【作者】 王悦；

【导师】 孙守慧；

【作者基本信息】 沈阳农业大学 ， 森林保护学， 2022， 硕士

【摘要】 美国白蛾Hyphantria cunea（Drury）是我国农林业检疫性有害生物。目前,国内长期使用化学农药防治美国白蛾导致了美国白蛾耐药性和许多环保问题,因此对美国白蛾绿色防控手段的研究是非常有必要的。本研究前期研究中结合已报道参与表皮碳氢化合物（CHC）合成的基因CYP4Gs,在美国白蛾转录组文库数据中筛选获得美国白蛾表皮碳氢化合物合成酶基因HcCYP4G1、HcCYP4G2和HcCYP4G3基因序列。本试验探索了HcCYP4Gs基因在美国白蛾幼虫中的时空表达模式,通过RNAi技术和干燥胁迫生物测定明确HcCYP4Gs基因美国白蛾耐旱生理中的作用。为基于抑制昆虫CHC合成的害虫治理方法提供探索性研究,对美国白蛾杀虫剂的发展提供理论基础。研究结论如下:1.美国白蛾HcCYP4Gs基因进行生物信息学分析结果显示,HcCYP4Gs氨基酸序列均与鳞翅目目蛾夜科的豹灯蛾（Arctia plantaginis）相似性最高,与鳞翅目夜蛾科昆虫的氨基酸序列相似性较高,与非鳞翅目昆虫CYP4G亚家族氨基酸序列相似性较低,结果符合形态分类学特征。HcCYP4Gs基因编码蛋白预测的分子量分别为63.03 k Da、64.32 k Da、64.83 k Da,p I分别为8.59、8.06、8.31。HcCYP4Gs编码蛋白的多肽链中含量最多的两种氨基酸为Leu和Lys,3个蛋白均属于不稳定性蛋白,且为可溶性蛋白。HcCYP4Gs均无信号肽,HcCYP4G1和HcCYP4G3具有1个跨膜区,分别存在41、57、40个潜在磷酸化位点。2.美国白蛾HcCYP4Gs基因时空表达的研究表明,4龄幼虫中HcCYP4G1和HcCYP4G3在不同日龄的表达量均无明显差异,HcCYP4G2在第3天和第4天的表达量显著低于第1天和第2的表达量。5龄幼虫中HcCYP4Gs在不同日龄的表达量在统计学上无明显差异。HcCYP4Gs在5龄幼虫各部位和组织中均有表达,HcCYP4G1和HcCYP4G2在腹部表达量最高,HcCYP4G3在头部表达量最高。结果表明HcCYP4G1和HcCYP4G2在4龄和5龄幼虫的表达量远高于HcCYP4G3。3.美国白蛾4龄RNAi的研究结果显示,4龄幼虫注射ds HcCYP4G1后,在第1天、第2天和第3天均对HcCYP4G1起到明显的抑制效果。注射后第1、2和3天沉默效率分别为69.30%、65.38%和78.03%,显著降低了HcCYP4G1的表达水平。注射ds HcCYP4G2后,经过检测没有对HcCYP4G2基因起到抑制效果,具体原因有待深入研究。结果表明,美国白蛾4龄幼虫对ds HcCYP4G1的响应程度较好,HcCYP4G1可作为美国白蛾遗传防控的新靶点。4.RNAi后4龄幼虫耐旱能力的研究结果显示,在RH=25%时注射ds HcCYP4G1后幼虫的死亡率明显高于注射ds GFP和dd H₂O的幼虫的死亡率,注射ds HcCYP4G2后幼虫的死亡率与注射ds GFP和dd H₂O的幼虫的死亡率无差别。在干燥环境下处理后死亡的幼虫,失水严重,体长明显小于正常生长的幼虫,虫体明显皱缩发黑。结合RNAi的结果同样表明,HcCYP4G1可作为美国白蛾遗传防控的良好靶标。更多还原

