【摘要】：鱗翅目昆蟲中的大多數(shù)種類都是植食性的農(nóng)林害蟲,常年對(duì)農(nóng)林業(yè)造成巨大的損失。目前防治此類害蟲仍以化學(xué)防治為主,仍然缺乏更為合理的防治方法,其中的一個(gè)原因在于此類害蟲的分子機(jī)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機(jī)理的研究。苜蓿銀紋夜蛾核型多角體病毒(Ac MNPV)是桿狀病毒中研究得最深入的一種,其宿主主要是鱗翅目昆蟲幼蟲。為更方便和高效地研究鱗翅目昆蟲的分子機(jī)理,實(shí)驗(yàn)嘗試對(duì)Ac MNPV復(fù)制必須基因進(jìn)行改造,使其復(fù)制受到調(diào)控,從而將Ac MNPV開發(fā)成能夠攜帶外源基因進(jìn)行過表達(dá)、基因沉默和加標(biāo)簽的病毒表達(dá)載體。實(shí)驗(yàn)選取一個(gè)Ac MNPV復(fù)制必須基因DNA聚合酶基因(dnapol),進(jìn)一步驗(yàn)證了其功能,證實(shí)其與復(fù)制密切相關(guān),之后通過Red重組系統(tǒng)將dnapol的一段序列敲除,同時(shí)插入誘導(dǎo)表達(dá)系統(tǒng)Tet-on的調(diào)節(jié)表達(dá)元件rt TA2s-M2。利用Bac-to-Bac系統(tǒng)將處于Ptight(Tet-on系統(tǒng)中的誘導(dǎo)型啟動(dòng)子)控制之下的dnapol重新插入Ac MNPV基因組中的多角體位點(diǎn)(polyhedrin,polh),同時(shí)插入增強(qiáng)型綠色熒光蛋白標(biāo)記基因(EGFP)完成載體的構(gòu)建,將構(gòu)建好的重組Ac MNPV基因組轉(zhuǎn)染Sf9細(xì)胞,在細(xì)胞水平檢測其復(fù)制可控性。實(shí)驗(yàn)構(gòu)建了多個(gè)不同的載體,結(jié)果表明,將dnapol序列重新插入polh位點(diǎn)之后,在重組Ac MNPV的DNA有一定量復(fù)制的基礎(chǔ)之上,dnapol位點(diǎn)的序列會(huì)回復(fù)為野生型序列,而polh位點(diǎn)的dnapol序列突變之后就可以解決這個(gè)問題,實(shí)驗(yàn)同時(shí)說明載體的背景復(fù)制水平與rt TA2s-M2的表達(dá)量密切相關(guān)。最終,實(shí)驗(yàn)構(gòu)建了polh位點(diǎn)為突變型dnapol的載體,同時(shí)通過更換啟動(dòng)子,降低了rt TA2s-M2的表達(dá)量。將載體轉(zhuǎn)染Sf9細(xì)胞之后,加入誘導(dǎo)劑強(qiáng)力霉素(Dox)的細(xì)胞表現(xiàn)出更強(qiáng)的綠色熒光,并且可以檢測到數(shù)目更多的重組載體的DNA,這說明重組載體DNA的復(fù)制可受到Dox的調(diào)控,然而重組病毒的滴度并沒有增加,這或許表明盡管許多DNA病毒,包括Ac MNPV,它們的包裝和DNA復(fù)制之間是緊密協(xié)調(diào)的,然而兩者之間并不是線性關(guān)系,存在著一定的緩沖。同時(shí)我們的系統(tǒng)中仍然存在一定的背景表達(dá),也即是在沒有Dox的情況下,載體仍然有一定量的復(fù)制,需進(jìn)一步的優(yōu)化。復(fù)制可控型Ac MNPV載體可作為一種研究鱗翅目昆蟲分子機(jī)理的工具,加快其分子機(jī)理研究進(jìn)程,從而對(duì)鱗翅目害蟲開發(fā)出更合理的防治方法,對(duì)農(nóng)林業(yè)生產(chǎn)具有重要意義。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4

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