RNAi介导抗WMV、ZYMV、CMV转基因甜瓜材料的研究
发布时间：2018-09-05 19:16
【摘要】：RNAi是指转入小片段双链RNA而引发目的基因表达沉默的现象,该现象可以抑制或减弱甜瓜病毒生理生化相关的基因表达,而达到抗病效果,本研究利用RNAi技术培育出转基因甜瓜苗,为了解新疆甜瓜上的抗病毒生理机制、抗病毒甜瓜的育种提供研究基础。本研究主要采用DNAMAN5.0、CLUSTAL X和MEGA5.O软件对新疆不同主栽区发生的WMV、CMV、ZYMV的cp基因进行序列比对,找出保守序列作为目标干扰片段,构建RNAi植物表达载体；利用农杆菌介导法将目的片段转入甜瓜,最终得到转基因甜瓜苗。本研究得出以下主要研究成果如下：(1)新疆不同地区WMV、ZYMA、CMVcp基因的碱基序列的同源性分别达到86.25%、93.37%、83.13%以上,同源相似性较高；通过进化关系分析表明WMV在南北疆均有分布,可分为2个亚组,第1亚组为南疆分离物,第2亚组为北疆分离物；CMV分为4个亚组,3株分离物与北京、山东、山西和台湾的进化关系较近；ZYMV可分为3个亚组,部分新疆分离物与3个国内外地区分离物的进化关系较近；新疆不同地区WMV、ZYMV、CMV cp基因均存在碱基突变现象。(2)建立WMV、ZYMV、CMV三种病毒多重RT-PCR反应体系(25μ1)：模板cDNA最适体积为1.8μ1；引物总体积为2μ1时,比例WMV：CMV：ZYMV=9:6:5较合适；dNTPs最适浓度为0.4 mmol/L；Taq DNA聚合酶单位活力为0.75 U；Mg2+最适浓度为1.5 mmol/L；最适退火温度为50℃。(3)建立'XHXC'和‘86-1’两种甜瓜以子叶为外植体的组织培养与再生体系。5%NaClO的浓度下消毒20 min条件下获得无菌苗最好；‘86-1’和'XHXC'分别在6-BA浓度为1.2 mg/L和1.0 mg/L时愈伤的长势最好；在MS10培养基上的不定芽诱导率较高；MS19为‘86-1’的最适芽伸长培养基,MS20为'XHXC'的最适芽伸长培养基；MS24为两种甜瓜最适生根培养基。(4)经过分子检测最终得到‘86-1’甜瓜转pCAMBIA2300WMV-SA基因苗2株,转pCAMBIA2300CMV-SA基因苗2株,转pCAMBIA2300ZYMV-SA基因苗4株；'XHXC'甜瓜转pCAMBIA2300WMV-SA基因苗2株,pCAMBIA2300CMV-SA基因苗3株,pCAMBIA2300ZYMV-SA基因苗2株。
[Abstract]:RNAi refers to the phenomenon of silencing gene expression induced by small fragment double-stranded RNA, which can inhibit or weaken the expression of genes related to physiology and biochemistry of muskmelon virus, and achieve the effect of disease resistance. In this study, transgenic muskmelon seedlings were cultivated by RNAi technique, which provided the basis for understanding the anti-virus physiological mechanism and breeding of anti-virus melon in Xinjiang. In this study, DNAMAN5.0,CLUSTAL X and MEGA5.O software were used to analyze the sequence alignment of WMV,CMV,ZYMV cp gene in different planting areas in Xinjiang, and the conservative sequence was identified as the target interference fragment to construct RNAi plant expression vector. The target fragment was transferred into muskmelon by Agrobacterium tumefaciens and transgenic melon seedlings were obtained. The main results of this study are as follows: (1) the homology of WMV,ZYMA,CMVcp gene in different regions of Xinjiang is over 86.250.373.13%, the homology is high, and the analysis of evolutionary relationship shows that WMV is distributed in both north and south Xinjiang. It can be divided into two subgroups, the first group is the south Xinjiang isolate, the second group is the northern Xinjiang isolate CMV is divided into 4 subgroups and 3 isolates, and the evolutionary relationship between Shandong, Shanxi and Taiwan can be divided into three subgroups. Some Xinjiang isolates were closely related to the evolution of the three isolates at home and abroad, and the WMV,ZYMV,CMV cp gene had base mutation in different regions of Xinjiang. (2) the multiplex RT-PCR reaction system of three WMV,ZYMV,CMV viruses was established (25 渭 1): the optimal volume of template cDNA was 1.8 渭 1; When the total volume of primer is 2 渭 1, the optimum concentration of WMV:CMV:ZYMV=9:6:5 is 0.4 mmol/L;Taq DNA polymerase unit activity, and the optimum concentration is 1.5 mmol/L;. The optimum annealing temperature was 50 鈩，
本文编号：2225217
[Abstract]:RNAi refers to the phenomenon of silencing gene expression induced by small fragment double-stranded RNA, which can inhibit or weaken the expression of genes related to physiology and biochemistry of muskmelon virus, and achieve the effect of disease resistance. In this study, transgenic muskmelon seedlings were cultivated by RNAi technique, which provided the basis for understanding the anti-virus physiological mechanism and breeding of anti-virus melon in Xinjiang. In this study, DNAMAN5.0,CLUSTAL X and MEGA5.O software were used to analyze the sequence alignment of WMV,CMV,ZYMV cp gene in different planting areas in Xinjiang, and the conservative sequence was identified as the target interference fragment to construct RNAi plant expression vector. The target fragment was transferred into muskmelon by Agrobacterium tumefaciens and transgenic melon seedlings were obtained. The main results of this study are as follows: (1) the homology of WMV,ZYMA,CMVcp gene in different regions of Xinjiang is over 86.250.373.13%, the homology is high, and the analysis of evolutionary relationship shows that WMV is distributed in both north and south Xinjiang. It can be divided into two subgroups, the first group is the south Xinjiang isolate, the second group is the northern Xinjiang isolate CMV is divided into 4 subgroups and 3 isolates, and the evolutionary relationship between Shandong, Shanxi and Taiwan can be divided into three subgroups. Some Xinjiang isolates were closely related to the evolution of the three isolates at home and abroad, and the WMV,ZYMV,CMV cp gene had base mutation in different regions of Xinjiang. (2) the multiplex RT-PCR reaction system of three WMV,ZYMV,CMV viruses was established (25 渭 1): the optimal volume of template cDNA was 1.8 渭 1; When the total volume of primer is 2 渭 1, the optimum concentration of WMV:CMV:ZYMV=9:6:5 is 0.4 mmol/L;Taq DNA polymerase unit activity, and the optimum concentration is 1.5 mmol/L;. The optimum annealing temperature was 50 鈩，
本文编号：2225217
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