【Abstract】 The Hyphantria cunea（Drury）is a quarantine pest of agriculture and forestry in my country.At present,the long-term use of chemical pesticides in China to control the white moth has led to drug resistance and many environmental problems.Therefore,it is very necessary to study the green control methods of H.cunea.Combined with the reported key genes CYP4Gs involved in CHC synthesis in the previous study of this study,the gene sequences of HcCYP4G1,HcCYP4G2 and HcCYP4G3 key genes of epidermal hydrocarbon synthase were obtained by screening from the transcriptome database of H.cunea.In this experiment,the spatiotemporal expression pattern of HcCYP4Gs gene in the larvae of H.cunea was explored,and the role of HcCYP4Gs gene in the drought tolerance physiology of H.cunea was clarified by RNAi technology and drying stress bioassay.To provide exploratory research for pest control methods based on the inhibition of insect CHC synthesis,and to provide theoretical basis for the development of biological preparations of H.cunea.The research conclusions are as follows:1.The results of bioinformatics analysis of the HcCYP4Gs gene of H.cunea showed that the amino acid sequence of HcCYP4Gs had the highest similarity with the A.plantaginis,and the amino acid sequence similarity with the Lepidoptera Spodoptera insects,and Low amino acid sequence similarity with other order insects CYP4G subfamily,and the results were consistent with the morphological taxonomy.The predicted molecular weights of the HcCYP4Gs gene-encoded proteins were 63.03 k Da,64.32 k Da,64.83 k Da,and the p I were8.59,8.06,and 8.31,respectively.The two most abundant amino acids in the polypeptide chain of HcCYP4Gs-encoded proteins were Leu and Lys.All three proteins were unstable and soluble proteins.HcCYP4Gs have no signal peptide,and HcCYP4G1 and HcCYP4G3 have a transmembrane region with 41,57,and 40 potential phosphorylation sites,respectively.2.The study of the spatiotemporal expression of HcCYP4Gs gene in H.cunea showed that there was no significant difference in the expression levels of HcCYP4G1 and HcCYP4G3 at different ages in the 4th instar larvae.The expression of HcCYP4G2 on the 3rd day and 4th day was significantly lower than that on the 1th day and 2nd day.The expression of HcCYP4Gs in 5th instar larvae at different ages was not significantly different.HcCYP4Gs were expressed in all parts and tissues of 5th instar larvae.HcCYP4G1 and HcCYP4G2 were most expressed in the abdomen,and HcCYP4G3 was the highest in the head.The results showed that the expression levels of HcCYP4G1 and HcCYP4G2 in the 4th and 5th instar larvae were much higher than those of HcCYP4G3.3.The results of the RNAi study of the 4th instar of H.cunea showed that after injection of ds HcCYP4G1 in 4th instar larvae,HcCYP4G1 was significantly inhibited on the 1st,2nd and 3rd days.The silencing efficiencies were 69.30%,65.38%and 78.03%on days 1,2 and 3after injection,respectively.And it has significantly reduced the expression level of HcCYP4G1.After injection of ds HcCYP4G2,it has no inhibitory effect on the HcCYP4G2gene after testing,and the specific reasons need to be further studied.The results showed that the 4th instar larvae of H.cunea had a good response to ds HcCYP4G1,and HcCYP4G1 could be a new target for genetic control of H.cunea.4.The results of the study on the drought tolerance of the 4th instar larvae after RNAi showed that the mortality of larvae injected with ds HcCYP4G1 at RH=25%was significantly higher than that of larvae injected with ds GFP and dd H₂O,and the mortality of larvae injected with ds HcCYP4G2 was not different from that of larvae injected with ds GFP and dd H₂O.The larvae that died after being treated in a dry environment suffered from severe water loss,the body length was significantly smaller than that of the normally grown larvae,and the larvae were obviously shriveled and blackened.The results combined with RNAi also indicated that HcCYP4G1 could serve as a good target for the genetic control of H.cunea.更多还原