基于黄瓜花叶病毒的基因沉默载体优化
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摘要
黄瓜花叶病毒(Cucumber mosaic virus, CMV)是雀麦花叶病毒科(Bromoviridae)黄瓜花叶病毒属(Cucumovirus)的典型成员,是世界上寄主范围最广的病毒。病毒诱导的基因沉默(Virus Induced Gene Silencing, VIGS)是一快速高效的研究植物基因功能工具。本研究在前人的基础上,基于CMV-Tsh株系的RNA2构建基因沉默载体,选择不同的指示基因进行沉默效果分析,并成功构建CMV的农杆菌侵染性克隆。主要内容如下：
一、CMV-Fny假重组弱病毒组合的筛选
以CMV-Fny与CMV亚组Ⅰ中不同株系的NA2(Cb7RNA2、CNa RNA2和Tsh RNA2)构建RNA2不同的假重组病毒,接种本氏烟。接种7天后,F1F2F3、F1C2F3、F1CNa2F3在寄主上均引起叶片皱缩、蜷曲的症状,而F1Tsh2F3在本氏烟上则引起轻花叶的症状反应,因此以CMV-Tsh RNA2作为构建VIGS载体母本。
二、基于CMV-Tsh RNA2的2b构建基因沉默载体
采用PCR以及双酶切等手段对CMV-Tsh RNA2进行了三种改造,构建Tsh的2b基因缺失95nt、完全缺失的Tsh RNA2,及在2b的终止密码子后,RNA2的UTR前引入Mlu Ⅰ和SnaBⅠ酶切等酶切位点,前两个载体分别命名为Tsh R23/42b、Tsh R2Δ2b,而Tsh RNA2的终止密码子后,RNA2的UTR前直接引入酶切位点构建而来的载体则命名为Tsh R22b。 pTsh R23/42b、pTsh R2Δ2b以及pTsh R22b体外转录产物分别与CMV-Fny RNA1和FnyRNA3混合接种本氏烟,证实假重组病毒具有侵染活性后；在上述的克隆位点分别插入354pb大小的本氏烟PDS片段,体外转录接种后比较本氏烟的光漂白程度,最终确定pTshR23/42b为本研究的基因沉默载体作为进一步分析对象。
三、不同指示基因沉默效果的研究
pTsh R23/42b载体中插入PDS或Su基因部分片段以及绿色荧光蛋白基因(GFP)部分片段,分析寄主的光漂白、叶表黄化和转GFP本氏烟(16c)变红程度,并采用semi RT-PCR分析靶标基因在植株中的表达量。结果表明：F1Tsh R23/42b-NbPDS354F3接种本氏烟后,植株叶表呈现明显的光漂白,F1Tsh R23/42b-Su351F3诱导本氏烟产生黄化现象,相应的植株中PDS和Su的表达量均下调；在UV lamp照射下,接种F1Tsh R23/42b-GFP217F3的16C植株则呈现发红,而接种F1Tsh R23/42bF3植株呈绿色,semi RT-PCR检测到植株中的GFP表达量下调。采用汁液摩擦接种的方法,分析F1Tsh R23/42b-NbPDS354F3在本氏烟的稳定性,结果表明,转接15次,F1Tsh R23/42b-NbPDS354F3均能稳定存在于寄主中,并使得本氏烟产生光漂白现象。
四、以启动子序列和大豆PDS为插入片段的沉默效应以及下调Beclin基因后转NN基因烟草对TMV的症状反应
在pTsh R23/42b载体中插入本氏烟PDS或GFP的启动子部分片段、大豆PDS部分片段(分别命名为proPDS、35s、GmPDS)以及Beclin基因部分片段,分析本氏烟的光漂白、16C变红程度、大豆的光漂白和NN基因烟草对TMV的症状反应。结果表明：本氏烟接种F1Tsh R23/42b-proPDS223F3后出现明显光漂白；16C接种F1Tsh R23/42b-35s120F3后,在UVlamp照射下呈现发红；在大豆的PDS被F1Tsh R23/42bGmPDS277F3被下调表达后,植株呈现光漂白表型。转NN基因烟草(本氏烟和三生烟)接种F1Tsh R23/42b-Beclin346F3后,经过7天再接种TMV,转NN基因烟草出现系统性坏死,而对照病毒F1Tsh R23/42bF3接种的植株,TMV则引起枯斑症状。
五、CMV农杆菌侵染性克隆的构建以及应用
PCR扩增TshR23/42b、Tsh R23/42b-NbPDS354、Tsh R23/42b-Su351、Fny RNA1、RNA2、RNA3全长序列,目的片段回收后将其克隆至瞬时表达载体pCB301中,阳性克隆转入农杆菌GV3101。通过农杆菌浸润接种本氏烟,分析农杆菌CMV-Fny,F1Tsh R23/42b-NbPDS353F3和F1Tsh R23/42b-PDS353F3在本氏烟上引起的光漂白和叶表黄化现象。结果显示：农杆菌CMV-Fny引起的症状反应跟体外转录接种CMV-Fny相一致,而浸润接种F1Tsh R23/42b-NbPDS353F3和F1pCB301-TshR23/42b-Su351F3的本氏烟分别出现光漂白和叶表黄化表型。
As a typical member of the genus Cucumovirus in the family Bromoviridae, Cucumber mosaic virus (CMV) has the most widespread host range in the world. Virus-induced gene silencing (VIGS) is an efficient tool to study the functions of plant gene. In this study, we constructed a gene silencing vector based on CMV-Tsh RNA2. In order to analyze gene silencing efficency, different reporter genes were cloned into CMV-Tsh RNA2, accumulation of target gene was assessed by semi-RT-PCR, and infectious clones of CMV of Agrobacterium were constructed successfully. Main results in this study followed as:
Firstly, pseudo-recombination viruses were constructed, with RNA1and RNA3from CMV-Fny and RNA2from different strains of CMV subgroup I including Cb7RNA2, CNa RNA2and Tsh RNA2. The pseudo-recombination viruses were mechanically inculated onto Nicotiana. benthamiana7days post-inoculation(dpi), syptoms of crimple and twist were obsvered on host plants inoculated with F1F2F3, F1C2F3, F1CNa2F3, but light mosaic apperard on the seedlings of host plant infected by F1Tsh2F3. Thus, CMV-Tsh RNA2was chosen to construct a gene silencing vector.
