本文選題：桔小實(shí)蠅 + 雌性特異致死品系��； 參考：《中國農(nóng)業(yè)科學(xué)院》2015年博士論文

【摘要】：昆蟲不育技術(shù)(Sterile insect technique,SIT)是根除實(shí)蠅類害蟲和預(yù)防害蟲在高風(fēng)險(xiǎn)地區(qū)定殖的最有效的防治方法。本研究以世界性農(nóng)業(yè)害蟲桔小實(shí)蠅Bactrocera dorsalis(Hendel)為對象,通過分離、鑒定桔小實(shí)蠅雌性特異胚胎致死的遺傳性別品系特異調(diào)控元件,構(gòu)建桔小實(shí)蠅雌性特異胚胎致死驅(qū)動(dòng)載體和效應(yīng)載體,建立桔小實(shí)蠅遺傳轉(zhuǎn)化技術(shù),以期獲得桔小實(shí)蠅雌性特異胚胎致死品系,實(shí)現(xiàn)在桔小實(shí)蠅飼養(yǎng)階段清除所有雌蟲,為不育雄蟲釋放的昆蟲不育技術(shù)項(xiàng)目提供技術(shù)支撐。主要研究結(jié)果如下:(1)利用實(shí)時(shí)熒光定量PCR分析Bdvasa的時(shí)空表達(dá)譜,反向PCR分離Bdvasa 5’側(cè)翼的上游啟動(dòng)子序列,結(jié)果表明Bdvasa是一種在胚胎早期高水平表達(dá)的母體效應(yīng)基因,分離得到Bdvasa翻譯起始密碼子上游1996 bp序列,成功用于構(gòu)建驅(qū)動(dòng)t TA表達(dá)的驅(qū)動(dòng)載體2個(gè)。(2)為分離候選的桔小實(shí)蠅顯性致死基因,通過同源基因克隆和RACE技術(shù)克隆桔小實(shí)蠅細(xì)胞凋亡效應(yīng)酶caspase-1,實(shí)時(shí)熒光定量PCR分析該基因的時(shí)空表達(dá)譜,腹部RNAi研究該基因在雌蟲生殖過程中的功能,結(jié)果為:克隆得到桔小實(shí)蠅細(xì)胞凋亡效應(yīng)酶caspase-1的c DNA全長1679 bp,編碼328個(gè)氨基酸殘基,并將該基因命名為Bdcp-1;Bdcp-1在桔小實(shí)蠅的不同發(fā)育時(shí)期和成蟲的不同組織間均有表達(dá),其表達(dá)量與桔小實(shí)蠅生長發(fā)育過程密切相關(guān);適量的UV照射可以提高Bdcp-1 m RNA轉(zhuǎn)錄水平,Bdcp-1在UV照射1 h的8日齡蛹中的表達(dá)量是未照射的5.6倍,照射1.5 h是未照射的5.2倍,暗示細(xì)胞通過caspase級聯(lián)反應(yīng)調(diào)控受損細(xì)胞凋亡,維持細(xì)胞正常的生命活動(dòng),但照射超過2 h后,這種凋亡機(jī)制被破壞;Bdcp-1在雌蟲生殖過程中具有重要作用,該基因沉默后,卵巢發(fā)育和卵黃蛋白基因Bdyp1的表達(dá)受到抑制,產(chǎn)卵前期延長,單頭雌蟲累計(jì)產(chǎn)卵量顯著降低。(3)為了分離桔小實(shí)蠅性別特異剪接調(diào)控元件,利用PCR結(jié)合RACE技術(shù)分析桔小實(shí)蠅Bdtra和Bdtra-2的基因組和c DNA結(jié)構(gòu),半定量PT-PCR研究這2個(gè)基因的表達(dá)模式,胚胎RNAi研究這2個(gè)基因的功能。結(jié)果為:桔小實(shí)蠅Bdtra存在1個(gè)雌性特異剪接體和2個(gè)雄性特異剪接體,Bdtra-2不存在性別特異剪接;Bdtra雌性特異剪接體和Bdtra-2基因具有母體遺傳表達(dá)特性,Bdtra雄性特異剪接體在產(chǎn)后1 h后的胚胎中才能檢測到;胚胎RNAi Bdtra或Bdtra-2均能高效誘導(dǎo)雌性胚胎發(fā)育為表型雄蟲,證明Bdtra和Bdtra-2在雌蟲體性發(fā)育過程中具有重要作用;分離得到Bdtra雌性特異剪接內(nèi)含子序列Bdtra I1068 bp,成功用于構(gòu)建雌性特異致死效應(yīng)載體。(4)利用胚胎顯微注射的方法,遺傳轉(zhuǎn)化地中海實(shí)蠅精子熒光標(biāo)記質(zhì)粒1260,熒光顯微鏡篩選得到2種不同熒光表達(dá)模式的桔小實(shí)蠅轉(zhuǎn)化個(gè)體,RT-PCR檢測到熒光蛋白基因t GEP和Ds Red在轉(zhuǎn)化個(gè)體中有表達(dá),成功建立桔小實(shí)蠅遺傳轉(zhuǎn)化技術(shù)體系。
[Abstract]:Sterile insect technique site is the most effective control method for the eradication of fly pests and the prevention of colonization of insect pests in high-risk areas.In this study, the worldwide agricultural pest Bactrocera dorsalis Hendeler was used to identify the genetic and sex-specific regulatory elements of female specific embryo death.The driving vector and effect vector of female specific embryo death were constructed, and the genetic transformation technology of fruit fly was established in order to obtain the female specific embryo lethal strain of fruit fly and to eliminate all female insects in the rearing stage of fruit fly.To provide technical support for the male sterile male release insect sterility technology project.The main results are as follows: (1) using real-time fluorescence quantitative PCR to analyze the temporal