家蚕核型多角体病毒lef-11基因RNA干扰策略的优化

发布时间：2018-01-31 02:54

  本文关键词： 家蚕 家蚕核型多角体病毒 RNA干扰 lef- 载体优化 启动子　出处：《微生物学报》2016年10期 　论文类型：期刊论文

【摘要】：【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)lef-11基因作为杆状病毒高度保守的晚期表达因子之一,对病毒晚期基因的表达极其重要。因此对lef-11基因进行有效RNA干扰将提高宿主细胞对BmNPV的抗性。【方法】本文基于经典的sh-RNA-loop骨架及家蚕内源性的miRNA骨架,构建以BmNPV lef-11基因为靶标的干扰载体pIZ-shRNA1、pIZ-shRNA2和pIZ-Ds RedamiR279;分别构建基于A4、IE1、IE1-295、IE2、IE2-339、hr3A4和hsp70启动子驱动的干扰载体,用以评价不同启动子驱动的干扰载体的抗病毒效果,并对干扰载体进行优化。【结果】首先,本文通过比较miRNA-based和shRNA-based RNAi载体对同一靶基因同一位点的干扰效率,发现pIZ-Ds Red-amiR279对BmNPV lef11基因的干扰效率超过90%,远优于shRNA-based RNAi载体的干扰效果;其次,通过对干扰载体进行优化,发现IE1启动子的效果最优,由其驱动的pIZ-Ds Red-milef11干扰载体也是本研究中的最优干扰载体,对病毒的增殖抑制效果最为明显。【结论】对目的基因的干扰效果并非启动子的活性越高、miRNA积累得越多就越好,只有综合考虑干扰片段的选择、启动子的活性以及靶基因自身的功能等多方面的因素,才能提高干扰效率,达到干扰目的。
[Abstract]:[objective] Bombyx mori nucleopolyhedrovirus. BmNPV)lef-11 gene is one of the highly conserved late expression factors of baculovirus. It is very important to express the advanced gene of virus. Therefore, the effective RNA interference of lef-11 gene will enhance the resistance of host cells to BmNPV. [methods] this paper is based on the classical sh-RNA-. Loop skeleton and endogenous miRNA skeleton of Bombyx mori. The interference vectors pIZ-shRNA1pIZ-shRNA2 and pIZ-Ds RedamiR279 were constructed with BmNPV lef-11 as the target. The interference vectors were constructed based on A4CIE1 (IE1-295) IE2-339G3A4 and hsp70 promoter-driven interference vectors, respectively. Used to evaluate the antiviral effect of different promoter driven interference vectors and optimize the interference vectors. [results] first. In this paper, we compared the interference efficiency of miRNA-based and shRNA-based RNAi vectors to the same target gene at the same site. It was found that the interference efficiency of pIZ-Ds Red-amiR279 to BmNPV lef11 gene was more than 90%. It is much better than the interference effect of shRNA-based RNAi carrier. Secondly, by optimizing the interference vector, it is found that the IE1 promoter has the best effect, and the pIZ-Ds Red-milef11 interference vector driven by the interference vector is also the optimal interference carrier in this study. The inhibitory effect on virus proliferation is most obvious. [conclusion] the interference effect on the target gene is not the higher the promoter activity is, the more miRNA accumulation is, only the selection of interference fragments is considered synthetically. The activity of promoter and the function of target gene can improve the interference efficiency and achieve the interference purpose.
【作者单位】： 西南大学家蚕基因组生物学国家重点实验室;西南大学农业部家蚕功能基因组和生物学重点实验室;
【基金】：国家自然科学基金(31472152) 国家蚕桑产业技术体系(CARS-22)~~
【分类号】：Q78;S884.51
【正文快照】： 蚕丝业作为我国传统的优势产业,在国际市场上具有主导地位。然而,蚕病的发生每年都会给蚕丝业造成巨大的经济损失。其中,家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)是蚕业生产上危害最严重的病原微生物之一,其引起的家蚕核型多角体病占蚕病损失70%左右。因，

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