Secondly, three candidates of gene silencing vector based on2b of CMV-Tsh RNA2were constructed. The first one was named Tsh R23/42b whose2b was shorter than normal by95nt; the second one was named Tsh R2A2b whose2b was deleted completely; Tsh R22b was construed by inserting by Mlu Ⅰ and SnaB Ⅰ between stop codon of2b and UTR of RNA2. In vitro transcripts of pTsh R23/42b、pTsh R2Δ2b and pTsh R22b were mixed with CMV-Fny RNA1, Fny RNA3respectively to inoculate onto N. benthamiana, and infectivity of pseu-recombinant virus were dected by RT-PCR.353bp PDS fragment of N. benthamiana(named NbPDS) was inserted into pTsh R23/42b、pTsh R2Δ2b and pTsh R22b respectively to compare photobleaching on N. benthamiana after inoculation. pTsh R23/42b was chosed as a gene silencing vector to do further analysis.
Thirdly, the effect of gene silencing of different reporter genes were tested. Fragments of PDS, Su or Green fluorescent protein gene(GFP) were inserted into pTsh R23/42b to analyze phenomenon of photobleaching, yellow leaf table on N. benthamiana or red level on N. benthamiana16c. Besides, semi RT-PCR was used to analyze expression level of target gene in plants. The resluts showed that obviorus photobleaching was observed on N. benthamiana inoculated with F1Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana infected by F1Tsh R23/42b-Su351F3; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; the accumulation of PDS,Su and GFP mRNA all declined. Sap inoculation was adopted to analyze stability of F1Tsh R23/42b-NbPDS354F3in N. benthamiana. The results indicated that F1Tsh R23/42b-NbPDS354F3could exist in N. benthamiana steadily and induce photobleaching in host plants.
Fourthly, partial promoters of PDS, GFP of N. benthamiana, fragments of soybean PDS(named proPDS,35s and GmPDS) and part of Beclin were inserted into pTsh R23/42b to analyze photobleaching on N. benthamiana and soybean, red level on16C and symptoms on NN transgene tobacco inoculated with TMV. The resluts showed that:significant photobleaching was observed on N. benthamiana and soybean; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; Systemic necrosis emerged on NN transgene tobacco which was inoculated with TMV, after being inoculated with F1Tsh R23/42b-Beclin346F37dpi.
Fifthly, CMV infectious clones of Agrobacterium were constructed. Full-length sequences of Tsh R23/42b, Tsh R23/42b-NbPDS354, Tsh R23/42b-Su351, Fny RNA1, RNA2, RNA3were inserted into transient expression vector pCB301to transform into Agrobacterium. Photobleaching and yellow leaf table on N. benthamiana induced by F1Tsh R23/42b-NbPDS354F3and F1Tsh R23/42b-Su351F3were analyzed through inoculating N. benthamiana with Agrobacterium infiltration inoculation. The resluts showed that:symptoms induced by CMV-Fny of Agrobacterium were in keeping with ones induced by in vitro transcripts RNA; significant photobleaching was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-Su351F3.