and spatial expression profiles of Bdvasa, reverse PCR was used to isolate the upstream promoter sequence of Bdvasa 5'flanking. The results showed that Bdvasa was a high level maternal effector gene expressed at early embryonic stage.The upstream 1996 BP sequence of Bdvasa translation initiation codon was isolated and successfully used to construct two driving vectors for TTA expression.The apoptotic effector enzyme caspase-1 was cloned by homologous gene cloning and RACE technique. The temporal and spatial expression profiles of the gene were analyzed by real-time fluorescence quantitative PCR, and the function of the gene during female reproduction was studied by abdominal RNAi.The results showed that the total length of c DNA of the apoptosis effector enzyme caspase-1 was 1679 BP, encoding 328 amino acid residues, and Bdcp-1 Bdcp-1 was expressed in different stages of development and in different tissues of adults.It was suggested that the apoptosis of damaged cells was regulated by caspase cascade reaction and maintained normal life activity. However, after irradiation for more than 2 hours, this mechanism of apoptosis was destroyed and Bdcp-1 played an important role in the reproductive process of female worms, and the gene was silenced.The ovarian development and the expression of yolk protein gene Bdyp1 were inhibited, the pre-oviposition was prolonged, the accumulative oviposition of single female decreased significantly.The genomes and c DNA structures of Bdtra and Bdtra-2 were analyzed by PCR and RACE, the expression patterns of these two genes were studied by semi-quantitative PT-PCR, and the functions of these two genes were studied by embryonic RNAi.The results showed that there was one female specific splice and two male specific splicing bodies in Bdtra, and there were no sex specific splicing female splicing bodies and Bdtra-2 genes had maternal genetic expression characteristics.Only one hour after delivery could be detected in embryos.RNAi Bdtra or Bdtra-2 could efficiently induce female embryos to develop into phenotypic males, indicating that Bdtra and Bdtra-2 play an important role in the development of females.Bdtra female specific splicing intron sequence Bdtra I1068 BP was isolated and successfully used to construct female specific lethal effect vector.Fluorescence labeling plasmid 1260 was obtained from transgenic spermatozoa of Drosophila morifolia. Two different fluorescent expression patterns were obtained by RT-PCR. The expression of fluorescent protein genes t GEP and Ds Red in transformed individuals was detected by RT-PCR.The technique system of genetic transformation of fruit fly was established successfully.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S433

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本文編號：1754434