【摘要】：橘小實(shí)蠅Bactrocera dorsalis(Hendel)是一種世界性分布、具有重要經(jīng)濟(jì)意義的實(shí)蠅類害蟲,食性雜,繁殖力強(qiáng),每年給果蔬業(yè)造成重大經(jīng)濟(jì)損失�，F(xiàn)階段呈現(xiàn)發(fā)生范圍廣、危害加重、連年大發(fā)生的特點(diǎn),原有的以化學(xué)防治為主的防控措施已不能控制橘小實(shí)蠅的擴(kuò)散和危害,迫切需要建立新的防控策略。本文以精子調(diào)控為切入點(diǎn),利用RNAi技術(shù)研究橘小實(shí)蠅雄蟲精子發(fā)生相關(guān)基因的功能及對(duì)成蟲生殖力的影響,將為探索新的防控措施提供新思路、新方法。本研究在獲得的橘小實(shí)蠅轉(zhuǎn)錄組數(shù)據(jù)庫基礎(chǔ)上,選取了gld2、tim、rae1、gudu四個(gè)精子發(fā)生過程基因?yàn)榘袠?biāo)基因,分別用飼喂和顯微注射ds RNA的方法將其分別沉默,研究gld2、tim、rae1、gudu基因?qū)﹂傩?shí)蠅生殖的影響,主要結(jié)果如下:1.成功克隆橘小實(shí)蠅gld2、tim、rae1、gudu基因片段,其c DNA序列分別為505bp、642bp、866bp、585bp。通過序列比對(duì),gld2、tim、rae1、gudu均與瓜實(shí)蠅同源性最高,相似性分別達(dá)到91%、91%、91%、89%。2.飼喂處理使gld2、tim、rae1、gudu基因沉默后,各基因均能導(dǎo)致單雌產(chǎn)卵量降低。對(duì)照組為25.2粒,而處理組依此為11粒、17.9粒、11.4粒、12.3粒;也均能導(dǎo)致卵的孵化率顯著降低。對(duì)照組孵化率為83.3%,而處理組孵化率降至70.6%、70.7%、67.3%、69.9%;gld2、tim、rae1飼喂處理后基因相對(duì)表達(dá)量顯著下調(diào),分別下調(diào)至55%、39%、38%。而dsgudu處理導(dǎo)致基因表達(dá)量上調(diào)至430%。3.注射處理使gld2、tim、rae1、gudu基因沉默后,交配比率與對(duì)照組相比具顯著降低;其中g(shù)ld2、rae1、gudu三個(gè)基因沉默后能導(dǎo)致單雌產(chǎn)卵量顯著降低。對(duì)照組孵化率為12.8粒,而處理組降至8.1粒、5.5粒、6.3粒。另外,這四個(gè)基因分別沉默后均能導(dǎo)致卵的孵化率顯著降低。對(duì)照組孵化率為84.2%,而處理組孵化率降至58.5%、65.1%、52.9%、69.1%。在分子水平,dsgld2、dsrae1處理后,基因表達(dá)量出現(xiàn)顯著下調(diào),分別下調(diào)至57%和52%;dstim、dsgudu處理后表達(dá)量則明顯上調(diào),分別上調(diào)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.
【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

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

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本文編號(hào)：2137399