一、CMV-Fny假重组弱病毒组合的筛选
以CMV-Fny与CMV亚组Ⅰ中不同株系的NA2(Cb7RNA2、CNa RNA2和Tsh RNA2)构建RNA2不同的假重组病毒,接种本氏烟。接种7天后,F1F2F3、F1C2F3、F1CNa2F3在寄主上均引起叶片皱缩、蜷曲的症状,而F1Tsh2F3在本氏烟上则引起轻花叶的症状反应,因此以CMV-Tsh RNA2作为构建VIGS载体母本。
二、基于CMV-Tsh RNA2的2b构建基因沉默载体
采用PCR以及双酶切等手段对CMV-Tsh RNA2进行了三种改造,构建Tsh的2b基因缺失95nt、完全缺失的Tsh RNA2,及在2b的终止密码子后,RNA2的UTR前引入Mlu Ⅰ和SnaBⅠ酶切等酶切位点,前两个载体分别命名为Tsh R23/42b、Tsh R2Δ2b,而Tsh RNA2的终止密码子后,RNA2的UTR前直接引入酶切位点构建而来的载体则命名为Tsh R22b。 pTsh R23/42b、pTsh R2Δ2b以及pTsh R22b体外转录产物分别与CMV-Fny RNA1和FnyRNA3混合接种本氏烟,证实假重组病毒具有侵染活性后；在上述的克隆位点分别插入354pb大小的本氏烟PDS片段,体外转录接种后比较本氏烟的光漂白程度,最终确定pTshR23/42b为本研究的基因沉默载体作为进一步分析对象。
三、不同指示基因沉默效果的研究
pTsh R23/42b载体中插入PDS或Su基因部分片段以及绿色荧光蛋白基因(GFP)部分片段,分析寄主的光漂白、叶表黄化和转GFP本氏烟(16c)变红程度,并采用semi RT-PCR分析靶标基因在植株中的表达量。结果表明：F1Tsh R23/42b-NbPDS354F3接种本氏烟后,植株叶表呈现明显的光漂白,F1Tsh R23/42b-Su351F3诱导本氏烟产生黄化现象,相应的植株中PDS和Su的表达量均下调；在UV lamp照射下,接种F1Tsh R23/42b-GFP217F3的16C植株则呈现发红,而接种F1Tsh R23/42bF3植株呈绿色,semi RT-PCR检测到植株中的GFP表达量下调。采用汁液摩擦接种的方法,分析F1Tsh R23/42b-NbPDS354F3在本氏烟的稳定性,结果表明,转接15次,F1Tsh R23/42b-NbPDS354F3均能稳定存在于寄主中,并使得本氏烟产生光漂白现象。
四、以启动子序列和大豆PDS为插入片段的沉默效应以及下调Beclin基因后转NN基因烟草对TMV的症状反应
在pTsh R23/42b载体中插入本氏烟PDS或GFP的启动子部分片段、大豆PDS部分片段(分别命名为proPDS、35s、GmPDS)以及Beclin基因部分片段,分析本氏烟的光漂白、16C变红程度、大豆的光漂白和NN基因烟草对TMV的症状反应。结果表明：本氏烟接种F1Tsh R23/42b-proPDS223F3后出现明显光漂白；16C接种F1Tsh R23/42b-35s120F3后,在UVlamp照射下呈现发红；在大豆的PDS被F1Tsh R23/42bGmPDS277F3被下调表达后,植株呈现光漂白表型。转NN基因烟草(本氏烟和三生烟)接种F1Tsh R23/42b-Beclin346F3后,经过7天再接种TMV,转NN基因烟草出现系统性坏死,而对照病毒F1Tsh R23/42bF3接种的植株,TMV则引起枯斑症状。
五、CMV农杆菌侵染性克隆的构建以及应用
PCR扩增TshR23/42b、Tsh R23/42b-NbPDS354、Tsh R23/42b-Su351、Fny RNA1、RNA2、RNA3全长序列,目的片段回收后将其克隆至瞬时表达载体pCB301中,阳性克隆转入农杆菌GV3101。通过农杆菌浸润接种本氏烟,分析农杆菌CMV-Fny,F1Tsh R23/42b-NbPDS353F3和F1Tsh R23/42b-PDS353F3在本氏烟上引起的光漂白和叶表黄化现象。结果显示：农杆菌CMV-Fny引起的症状反应跟体外转录接种CMV-Fny相一致,而浸润接种F1Tsh R23/42b-NbPDS353F3和F1pCB301-TshR23/42b-Su351F3的本氏烟分别出现光漂白和叶表黄化表型。
As a typical member of the genus Cucumovirus in the family Bromoviridae, Cucumber mosaic virus (CMV) has the most widespread host range in the world. Virus-induced gene silencing (VIGS) is an efficient tool to study the functions of plant gene. In this study, we constructed a gene silencing vector based on CMV-Tsh RNA2. In order to analyze gene silencing efficency, different reporter genes were cloned into CMV-Tsh RNA2, accumulation of target gene was assessed by semi-RT-PCR, and infectious clones of CMV of Agrobacterium were constructed successfully. Main results in this study followed as:
Firstly, pseudo-recombination viruses were constructed, with RNA1and RNA3from CMV-Fny and RNA2from different strains of CMV subgroup I including Cb7RNA2, CNa RNA2and Tsh RNA2. The pseudo-recombination viruses were mechanically inculated onto Nicotiana. benthamiana7days post-inoculation(dpi), syptoms of crimple and twist were obsvered on host plants inoculated with F1F2F3, F1C2F3, F1CNa2F3, but light mosaic apperard on the seedlings of host plant infected by F1Tsh2F3. Thus, CMV-Tsh RNA2was chosen to construct a gene silencing vector.
