HaRLI基因的烟草遗传转化分析

发布时间：2018-06-23 13:15

  本文选题：棉铃虫 + 核糖核酸酶L抑制剂　； 参考：《河北大学》2017年硕士论文

【摘要】：RNase L途径是由干扰素引起的一种抗病毒途径,RLI基因作为一种RNase L通路的负调节因子,被越来越多的研究。本研究以HaRLI基因作为研究对象,探究该基因在病毒处理以及植物介导的昆虫RNAi中的功能。具体的研究内容如下:1.HaRLI基因的克隆及序列分析采用同源搜索的方法,从棉铃虫转录组中得到BmRLI同源基因序列。利用RT-PCR方法在棉铃虫中克隆得到全长基因序列,命名为HaRLI。该基因包含一个1824bp的ORF,编码608个氨基酸。NCBI Blast显示其编码产物存在三个保守结构域,一个铁硫结合域和两个ATP结合盒。2.HaNPV病毒处理后HaRLI基因的表达情况利用RT-qPCR方法对正常饲养和病毒处理后虫子体内的HaRLI基因表达情况进行分析。结果表明:相对于正常饲养的虫子,HaNPV病毒处理24 h后HaRLI基因表达水平显著下降,达60%。3.转基因植株的获得及后代遗传分析HaRLI基因上存在一个BamH I位点,因此我们在两条引物的5′端分别设计Bsa I位点嵌套BamH I或者Sal I酶切位点,构建植物表达载体pBin438-HaRLI,农杆菌介导的方法将HaRLI基因导入到烟草基因组中。通过对Kan抗性平皿上种植的转基因T0和T1代植株种子进行遗传分析,结果发现:T0-4植株种子的Kan抗性符合孟德尔3:1分离比,推测第4株转基因植株为单一拷贝插入;该植株后代17株T1代植株的种子Kan抗性结果显示,5株为全抗型(纯和)、10株呈现大约3:1的分离比(杂合)、另外有2株分离比并不符合孟德尔的分离比,其抗性苗大约为敏感苗的4倍。4.叶片饲喂RNAi效率的影响构建了几丁质合酶1(Hachs1)、几丁质合酶2(Hachs2)和绿色荧光蛋白(GFP)基因的RNAi载体,并转化至HT115感受态中。用涂抹表达dsRNA菌液的野生型和T1-8植株叶片饲喂棉铃虫,RT-qPCR检测饲喂24 h的幼虫发现,野生型叶片饲喂后,Hachs2基因表达量出现下调,而Hachs1基因并没有被沉默,反而表达量有所升高;转基因叶片饲喂后,Hachs1和Hachs2基因表达量均下调,且相对于对照组,Hachs2基因下调幅度具有极显著的差异。对比发现,相对于喂食涂抹细菌dsRNA的野生型烟草叶片,Hachs1和Hachs2基因在喂食转基因叶片的棉铃虫中沉默效率更高。5.转HaRLI基因叶片抗虫性分析选取WT、T1-8和T1-20叶片进行棉铃虫的饲喂,12 h后叶片之间未观察到明显的区别,经36 h的喂食,虫子对WT叶片的咬噬情况相对较严重,但并没有明显的致死现象,说明转基因烟草对棉铃虫具有一定的抗性。以上研究为HaRLI基因的植物基因工程研究提供一些参考。
[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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】

相关期刊论文 前10条

1 林馥芬;侯红利;刘晋;王学林;邓汉超;周向阳;;转基因植物DNA成分检测技术研究进展[J];中国种业;2016年11期

2 黄诗迪;黄彩萍;于欢;王敦;;棉铃虫核型多角体病毒感染对宿主昆虫GST活性及其表达水平的影响[J];西北农林科技大学学报(自然科学版);2015年11期

3 董福双;张立华;吕孟雨;;一种简单有效的小麦转基因后代卡那霉素筛选方法[J];河北农业科学;2015年04期

4 杨芳;李芳功;冯振群;;棉铃虫核型多角体病毒杀虫剂防治烟田烟青虫田间药效试验[J];中国农技推广;2014年08期

5 田宏刚;刘同先;张文庆;;昆虫RNAi技术与方法[J];应用昆虫学报;2013年05期

6 李良德;刘婕;姜春来;赖先文;;昆虫RNAi效率的影响因素研究进展[J];生物技术通报;2012年11期

7 于欢;吕婷婷;李晓峰;王敦;;HearNPV侵染对棉铃虫中肠碱性磷酸酶活性及表达水平的影响[J];西北农林科技大学学报(自然科学版);2012年12期

8 吕孟雨;董福双;张俊敏;王海波;;转基因玉米抗卡那霉素筛选方法研究[J];西南农业学报;2010年06期

9 燕丽萍;夏阳;梁慧敏;王友平;刘翠兰;王振猛;李丽;;卡那霉素叶片涂抹法田间筛选转基因苜蓿的研究[J];中国农学通报;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

相关会议论文 前1条

1 张光裕;白传珍;;我国第一个商品病毒杀虫剂—棉铃虫核型多角体病毒杀虫剂的研究与开发[A];全国生物防治学术讨论会论文集[C];1991年

，

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