茶尺蠖几丁质合成酶基因克隆及其双链干涉重组病毒毒力研究
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摘要
茶尺蠖等鳞翅目害虫是茶园的重要害虫,传统方法使用化学农药进行害虫防治导致成品茶农药残留等一系列问题；NPV病毒(Nuclear polyhedrosis virus)是专一防治茶尺蠖的重要生物农药,但因其毒力和有效期等局限性而未大范围的应用。针对这一问题,本研究以茶尺蠖几丁质合成酶(Chitin synthase, CS)CS1基因为基础,借助RNAi技术和NPV病毒具有外源基因插入位点表达外源基因的特性,克隆了CSl基因全长序列,构建了包含CS保守基因双链干涉序列的重组NPV病毒,并对其毒性进行了评估。为今后开发高毒力的NPV病毒生物农药提供了一个新思路。同时,本研究还克隆了一部分茶尺蠖其他功能基因,为下一步建立茶尺蠖功能基因双链干涉重组病毒库奠定基础。
利用RT-PCR技术,克隆了茶尺蠖CS1基因全长序列,共5496bp,其中编码区长度为4692bp。根据该基因编码的1563个氨基酸序列进行预测分析,初步了解了茶尺蠖CS1基因的催化和代谢机理。明确了茶尺蠖CS1基因的11个保守位点；证明CS1基因的表达在茶尺蠖各个龄期对消化系统、呼吸系统及表皮等重要器官和组织的形成具有重要影响,为确定重组NPV病毒防治茶尺蠖适宜防治期的确定提供了理论依据。
利用Bac-to-Bac system的NPV病毒外源基因表达系统,将构包含茶尺蠖CS1保守基因双链干涉序列转化NPV病毒,获得重组NPV病毒。重组NPV病毒以SF9细胞为寄主进行繁殖扩增,获得重组NPV病毒生物农药制备的适宜条件。证明被重组NPV病毒感染的SF9细胞的CS基因表达受到抑制,阐明重组NPV病毒可能通过抑制昆虫CS基因表达和几丁质生物合成,从而达到控制害虫的目的。
以重组NPV病毒注射和饲喂茶尺蠖幼虫,证明重组NPV病毒能够有效抑制茶尺蠖CS基因的表达,阻碍几丁质的生物合成,导致茶尺蠖幼虫取食、蜕皮、羽化等生理机能障碍。重组NPV病毒的杀虫效率明显高于野生型NPV病毒,尤其对低龄茶尺蠖；筛选获得高毒力的重组病毒dsCS2;用其饲喂幼虫,第1天的死亡率即达到65%,前3天的累计死亡率为95%。研究结果表明,田间使用重组病毒控制茶尺蠖时,在低龄时使用效果较好。另外对蚕和石榴尺蠖进行重组病毒注射和饲喂处理,证明dsCS2重组病毒对蚕和石榴尺蠖也具有一定毒杀作用。据此认为,利用dsCS2重组病毒防治茶园茶尺蠖,还有可能对其他鳞翅目等具有较高CS基因同源性的害虫具有控制效果。
Ectropis obliqua Prout is a major pest in tea fields. Application of chemical pesticides in controlling the pests makes the pesticide residues in tea hang-up. Nucleopolyhedrovirus (NPV) is an effective biological pesticide to control the E. obliqua in tea field. However, short term and low efficacy limited the wide application of NPV in tea field. In this study, a full-length sequence of chitin synthase-1 (CS-1) cDNA from E. obliqua was cloned. A 192 bp conserved domain cloned from E. obliqua CS-1 gene was used to construct recombined Autographa californica M nucleopolyhedrovirus (AcMNPV) using double-stranded RNA interference (dsRNAi) method. The toxicity assessment of the recombinant showed that the CS-1 dsRNAi mediated by the NPV is peomising for development of alternative bio-pesticide to control the tea pest E. obliqua. Some other function genes were cloned for E. obliqua for further research.
The cDNA sequence encoding the CS-1 and its expression pattern during development of E. obliqua were investigated. The CS-1 was expressed during the development of E. obliqua., and strong expressions were found in the third and fourth instar larvae, during which the growth rate of E. obliqua larvae was the rapidest. Catalysis model of CS enzyme in E. obliqua was also predicted according to the specific motifs and topological structure prediction of the protein. This study provided an important information for the further research on development of RNAi technology to control E. obliqua.
The recombined AcMNPV was constructed with dsRNAi method using Bac-to-Bac system. The recombined AcMNPV virus could propagate in host cells sf9 and this can be used to develop an alternative method to control the E. obliqua in tea field. It is considered that the recombined AcMNPV virus control the E. obliqua by inhibiting the CS gene expression and chitin synthesis of the pest.
