橘小实蝇精子发生相关基因功能研究

发布时间：2018-07-22 12:00

【摘要】：橘小实蝇Bactrocera dorsalis(Hendel)是一种世界性分布、具有重要经济意义的实蝇类害虫,食性杂,繁殖力强,每年给果蔬业造成重大经济损失。现阶段呈现发生范围广、危害加重、连年大发生的特点,原有的以化学防治为主的防控措施已不能控制橘小实蝇的扩散和危害,迫切需要建立新的防控策略。本文以精子调控为切入点,利用RNAi技术研究橘小实蝇雄虫精子发生相关基因的功能及对成虫生殖力的影响,将为探索新的防控措施提供新思路、新方法。本研究在获得的橘小实蝇转录组数据库基础上,选取了gld2、tim、rae1、gudu四个精子发生过程基因为靶标基因,分别用饲喂和显微注射ds RNA的方法将其分别沉默,研究gld2、tim、rae1、gudu基因对橘小实蝇生殖的影响,主要结果如下:1.成功克隆橘小实蝇gld2、tim、rae1、gudu基因片段,其c DNA序列分别为505bp、642bp、866bp、585bp。通过序列比对,gld2、tim、rae1、gudu均与瓜实蝇同源性最高,相似性分别达到91%、91%、91%、89%。2.饲喂处理使gld2、tim、rae1、gudu基因沉默后,各基因均能导致单雌产卵量降低。对照组为25.2粒,而处理组依此为11粒、17.9粒、11.4粒、12.3粒;也均能导致卵的孵化率显著降低。对照组孵化率为83.3%,而处理组孵化率降至70.6%、70.7%、67.3%、69.9%;gld2、tim、rae1饲喂处理后基因相对表达量显著下调,分别下调至55%、39%、38%。而dsgudu处理导致基因表达量上调至430%。3.注射处理使gld2、tim、rae1、gudu基因沉默后,交配比率与对照组相比具显著降低;其中gld2、rae1、gudu三个基因沉默后能导致单雌产卵量显著降低。对照组孵化率为12.8粒,而处理组降至8.1粒、5.5粒、6.3粒。另外,这四个基因分别沉默后均能导致卵的孵化率显著降低。对照组孵化率为84.2%,而处理组孵化率降至58.5%、65.1%、52.9%、69.1%。在分子水平,dsgld2、dsrae1处理后,基因表达量出现显著下调,分别下调至57%和52%;dstim、dsgudu处理后表达量则明显上调,分别上调至890%和230%。
[Abstract]:Bactrocera dorsalis (Hendel) is a kind of worldwide distribution, which has important economic significance. It has a mixed diet and strong fecundity, which causes a great economic loss to fruit and vegetable industry every year. At the present stage, the occurrence range is wide, the harm is aggravated, and the main prevention and control measures based on chemical control are unable to control the spread and harm of the fruit fly. It is urgent to establish a new control strategy. By using RNAi technique to study the function of spermatogenesis related genes and their effects on the fecundity of adults, this paper will provide new ideas and methods for exploring new prevention and control measures. On the basis of the transcriptome database of small fruit fly, four spermatogenesis process bases of gld2timtimrae1gudu were selected as target genes, which were silenced by feeding and microinjection of DS RNA, respectively. The effect of gld2timtima rae1 Gudu gene on the reproduction of Orange fruit fly was studied. The main results are as follows: 1. The gene fragment of gld2timtimrae1 (Gudu) was cloned successfully, and the sequence of the cDNA was 505bpn866bp5bp5bp. The sequence alignment of Gld2timrae1 Gudu was the highest homology with the fruit fly, and the similarity reached 91g / 91g / 91g ~ (89. 2), respectively. After the silencing of gld2 timrae1 Gudu gene, each gene could decrease the oviposition of single female. The control group was 25.2, while the treatment group was 11, 17.9, 11.4, and 12.3, which also resulted in a significant decrease in the hatching rate of eggs. The hatching rate of the control group was 83.3, while that of the treatment group decreased to 70.6 and 70.7 and 67.3% and 69.3%, respectively, and the relative expression of the gene was significantly down-regulated to 5550%. Dsgudu treatment resulted in an up-regulation of gene expression to 430. 3. After the silencing of gld2timtimrae1gudu gene, the mating rate was significantly lower than that of the control group, and the silencing of gld2timrae1gudu gene resulted in a significant decrease in the number of single female oviposition. The hatching rate of the control group was 12.8, while that of the treatment group decreased to 8.1, 5.5, and 6.3, respectively. In addition, the silencing of the four genes resulted in a significant decrease in the hatching rate of eggs. The hatching rate of the control group was 84.2%, while that of the treatment group decreased to 58.5% and 65.1%, 52.9% and 69.1% respectively. After treatment with dsgld2dsrae1 at molecular level, the gene expression level was significantly down-regulated to 57% and 52dstimdsgudu, respectively, to 890% and 230%, respectively.
【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

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