本文選題：棉鈴蟲 + 核糖核酸酶L抑制劑�。� 參考：《河北大學(xué)》2017年碩士論文

【摘要】：RNase L途徑是由干擾素引起的一種抗病毒途徑,RLI基因作為一種RNase L通路的負(fù)調(diào)節(jié)因子,被越來越多的研究。本研究以HaRLI基因作為研究對象,探究該基因在病毒處理以及植物介導(dǎo)的昆蟲RNAi中的功能。具體的研究內(nèi)容如下:1.HaRLI基因的克隆及序列分析采用同源搜索的方法,從棉鈴蟲轉(zhuǎn)錄組中得到BmRLI同源基因序列。利用RT-PCR方法在棉鈴蟲中克隆得到全長基因序列,命名為HaRLI。該基因包含一個1824bp的ORF,編碼608個氨基酸。NCBI Blast顯示其編碼產(chǎn)物存在三個保守結(jié)構(gòu)域,一個鐵硫結(jié)合域和兩個ATP結(jié)合盒。2.HaNPV病毒處理后HaRLI基因的表達(dá)情況利用RT-qPCR方法對正常飼養(yǎng)和病毒處理后蟲子體內(nèi)的HaRLI基因表達(dá)情況進(jìn)行分析。結(jié)果表明:相對于正常飼養(yǎng)的蟲子,HaNPV病毒處理24 h后HaRLI基因表達(dá)水平顯著下降,達(dá)60%。3.轉(zhuǎn)基因植株的獲得及后代遺傳分析HaRLI基因上存在一個BamH I位點,因此我們在兩條引物的5′端分別設(shè)計Bsa I位點嵌套BamH I或者Sal I酶切位點,構(gòu)建植物表達(dá)載體pBin438-HaRLI,農(nóng)桿菌介導(dǎo)的方法將HaRLI基因?qū)氲綗煵莼蚪M中。通過對Kan抗性平皿上種植的轉(zhuǎn)基因T0和T1代植株種子進(jìn)行遺傳分析,結(jié)果發(fā)現(xiàn):T0-4植株種子的Kan抗性符合孟德爾3:1分離比,推測第4株轉(zhuǎn)基因植株為單一拷貝插入;該植株后代17株T1代植株的種子Kan抗性結(jié)果顯示,5株為全抗型(純和)、10株呈現(xiàn)大約3:1的分離比(雜合)、另外有2株分離比并不符合孟德爾的分離比,其抗性苗大約為敏感苗的4倍。4.葉片飼喂RNAi效率的影響構(gòu)建了幾丁質(zhì)合酶1(Hachs1)、幾丁質(zhì)合酶2(Hachs2)和綠色熒光蛋白(GFP)基因的RNAi載體,并轉(zhuǎn)化至HT115感受態(tài)中。用涂抹表達(dá)dsRNA菌液的野生型和T1-8植株葉片飼喂棉鈴蟲,RT-qPCR檢測飼喂24 h的幼蟲發(fā)現(xiàn),野生型葉片飼喂后,Hachs2基因表達(dá)量出現(xiàn)下調(diào),而Hachs1基因并沒有被沉默,反而表達(dá)量有所升高;轉(zhuǎn)基因葉片飼喂后,Hachs1和Hachs2基因表達(dá)量均下調(diào),且相對于對照組,Hachs2基因下調(diào)幅度具有極顯著的差異。對比發(fā)現(xiàn),相對于喂食涂抹細(xì)菌dsRNA的野生型煙草葉片,Hachs1和Hachs2基因在喂食轉(zhuǎn)基因葉片的棉鈴蟲中沉默效率更高。5.轉(zhuǎn)HaRLI基因葉片抗蟲性分析選取WT、T1-8和T1-20葉片進(jìn)行棉鈴蟲的飼喂,12 h后葉片之間未觀察到明顯的區(qū)別,經(jīng)36 h的喂食,蟲子對WT葉片的咬噬情況相對較嚴(yán)重,但并沒有明顯的致死現(xiàn)象,說明轉(zhuǎn)基因煙草對棉鈴蟲具有一定的抗性。以上研究為HaRLI基因的植物基因工程研究提供一些參考。
[Abstract]:RNase L pathway, an antiviral pathway induced by interferon, has been studied more and more as a negative regulator of RNase L pathway. In this study, HaRLI gene was used to study the function of HaRLI gene in virus treatment and plant mediated insect RNAi. The specific contents were as follows: 1. Cloning and sequence analysis of HaRLI gene. BmRLI homologous gene sequence was obtained from Helicoverpa armigera transcriptome by homology search method. The full-length gene was cloned from Helicoverpa armigera by RT-PCR and named HaRLI. The gene contains an ORF of 1824bp and encodes 608 amino acids. NCBI blast shows that there are three conserved domains in the encoding product. One iron-sulfur binding domain and two ATP-binding cassette. 2. HaRLI gene expression after treatment with HaNPV virus was analyzed by RT-qPCR. The results showed that the expression level of HaRLI gene was significantly decreased to 60.3. There was a BamHI locus on HaRLI gene, so we designed BSAI locus nested BamH I or Sal I sites at the 5'end of two primers. The plant expression vector pBin438-HaRLI was constructed, and the HaRLI gene was transfected into tobacco genome by Agrobacterium tumefaciens. Based on the genetic analysis of transgenic T0 and T1 plants planted on Kan resistance dishes, it was found that the Kan resistance of the seeds of 10 T0-4 plants was in accordance with the Mendelian segregation ratio at 3:1, and the fourth transgenic plant was assumed to be a single copy insertion. The results of Kan resistance in the seeds of 17 T1 generation plants of this plant showed that 5 plants were totally resistant (pure sum) and 10 plants showed about 3:1 segregation ratio (heterozygosity), and the other 2 plants did not accord with Mendelian segregation ratio. The resistant seedlings were about 4 times of that of the sensitive seedlings. The RNAi vectors of chitinase 1 (Hachs1), chitin synthase 2 (Hachs2) and green fluorescent protein (GFP) genes were constructed and transformed into HT115 receptive state. The wild type and T1-8 plant leaves were fed with cotton bollworm RT-qPCR for 24 h. The results showed that the expression of Hachs2 gene in wild type leaves was down-regulated, but Hachs1 gene was not silenced, but the expression of Hachs1 gene was increased. The expression of Hachs1 and Hachs2 genes were down-regulated in transgenic leaves, and the down-regulation range of Hachs2 gene was significantly different from that of control group. The results showed that the silencing efficiency of Hachs1 and Hachs2 genes was higher in Helicoverpa armigera than that in wild tobacco leaves fed with bacterial dsRNA. No significant difference was observed between the leaves of WTT1-8 and T1-20 after feeding for 12 h. After 36 h feeding, the bite rate of WT leaves was relatively serious. But there was no obvious death phenomenon, which indicated that transgenic tobacco had certain resistance to Helicoverpa armigera. These studies provide some references for the plant genetic engineering of HaRLI gene.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 林馥芬;侯紅利;劉晉;王學(xué)林;鄧漢超;周向陽;;轉(zhuǎn)基因植物DNA成分檢測技術(shù)研究進(jìn)展[J];中國種業(yè);2016年11期