Injecting and feeding tests using the recombined AcMNPV virus showed that the growth and development of both larvae and pupae of E. oblique were inhibited and the virus efficacy of the recombined AcMNPV on E. obliqua larvae was significantly higher than that of wild NPV. A dsCS2 recombined AcMNPV with the highest virus efficacy was screened from the tested viruses. The cumulative mortality of E. obliqua Ist instar larvae who were fed with the dsCS2 recombined AcMNPV virus was 95% on third day. It is considered that the dsRNAi mediated inhibition of the expression of gene CS in the pests. The study also showed that dsCS2 could infect other insects such as Bombyx mori and pomegranate-cankerworm. It shows that a foreign dsRNAi gene from one species can interference CS gene expression of a different species. The bio-pesticides developed by dsRNAi may have a broad-spectrum in pest-control.
利用RT-PCR技术,克隆了茶尺蠖CS1基因全长序列,共5496bp,其中编码区长度为4692bp。根据该基因编码的1563个氨基酸序列进行预测分析,初步了解了茶尺蠖CS1基因的催化和代谢机理。明确了茶尺蠖CS1基因的11个保守位点；证明CS1基因的表达在茶尺蠖各个龄期对消化系统、呼吸系统及表皮等重要器官和组织的形成具有重要影响,为确定重组NPV病毒防治茶尺蠖适宜防治期的确定提供了理论依据。
利用Bac-to-Bac system的NPV病毒外源基因表达系统,将构包含茶尺蠖CS1保守基因双链干涉序列转化NPV病毒,获得重组NPV病毒。重组NPV病毒以SF9细胞为寄主进行繁殖扩增,获得重组NPV病毒生物农药制备的适宜条件。证明被重组NPV病毒感染的SF9细胞的CS基因表达受到抑制,阐明重组NPV病毒可能通过抑制昆虫CS基因表达和几丁质生物合成,从而达到控制害虫的目的。
以重组NPV病毒注射和饲喂茶尺蠖幼虫,证明重组NPV病毒能够有效抑制茶尺蠖CS基因的表达,阻碍几丁质的生物合成,导致茶尺蠖幼虫取食、蜕皮、羽化等生理机能障碍。重组NPV病毒的杀虫效率明显高于野生型NPV病毒,尤其对低龄茶尺蠖；筛选获得高毒力的重组病毒dsCS2;用其饲喂幼虫,第1天的死亡率即达到65%,前3天的累计死亡率为95%。研究结果表明,田间使用重组病毒控制茶尺蠖时,在低龄时使用效果较好。另外对蚕和石榴尺蠖进行重组病毒注射和饲喂处理,证明dsCS2重组病毒对蚕和石榴尺蠖也具有一定毒杀作用。据此认为,利用dsCS2重组病毒防治茶园茶尺蠖,还有可能对其他鳞翅目等具有较高CS基因同源性的害虫具有控制效果。
Ectropis obliqua Prout is a major pest in tea fields. Application of chemical pesticides in controlling the pests makes the pesticide residues in tea hang-up. Nucleopolyhedrovirus (NPV) is an effective biological pesticide to control the E. obliqua in tea field. However, short term and low efficacy limited the wide application of NPV in tea field. In this study, a full-length sequence of chitin synthase-1 (CS-1) cDNA from E. obliqua was cloned. A 192 bp conserved domain cloned from E. obliqua CS-1 gene was used to construct recombined Autographa californica M nucleopolyhedrovirus (AcMNPV) using double-stranded RNA interference (dsRNAi) method. The toxicity assessment of the recombinant showed that the CS-1 dsRNAi mediated by the NPV is peomising for development of alternative bio-pesticide to control the tea pest E. obliqua. Some other function genes were cloned for E. obliqua for further research.
The cDNA sequence encoding the CS-1 and its expression pattern during development of E. obliqua were investigated. The CS-1 was expressed during the development of E. obliqua., and strong expressions were found in the third and fourth instar larvae, during which the growth rate of E. obliqua larvae was the rapidest. Catalysis model of CS enzyme in E. obliqua was also predicted according to the specific motifs and topological structure prediction of the protein. This study provided an important information for the further research on development of RNAi technology to control E. obliqua.
The recombined AcMNPV was constructed with dsRNAi method using Bac-to-Bac system. The recombined AcMNPV virus could propagate in host cells sf9 and this can be used to develop an alternative method to control the E. obliqua in tea field. It is considered that the recombined AcMNPV virus control the E. obliqua by inhibiting the CS gene expression and chitin synthesis of the pest.
Injecting and feeding tests using the recombined AcMNPV virus showed that the growth and development of both larvae and pupae of E. oblique were inhibited and the virus efficacy of the recombined AcMNPV on E. obliqua larvae was significantly higher than that of wild NPV. A dsCS2 recombined AcMNPV with the highest virus efficacy was screened from the tested viruses. The cumulative mortality of E. obliqua Ist instar larvae who were fed with the dsCS2 recombined AcMNPV virus was 95% on third day. It is considered that the dsRNAi mediated inhibition of the expression of gene CS in the pests. The study also showed that dsCS2 could infect other insects such as Bombyx mori and pomegranate-cankerworm. It shows that a foreign dsRNAi gene from one species can interference CS gene expression of a different species. The bio-pesticides developed by dsRNAi may have a broad-spectrum in pest-control.
引文
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