Secondly, three candidates of gene silencing vector based on2b of CMV-Tsh RNA2were constructed. The first one was named Tsh R23/42b whose2b was shorter than normal by95nt; the second one was named Tsh R2A2b whose2b was deleted completely; Tsh R22b was construed by inserting by Mlu Ⅰ and SnaB Ⅰ between stop codon of2b and UTR of RNA2. In vitro transcripts of pTsh R23/42b、pTsh R2Δ2b and pTsh R22b were mixed with CMV-Fny RNA1, Fny RNA3respectively to inoculate onto N. benthamiana, and infectivity of pseu-recombinant virus were dected by RT-PCR.353bp PDS fragment of N. benthamiana(named NbPDS) was inserted into pTsh R23/42b、pTsh R2Δ2b and pTsh R22b respectively to compare photobleaching on N. benthamiana after inoculation. pTsh R23/42b was chosed as a gene silencing vector to do further analysis.
Thirdly, the effect of gene silencing of different reporter genes were tested. Fragments of PDS, Su or Green fluorescent protein gene(GFP) were inserted into pTsh R23/42b to analyze phenomenon of photobleaching, yellow leaf table on N. benthamiana or red level on N. benthamiana16c. Besides, semi RT-PCR was used to analyze expression level of target gene in plants. The resluts showed that obviorus photobleaching was observed on N. benthamiana inoculated with F1Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana infected by F1Tsh R23/42b-Su351F3; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; the accumulation of PDS,Su and GFP mRNA all declined. Sap inoculation was adopted to analyze stability of F1Tsh R23/42b-NbPDS354F3in N. benthamiana. The results indicated that F1Tsh R23/42b-NbPDS354F3could exist in N. benthamiana steadily and induce photobleaching in host plants.
Fourthly, partial promoters of PDS, GFP of N. benthamiana, fragments of soybean PDS(named proPDS,35s and GmPDS) and part of Beclin were inserted into pTsh R23/42b to analyze photobleaching on N. benthamiana and soybean, red level on16C and symptoms on NN transgene tobacco inoculated with TMV. The resluts showed that:significant photobleaching was observed on N. benthamiana and soybean; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; Systemic necrosis emerged on NN transgene tobacco which was inoculated with TMV, after being inoculated with F1Tsh R23/42b-Beclin346F37dpi.
Fifthly, CMV infectious clones of Agrobacterium were constructed. Full-length sequences of Tsh R23/42b, Tsh R23/42b-NbPDS354, Tsh R23/42b-Su351, Fny RNA1, RNA2, RNA3were inserted into transient expression vector pCB301to transform into Agrobacterium. Photobleaching and yellow leaf table on N. benthamiana induced by F1Tsh R23/42b-NbPDS354F3and F1Tsh R23/42b-Su351F3were analyzed through inoculating N. benthamiana with Agrobacterium infiltration inoculation. The resluts showed that:symptoms induced by CMV-Fny of Agrobacterium were in keeping with ones induced by in vitro transcripts RNA; significant photobleaching was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-Su351F3.
引文
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