2 黃詩迪;黃彩萍;于歡;王敦;;棉鈴蟲核型多角體病毒感染對宿主昆蟲GST活性及其表達(dá)水平的影響[J];西北農(nóng)林科技大學(xué)學(xué)報(自然科學(xué)版);2015年11期

3 董福雙;張立華;呂孟雨;;一種簡單有效的小麥轉(zhuǎn)基因后代卡那霉素篩選方法[J];河北農(nóng)業(yè)科學(xué);2015年04期

4 楊芳;李芳功;馮振群;;棉鈴蟲核型多角體病毒殺蟲劑防治煙田煙青蟲田間藥效試驗[J];中國農(nóng)技推廣;2014年08期

5 田宏剛;劉同先;張文慶;;昆蟲RNAi技術(shù)與方法[J];應(yīng)用昆蟲學(xué)報;2013年05期

6 李良德;劉婕;姜春來;賴先文;;昆蟲RNAi效率的影響因素研究進(jìn)展[J];生物技術(shù)通報;2012年11期

7 于歡;呂婷婷;李曉峰;王敦;;HearNPV侵染對棉鈴蟲中腸堿性磷酸酶活性及表達(dá)水平的影響[J];西北農(nóng)林科技大學(xué)學(xué)報(自然科學(xué)版);2012年12期

8 呂孟雨;董福雙;張俊敏;王海波;;轉(zhuǎn)基因玉米抗卡那霉素篩選方法研究[J];西南農(nóng)業(yè)學(xué)報;2010年06期

9 燕麗萍;夏陽;梁慧敏;王友平;劉翠蘭;王振猛;李麗;;卡那霉素葉片涂抹法田間篩選轉(zhuǎn)基因苜蓿的研究[J];中國農(nóng)學(xué)通報;2009年14期

10 ;The ORF 113 of Heliocoverpa armigera Single Nucleopolyhedrovirus Encodes a Functional Fibroblast Growth Factor[J];Virologica Sinica;2008年05期

相關(guān)會議論文 前1條

1 張光裕;白傳珍;;我國第一個商品病毒殺蟲劑—棉鈴蟲核型多角體病毒殺蟲劑的研究與開發(fā)[A];全國生物防治學(xué)術(shù)討論會論文集[C];1991年

，

本文編號：2057232