复制可控型基因表达载体AcMNPV构建的探索
发布时间：2018-12-20 10:57
【摘要】：鳞翅目昆虫中的大多数种类都是植食性的农林害虫,常年对农林业造成巨大的损失。目前防治此类害虫仍以化学防治为主,仍然缺乏更为合理的防治方法,其中的一个原因在于此类害虫的分子机理未得以深入的研究,而缺乏高效的研究工具和方法又限制了其分子机理的研究。苜蓿银纹夜蛾核型多角体病毒(Ac MNPV)是杆状病毒中研究得最深入的一种,其宿主主要是鳞翅目昆虫幼虫。为更方便和高效地研究鳞翅目昆虫的分子机理,实验尝试对Ac MNPV复制必须基因进行改造,使其复制受到调控,从而将Ac MNPV开发成能够携带外源基因进行过表达、基因沉默和加标签的病毒表达载体。实验选取一个Ac MNPV复制必须基因DNA聚合酶基因(dnapol),进一步验证了其功能,证实其与复制密切相关,之后通过Red重组系统将dnapol的一段序列敲除,同时插入诱导表达系统Tet-on的调节表达元件rt TA2s-M2。利用Bac-to-Bac系统将处于Ptight(Tet-on系统中的诱导型启动子)控制之下的dnapol重新插入Ac MNPV基因组中的多角体位点(polyhedrin,polh),同时插入增强型绿色荧光蛋白标记基因(EGFP)完成载体的构建,将构建好的重组Ac MNPV基因组转染Sf9细胞,在细胞水平检测其复制可控性。实验构建了多个不同的载体,结果表明,将dnapol序列重新插入polh位点之后,在重组Ac MNPV的DNA有一定量复制的基础之上,dnapol位点的序列会回复为野生型序列,而polh位点的dnapol序列突变之后就可以解决这个问题,实验同时说明载体的背景复制水平与rt TA2s-M2的表达量密切相关。最终,实验构建了polh位点为突变型dnapol的载体,同时通过更换启动子,降低了rt TA2s-M2的表达量。将载体转染Sf9细胞之后,加入诱导剂强力霉素(Dox)的细胞表现出更强的绿色荧光,并且可以检测到数目更多的重组载体的DNA,这说明重组载体DNA的复制可受到Dox的调控,然而重组病毒的滴度并没有增加,这或许表明尽管许多DNA病毒,包括Ac MNPV,它们的包装和DNA复制之间是紧密协调的,然而两者之间并不是线性关系,存在着一定的缓冲。同时我们的系统中仍然存在一定的背景表达,也即是在没有Dox的情况下,载体仍然有一定量的复制,需进一步的优化。复制可控型Ac MNPV载体可作为一种研究鳞翅目昆虫分子机理的工具,加快其分子机理研究进程,从而对鳞翅目害虫开发出更合理的防治方法,对农林业生产具有重要意义。
[Abstract]:Most species of Lepidoptera insects are herbivorous pests, which cause great losses to agriculture and forestry. At present, chemical control is still the main method to control such pests, and there is still a lack of more reasonable control methods. One of the reasons is that the molecular mechanism of these pests has not been further studied. However, the lack of efficient research tools and methods limits the study of its molecular mechanism. The nuclear polyhedrosis virus (Ac MNPV) of Spodoptera sativa is one of the most widely studied baculoviruses, and its host is mainly Lepidoptera larvae. In order to study the molecular mechanism of Lepidoptera insects more conveniently and efficiently, the experiment attempted to modify the necessary genes of Ac MNPV replication, so that the replication of Lepidoptera could be regulated, so that Ac MNPV could be developed to carry foreign genes for overexpression. Gene silencing and tagging virus expression vector. A DNA polymerase gene (dnapol), a necessary gene for Ac MNPV replication, was selected to further verify its function, which was confirmed to be closely related to replication. Then, a sequence of dnapol was knocked out by Red recombination system. Simultaneous insertion of the regulatory expression element rt TA2s-M2. of the inducible expression system Tet-on The dnapol under the control of Ptight (inducible promoter in Tet-on system) was inserted into the polyhedron (polyhedrin,polh) of the Ac MNPV genome by Bac-to-Bac system. At the same time, an enhanced green fluorescent protein labeled gene (EGFP) was inserted to complete the construction of the vector. The constructed recombinant Ac MNPV genome was transfected into Sf9 cells and its replication controllability was detected at the cell level. Several different vectors were constructed. The results showed that after the dnapol sequence was inserted into the polh site, the sequence of the dnapol site returned to the wild-type sequence on the basis of a certain amount of replication of the DNA of the recombinant Ac MNPV. The mutation of dnapol sequence at polh site can solve this problem. The experiment also shows that the level of background replication of the vector is closely related to the expression of rt TA2s-M2. Finally, a mutant dnapol vector with polh locus was constructed, and the expression of rt TA2s-M2 was reduced by changing the promoter. After transfection of the vector into Sf9 cells, the cells added with doxycycline (Dox) showed stronger green fluorescence, and more DNA, of the recombinant vector could be detected, which indicated that the replication of the recombinant vector DNA could be regulated by Dox. However, the titer of recombinant viruses did not increase, which may suggest that although many DNA viruses, including Ac MNPV, are closely coordinated between their packaging and DNA replication, the relationship between them is not linear and there is a buffer between them. At the same time, there is still a certain background expression in our system, that is, in the absence of Dox, the vector still has a certain amount of replication, which needs further optimization. Replication of controllable Ac MNPV vector can be used as a tool to study the molecular mechanism of Lepidoptera insects and accelerate the process of molecular mechanism research, thus developing a more reasonable control method for Lepidoptera pests, which is of great significance to agricultural and forestry production.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4
