【作者】 崔丽；

【导师】 袁会珠； 芮昌辉；

【作者基本信息】 中国农业科学院 ， 农药学， 2014， 博士

【摘要】 NK130102是新型邻苯二甲酰胺类化合物，通过转录组测序的方法测定了NK130102和氟苯虫酰胺处理的亚洲玉米螟的转录组，在此基础上克隆了亚洲玉米螟鱼尼丁受体（OfRyR）cDNA，并深入研究了不同浓度的氟苯虫酰胺在不同作用时间下对OfRyR cDNA表达量的影响。此外，我们在OfRyR cDNA的基础上进一步克隆了OfRyR的gDNA序列。结论如下：1) NK130102和氟苯虫酰胺对亚洲玉米螟2龄幼虫的LC50分别为0.2和0.17μg g-1，两种药剂的LC95分别为8.8和3.8μg g-1。亚洲玉米螟经8.8μg g-1的NK130102处理48h后，钙信号途径中的鱼尼丁受体基因表达量上调3.7倍，肌质网/内质网Ca2+ATPase基因上调2.8倍；与肌肉收缩相关的蛋白肌球蛋白，肌动蛋白，M-线蛋白unc-89，肌联蛋白的基因表达量分别上调7.4，2.8，26.9和3.7倍。因此，我们可以初步判断，NK130102可以作用于钙离子释放通道，引起肌质网/内质网钙泵中Ca2+的持续释放，导致细胞质中Ca2+浓度的升高，从而引起肌肉的持续收缩。此外，重要的杀虫剂靶标基因及解毒代谢酶基因序列的获得为基于靶标的新化合物的生物合理设计提供了基础。2)在此基础上，克隆了OfRyR cDNA，其开放阅读框为15324bp，可以编码5108个氨基酸，该氨基酸与其它昆虫的鱼尼丁受体氨基酸序列具有79–97%的相似性，其中与稻纵卷叶螟鱼尼丁受体氨基酸序列的相似性最高（97%）。序列分析结果表明OfRyR cDNA的C-末端存在6个跨膜区域，且这6个跨膜区恰好对应6个高疏水的区域。C-末端跨膜区存在一个构成钙离子通道的保守序列GVRAGGGIGD。实时荧光定量PCR的结果表明：OfRyRcDNA在卵中的表达水平最低，在成虫中的表达量最高，在1龄、3龄、5龄幼虫的相对表达量是卵中的1.19-1.99倍。此外，我们在OfRyR cDNA中发现了两种形式的可变剪接，一对位于第二个SPRY结构域上的互斥外显子（a/b），和一对位于第三至第四个RyR结构域上的选择性外显子（c）。诊断PCR结果表明：互斥外显子a和b在亚洲玉米螟所有龄期中均同时存在，但是外显子a和b各自所占的比例在不同龄期中却存在很大的差异。对于选择性外显子c，存在频率最高的龄期是三龄幼虫期，达到了47.5±16.3%。然而，卵期未检测到c的存在。3)形态学研究表明：氟苯虫酰胺和NK130102导致亚洲玉米螟出现拒食，呕吐，虫体收缩变短的中毒症状，并且体重明显变轻。实时荧光定量PCR的结果表明：氟苯虫酰胺和NK130102导致OfRyR cDNA表达量明显上调，而甲氨基阿维菌素苯甲酸盐却没有引起该基因表达量的显著变化。而且，药剂处理24h后，RyR基因的表达量随氟苯虫酰胺浓度的升高，大致呈现逐渐上调的趋势；随着时间的延长，较低浓度的氟苯虫酰胺使得RyR基因的表达量继续上调，药剂处理48h，1μg/g的氟苯虫酰胺导致RyR基因表达量上调的幅度最大，此时RyR基因的表达量是对照的2.96倍。4)我们在OfRyR cDNA序列的基础上，进一步克隆了该基因的gDNA序列。我们通过测序获得了长度为93575bp的DNA序列，该基因包含95个外显子，其中第25和26个外显子是一对互斥外显子，第59个外显子是一对选择性外显子。除了第21个内含子以GC开头外，其它内含子和外显子交界处都遵循GT/AG规律。综上所述，通过转录组测序初步明确了新化合物NK130102的作用机制，通过克隆OfRyR cDNA和gDNA，研究氟苯虫酰胺和NK130102对OfRyR cDNA表达量的影响，进一步明确了二酰胺类化合物的作用机制，为基于靶标的新农药分子生物合理设计提供理论依据。更多还原

