孕酮介导的卵母细胞成熟信号通路在棉蚜生殖方式转换中的作用研究

发布时间：2019-04-27 13:12

【摘要】：棉蚜(Aphis gossypii Glover)是重要的农业害虫,在我国北方它能随季节的变化进行周期性的孤雌生殖和有性生殖,即春夏以孤雌蚜进行无性生殖而大量繁殖,秋天产生性母,进而产生性雌,与雄蚜交配产卵,以卵过冬。虽然棉蚜的全生活史及生殖型转换(或生殖可塑性)方式已经明了,但其中的分子机制一直不清楚。目前研究蚜虫生殖型转换的一个模式是豌豆蚜(Acyrthosiphon pisum Harris),与豌豆蚜不同,棉蚜有明显的性母并且转换寄主植物。之前我们实验室利用RNA-seq测定了棉蚜孤雌、性母、性雌三种生殖型的差异基因表达谱,通过GO聚类和KEGG通路分析富集出了光传导、孕酮介导的卵母细胞成熟、内分泌调节、以及表皮构成信号通路。本论文选择孕酮介导的卵母细胞成熟信号通路和光传导信号通路进行了初步研究。首先利用qRT-PCR对孕酮介导的卵母细胞成熟信号通路中的b ubl、 ccna2、polo、plkl 和 mad2基因进行了表达验证,bub1、ccna2和polo在孤雌中的表达水平显著高于性母。通过注射dsRNA对bub1、ccna2和polo进行了初步的RNAi实验,发现bubl或ccna2RNAi能够影响孤雌蚜的卵巢发育,但是否影响生殖型转换目前还不能确定。同时,也通过qRT-PCR测定了光传导信号通路中的ninac、opsin、arr2、GNB2、Trp和ANA基因从孤雌向性母转化过程中的时序表达模式,发现ninac、opsin、arr2和GNB2的表达水平在性母4龄若虫期显著上升。棉蚜个体小、体壁薄、体液多,有关棉蚜RNAi的报道很少,尚没有注射和饲喂dsRNA的比较实验。本论文对注射和饲喂dsR NA进行了系统的比较研究,包括注射位点、注射剂量、饲喂剂量、组织摄取、干扰效率等,对注射和饲喂dsRNA的优缺点进行了讨论。实验结果表明,注射位点在中胸与后胸之间,注射剂量10ng,饲喂浓度400 ng/μ l为宜。注射和饲喂的荧光(Cy3)标记siRNA都能够在卵巢和肠中检测到。
[Abstract]:Cotton aphid (Aphis gossypii Glover) is an important agricultural pest. In northern China, it can periodically parthenogenesis and sexual reproduction with seasonal changes, that is, in spring and summer, parthenogenetic aphid propagates in large numbers, and in autumn it produces sexual mother. It then produces a sexual female, mating with the male aphid to spawn and wintering with the eggs. Although the whole life history of APHIS gossypii (APHIS gossypii) and the mode of reproductive transformation (or reproductive plasticity) have been known, the molecular mechanism is not clear. At present, one of the models to study the transformation of aphid reproductive type is that the aphid (Acyrthosiphon pisum Harris), is different from the pea aphid, the cotton aphid has obvious sexual mother and the host plant is changed. Previously, we used RNA-seq to determine the differential gene expression profiles of parthenogenetic, sexual and sexual aphids of APHIS gossypii. Light transduction, progesterone-mediated oocyte maturation and endocrine regulation were enriched by GO clustering and KEGG pathway analysis. And the epidermis constitutes a signaling pathway. In this paper, progesterone-mediated oocyte maturation signal pathway and light transduction signal pathway were studied. At first, the expression of b-ubl, ccna2,polo,plkl and mad2 genes in progesterone-mediated oocyte maturation signaling pathway was verified by qRT-PCR. The expression level of bub1,ccna2 and polo in parthenogenetic cells was significantly higher than that in parthenogenetic women. The preliminary RNAi tests of bub1,ccna2 and polo by injecting dsRNA showed that bubl or ccna2RNAi could affect the ovarian development of parthenogenetic aphid, but it was not clear whether bubl or ccna2RNAi affected the transformation of reproductive type. At the same time, the temporal expression patterns of ninac,opsin,arr2,GNB2,Trp and ANA genes in the light transduction signaling pathway from parthenogenetic to mother were also determined by qRT-PCR. It was found that ninac,opsin, was found to be in the process of transformation from parthenogenesis to parthenogenesis. The expression levels of arr2 and GNB2 increased significantly at the 4th instar nymph stage of the sexual mother. Cotton aphid (APHIS gossypii) is small in size, thin in body wall and abundant in body fluid. There are few reports about RNAi in cotton aphid, and there is no comparative experiment of injecting and feeding dsRNA. In this paper, a systematic comparative study of injection and feeding of dsRNA was carried out, including injection site, injection dose, feeding dose, tissue uptake, interference efficiency, etc. The advantages and disadvantages of injection and feeding of dsRNA were discussed. The results showed that the injection site was between the middle chest and the posterior chest, the injection dose was 10 ng, and the feeding concentration was 400 ng/ 渭 l. Both injected and fed Cy3 labeled siRNA can be detected in ovaries and intestines.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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