORF of15423bp encoding5140amino acid residues, which shares81%,79%and78%overall identity with Cnaphalocrocis medinalis, Plutella xylostella and Drosophila melanogaster RyR homologues, respectively. The composite HaRyR contains an ORF of15429bp encoding5142 amino acid residues, which shares94%,92%and78%overall identity with C. medinalis P. xylostella 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 NlRyR (4989-4998) and HaRyR(4994-5003), suggesting that NlRyR and HaRyR 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 NlRyR at positions4199-4226and4234-4261as well as in HaRyR at positions4218-4245and4253-4280, suggesting that this channel, similar to mammalian RyRs, may be regulated by cytosolic Ca2+. Analysis of conserved structural domains showed that both NIRyR and HaRyR contain20domains including six transmembrane domains.The mRNA levels of NlRyR and HaRyR in different developmental stages were analyzed using qRT-FCR. The results showed that NlRyR was expressed at all developmental stages tested. While there is no significant difference between egg and nymph stages, the mRNA expression levels of NlRyR gradually decreased during nymph development from1st instar to4th instar, and increased significantly from4th instar to5th instar. Remarkablely, the mRNA expression levels in macropterous female adults were significantly higher than that in brachypterous female adults as well as macroptereus and brachypterous male adults. In HaRyR, the lowest expression level was observed at egg stage. While there is no significant difference among third insrar larvae, female pupae, male pupae and female adults, and nymph stages, the mRNA expression level of male adults was significantly higher than other developmental stages. Anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Furthermore, three alternative splicing sites were identified in NIRyR, including two optional exons and one pair of mutually exclusive exons (A/B), while only one alternative splicing site was identified in HaRyR. Diagnostic PCR assays showed that exon a was present in all cDNA clones examined from the egg, fiset instar nymphs, third instar nymphs, macropterous male adults, and brachypterous female adults. In contrast, exon b was present at low frequencies (5%、23.8%and4.7%) in fifth instar nymphs, macropterous female adults, and brachypterous male adults.更多还原