【Abstract】 NK130102is a novel phthalic amide insecticide. In order to investigate the mechanism ofthis compound, transcriptome sequencing was used to evaluate changes in the globle geneexpression of O. furnacalis treated with NK130102and flubendiamide. Based on thetranscriptome sequencing results, the OfRyR cDNA was cloned. The effects of differentinsecticides and different concentration of flubendiamide on OfRyR cDNA relative expressionabundance wre carried out. We also cloned the gDNA of OfRyR based on the OfRyR cDNA.Conclusions are as follows:1) The LC50values of NK130102and flubendiamide against Ostrinia furnacalis were0.2and0.17mg g-1respectively, and the LC95values of two insecticeides against Ostrinia furnacalis were8.8and3.8μg g-1respectively. The ryanodine receptor (RyR) gene and sarco/endoplasmicreticulum calcium ATPase gene in the calcium signaling pathway were up-regulated for3.7and2.8folds, and the myosin, actin, muscle M-line assembly protein unc-89, titin genes involved invascular smooth muscle contraction were up-regulated for7.4,2.8,26.9and3.7folds in O.furnacalis treated with NK130102at the concentration of8.8mg g-1for48h. Therefore, we coulddeduce that NK130102acting on the calcium release channel, reduced the continuous Ca2+releasefrom calcium pump, and the increased Ca2+in cytoplasm caused the persistent muscle contraction.In particular, the insecticide targets and metabolism genes will facilitate the designing of newcompounds based on the insecticide target.2) Based on the transcriptome sequencing, we cloned and characterized the full-length RyRcDNA from Ostrinia furnacalis. This cDNA contained an ORF of15324bp encoding a protein of5108amino acid residues, which show79–97%amino acid identity with other insect RyRisoforms and share the greatest identity with Cnaphalocrocis medinalis RyR (97%). Quantitativereal-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highestlevel in adult. The relative expression levels of OfRyR in first, third and fifth-instar larva were1.19~1.99times of that in egg. Moreover, two alternative splicing sites were identified in theOfRyR gene. One pair of mutually exclusive exons (a/b) were present in the central part of thepredicted second SPRY domain, and an optional exon (c) was located between the third and fourthRyR domains. Diagnostic PCR showed that exons a and b existed in all developmental stages ofOfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of theseexons were different among different developmental stages.3) Morphological study demonstrated that the weight of O. furnacalis treated withflubendiamide and NK130102were much lighter than those treated with emamectin benzoate andDMSO. Flubendiamide and NK130102also caused the toxic symptom of antifeedant, vomiting,contract and shortening. Real-time fluorescent quantitative PCR showed the relative expressionquantity of RyR cDNA was increased when O. furnacalis treated with flubendiamide andNK130102, while emamectin benzoate has no effect on the OfRyR expression. The relative expression quantity of RyR cDNA from O. furnacalis treated with different concentration offlubendiamide was also carried out. The OfRyR cDNA increased with the increasingflubendiamide concentrations after treatment24h, and flubendiamide at lower concentrations canlead to continuing rise over time. The biggeat raise of OfRyR cDNA was2.96-fold than controlwhen Flubendiamide at1μg/g for48h.4) We cloned the gDNA of OfRyR based on OfRyR cDNA. The full length of93575bpOfRyR gDNA contains95exons, the25and26exons are a pair of exclusive exons and the59exon is an optional exon. The exon/intron boundaries assigned follow the GT/AG rule.In conclusion, transcriptome sequencing data provide useful information in understanding themechanisms of novel insecticide NK130102. We cloned cDNAand gDNA of OfRyR and analysedthe effects of diamides on the expression of OfRyR, which further clarify the mechanism ofdiamide insecticides. The results will facilitate the designing of new compounds based on theinsecticide targets and could provide the basis for further study of resisitance to diamideinsecticides.更多还原