小菜蛾innexin基因家族鉴定与innexin 2表达抑制

发布时间：2018-05-29 09:47

本文选题：小菜蛾 + 间隙连接蛋白　；参考：《福建农林大学》2016年硕士论文

【摘要】：小菜蛾Plutella xylostella (L.),是世界性十字花科植物的重要害虫,每年造成巨大的经济损失,且已经几乎对所有农药都产生了抗药性,亟待寻求更加有效的防治措施。间隙连接是细胞间通过细胞质膜相互联系,维持组织结构协调和完整性的联系结构,若阻断此连接,可以阻止细胞间的交流,影响生物体的生长发育,为制定有效的小菜蛾防治策略提供新思路。以小菜蛾基因组数据立库,与已知的innexin基因进行比对,筛选出了9个候选innexin基因,分别为Px009151、Px009152、Px009153、Px009154、Px009155、 Px009208、Px013282、Px013283和Px016319,长度分别为1080,1257,1086,1112,315,1161,1020,978,1113 bp；设计引物,经PCR扩增,克隆并测序获得预测的9个innexin基因CDS序列；将小菜蛾innexin蛋白家族与其他已知5种昆虫的innexin蛋白家族成员以最大似然法构建进化树,分析小菜蛾此蛋白家族成员的进化关系,结果表明Px016319蛋白序列与昆虫保守的zpg蛋白序列高度相似；9个innexin基因在不同发育阶段和组织的表达模式：Px016319、Px009151以及Px009208较其它innexin基因的相对表达量高；Px016319在卵和雌性成虫中大量表达,且在卵巢相对高表达,与果蝇中同属innexin基因家族的zpg具有相类似的表达模式；Px009151与Px009208在所有发育阶段均有表达,在卵、蛹和成虫中相对高表达。表达模式分析的结果表明小菜蛾Px016319可能参与生殖细胞的分化与形成,参与雌雄成虫的繁殖行为；进一步明确Px016319与其它昆虫zpg在功能上的一致性与分化,对其在小菜蛾中的表达进行抑制,探索并建立了利用CRISPR/Cas9系统在小菜蛾细胞系中敲除Px016319的方法,结果显示敲除后该基因的表达量显著降低；同时在小菜蛾雌蛹和雄蛹中对Px016319进行RNAi,干扰后该基因的表达量降低。综上所述,推测在小菜蛾中Px016319可能具有和zpg相似的功能,可作为防控小菜蛾的干预靶基因；结合RNAi技术和基因敲除技术抑制该基因在小菜蛾中表达,研究结果可为开发小菜蛾的遗传防治新策略提供科学依据。
[Abstract]:Plutella xylostella Plutella xylostella L.is an important pest of cruciferous plants in the world. It causes huge economic losses every year and has become resistant to almost all pesticides. It is urgent to seek more effective control measures. Intercellular junction is the connection between cells through the cytoplasmic membrane to maintain the coordination and integrity of the tissue structure. If this connection is blocked, it can prevent the communication between cells and affect the growth and development of organisms. To provide a new idea for the effective control strategy of Plutella xylostella. By comparing the genomic data of Plutella xylostella with known innexin genes, nine candidate innexin genes were screened out, namely Px009151Px009152Px009153Px009155, Px009208Px013282Px013283 and Px016319. the length of the primer was 108611123151061101109781313 BP, respectively. The primers were amplified by PCR. The predicted CDS sequences of 9 innexin genes were cloned and sequenced, and the phylogenetic relationship between the innexin family of Plutella xylostella and the innexin family members of other known five species of insects was constructed by maximum likelihood method, and the phylogenetic relationship of the members of the innexin protein family of Plutella xylostella was analyzed. The results showed that the sequence of Px016319 protein was highly similar to that of zpg protein conserved by insects, the expression patterns of 9 innexin genes in different developmental stages and tissues were: Px016319Px009151, and the relative expression of Px009208 was higher than that of other innexin genes. Px016319 was highly expressed in eggs and female adults. The expression patterns of Px009151 and Px009208 were similar to those of innexin gene family in Drosophila melanogaster. Px009151 and Px009208 were highly expressed in eggs, pupae and adults at all developmental stages. The results of expression pattern analysis showed that Px016319 might be involved in the differentiation and formation of germ cells and the reproductive behavior of female and male adults, and the functional consistency and differentiation between Px016319 and other insect zpg were further clarified. The method of knockout Px016319 using CRISPR/Cas9 system in Plutella xylostella cell line was explored and established. The results showed that the expression of the gene decreased significantly after knockout. At the same time, the expression of Px016319 in female and male pupae of Plutella xylostella was decreased after interference. In conclusion, Px016319 may have similar function to zpg in diamondback moth and can be used as an intervention target gene for controlling Plutella xylostella, and RNAi and gene knockout technology can inhibit the gene expression in Plutella xylostella. The results can provide scientific basis for developing new genetic control strategies of Plutella xylostella.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433.4

【相似文献】