本文编号：2387899
[Abstract]:Most species of Lepidoptera insects are herbivorous pests, which cause great losses to agriculture and forestry. At present, chemical control is still the main method to control such pests, and there is still a lack of more reasonable control methods. One of the reasons is that the molecular mechanism of these pests has not been further studied. However, the lack of efficient research tools and methods limits the study of its molecular mechanism. The nuclear polyhedrosis virus (Ac MNPV) of Spodoptera sativa is one of the most widely studied baculoviruses, and its host is mainly Lepidoptera larvae. In order to study the molecular mechanism of Lepidoptera insects more conveniently and efficiently, the experiment attempted to modify the necessary genes of Ac MNPV replication, so that the replication of Lepidoptera could be regulated, so that Ac MNPV could be developed to carry foreign genes for overexpression. Gene silencing and tagging virus expression vector. A DNA polymerase gene (dnapol), a necessary gene for Ac MNPV replication, was selected to further verify its function, which was confirmed to be closely related to replication. Then, a sequence of dnapol was knocked out by Red recombination system. Simultaneous insertion of the regulatory expression element rt TA2s-M2. of the inducible expression system Tet-on The dnapol under the control of Ptight (inducible promoter in Tet-on system) was inserted into the polyhedron (polyhedrin,polh) of the Ac MNPV genome by Bac-to-Bac system. At the same time, an enhanced green fluorescent protein labeled gene (EGFP) was inserted to complete the construction of the vector. The constructed recombinant Ac MNPV genome was transfected into Sf9 cells and its replication controllability was detected at the cell level. Several different vectors were constructed. The results showed that after the dnapol sequence was inserted into the polh site, the sequence of the dnapol site returned to the wild-type sequence on the basis of a certain amount of replication of the DNA of the recombinant Ac MNPV. The mutation of dnapol sequence at polh site can solve this problem. The experiment also shows that the level of background replication of the vector is closely related to the expression of rt TA2s-M2. Finally, a mutant dnapol vector with polh locus was constructed, and the expression of rt TA2s-M2 was reduced by changing the promoter. After transfection of the vector into Sf9 cells, the cells added with doxycycline (Dox) showed stronger green fluorescence, and more DNA, of the recombinant vector could be detected, which indicated that the replication of the recombinant vector DNA could be regulated by Dox. However, the titer of recombinant viruses did not increase, which may suggest that although many DNA viruses, including Ac MNPV, are closely coordinated between their packaging and DNA replication, the relationship between them is not linear and there is a buffer between them. At the same time, there is still a certain background expression in our system, that is, in the absence of Dox, the vector still has a certain amount of replication, which needs further optimization. Replication of controllable Ac MNPV vector can be used as a tool to study the molecular mechanism of Lepidoptera insects and accelerate the process of molecular mechanism research, thus developing a more reasonable control method for Lepidoptera pests, which is of great significance to agricultural and forestry production.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4
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