植物抗病毒基因工程研究 Ⅰ黄瓜花叶病毒复制酶基因表达载体构建和遗传转化 Ⅱ导入病毒基因的转基因植物释放的风险评估
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摘要
根据已发表的黄瓜花叶病毒（CMV）Fny RNA2基因序列，设计了全长的2a复制
酶（Replicase）基因的特异引物，以CMV Fny RNA2 cDNA的克隆pFny209为模板，
进行PCR扩增，得到全长2.5kb的2a复制酶基因扩增产物（RP）。对此产物进行纯化，
并用NcoⅠ和BspHⅠ进行双酶切，得到三个片段。将不含有GDD保守区的两个片段用
T4 DNA连接酶连接，并对连接产物进行PCR扩增，得到2.2kb左右的缺失GDD保守
区的CMV 2a复制酶基因扩增产物（RP△GDD）。以CMV P1 RNA2基因组为模板，
根据CMV Fny RNA2基因组的发表序列设计引物，进行PCR扩增，得到5'端及3'端
分别缺失的2a复制酶基因（P1-RP）。将三个不同长度的2a复制酶基因片断克隆到
pGEM-T Easy Vector上，筛选获得重组质粒pTEVRP、pTEVRP△GDD和pTEVP1-RP。
对重组质粒进行序列测定，结果表明序列正确。
将三个复制酶基因重组于植物表达载体中，构建植物基因转化载体系统。用SmaⅠ
和BamHⅠ对重组质粒pTEVRP和pTEVRP△GDD双酶切，获得全长的（RP）及缺失
GDD保守区的2a复制酶基因片段（RP△GDD）；用SmaⅠ和XbaⅠ对重组质粒
PTEVP1-RP双酶切获得5'端及3'端分别缺失的复制酶基因片段（P1-RP）。将这些目
的基因片段定向克隆到植物表达载体pBI121中，PCR及酶切鉴定重组质粒正确。由此
获得了不同长度的2a复制酶基因的植物表达载体pBIRP、pBIRP△GDD和pBIP1-RP。
通过三亲交配法分别将三个植物表达载休导入根癌农杆菌EHA105中，PCR及酶
切鉴定，证实质粒均已导入农杆菌菌株。
通过叶盘转化法，获得了转化CMV P1-RP基因的烟草植株。对10株转基因烟草
进行PCR检测，9株能够扩增出目的基因。取PCR鉴定为阳性的3个植株，进行Southern
blot印迹分析，其杂交结果均为阳性，该结果表明CMV P1-RP基因已整合于烟草基
因组DNA中。对3株烟草的Northern blot印迹杂交结果也为阳性，表明转入烟草的CMV
P1-RP基因已在受体植物中转录表达。
建立了番茄亲本B-1的遗传转化体系，找到了适宜番茄B-1分化的培养基及最佳
l 激素比率，芽的分化率（叶盘上形成的芽数与叶盘的比）可达500％以上。
l 通过叶盘转化法，同样获得了转化CMV Fnv全长的 Za复制酶基因的番茄植株。
l 对 10株转基因番茄进行 PCR检测，有 7株能够扩增出目的基因。取 3株 PCR鉴定为
l 阳性的植株，进行 S。uthm hi仇印迹分析，其杂交结果均为阳性，而且其中 1株存在
l 多拷贝，表明 CMV Ffiy Za复制酶基因已整合于番茄基因组 DNA中。对 3株番茄的
INorthern blot印迹分析发现2株杂交信号为阳性，表明转入番茄的CMV Ffly Za复制
D 酶基因已在部分受体植物中得到表达。
l 用 CMV PI株系和 CMV RB株系接种转基因植物，通过症状观察和 ELISA检测，
l 转基因植株有三种不同的抗性反应。即症状严重型；症状延迟型；无症状型。ELISA
l 测定结果也表明，一部分转基因植物对 CMV PI株系和 CMV RB株系没有抗性；一部
D 分转基因植物具有延迟发病作用；少部分转基因植物具有抗病作用。有关抗性机理的
0 研究正在进行中。
D 导入TOMV、TMV移动蛋白基因的烟草和导入CMV PI－RP复制酶基因的烟草，分
【别经组织培养扩繁并移栽于温室中。4－5叶期分别接种CMVRB株系、PVX、PVY、l
ITMV，并以非转化普通烟为对照。症状观察表明，病毒在转基因植株上的症状与对照植l
D 株上的症状无明显差异，ELISA测定也表明转化植株体内病毒的含量与对照相近。
将导入ToMV移动蛋白基因的烟草接种TMV，一个月后取其系统叶接种心叶烟D
和三生烟（NN基因型），并以TMV接种心叶烟和三生烟为对照。症状观察结果表明l
两者接种的JL’叶烟和三生烟病斑大小相近、病斑颜色相同。随后10个月中定期取系统D
叶接种心叶烟和三生烟，观察病斑大小的变化，没有发现所转基因与接种病毒之间的D
重组。将导入CMV PI．RP复制酶基因的烟草接种CMV RB株系，一个月后取其系统l
叶接种蚕豆，并以CMV RB株系接种蚕豆为对照。结果表明两者接种的蚕豆病斑大小 l
相同。随后6个月中定期取系统叶接种蚕豆，观察病斑大小的变化，也没有发现所转0
基因与接种病毒之间的重组。D
According to the published RNA2 sequence of Cucumber mosaic virus ( CMV ) Fny, the specific primers were designed. The entire CMV Fny 2a replicase gene (RP) with length of 2.5kb was amplified by polymerase chain reaction (PCR). This PCR product was digested with Nco I and BspH I, and three fragments were obtained. Two fragments which couldn't encode GDD motif were ligated and amplified by PCR. And a 2.2kb product with deletion of GDD motif was obtained. The specific primers were also designed for amplification of a 530bp fragment containing partial replicase gene with GDD motif using CMV PI as template. These three replicase genes of CMV were cloned into pGEM-T Easy Vector. DNA sequence analyses confirmed that the ORFs of the amplicated replicase gene were correct.
In order to construct expression vectors, the 2a replicase gene and the 2a replicase gene
without GDD motif were cleavaged from pGEM-T Easy Vector with Sma I and BamR I. The 530bp partial CMV PI 2a replicase gene fragment was also cleavaged from pGEM-T Easy Vector with Sma I and Xba I. These replicase genes were cloned into plant expression vector pBI121. PCR and double digestion confirmed that the recombination plasmids contain the target genes.
The three genes in plant expression vectors were transferred into Agrobacterium tumefaciens EHA105 by tri-parental mating method. PCR and double digestion also confirmed that the three of target genes were transferred into Agrobacterium tumefaciens.
The transformed tobacco plants with CMV Pl-RP gene were obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tobaccos showed CMV Pl-RP gene could be amplified from 9 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tobacco. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed all the samples had hybridization signal with CMV Pl-RP gene as probe, and no
hybridization signal was observed in control. This result indicated the exogenous CMV Pl-RP gene has been transcribed hi the transformed tobaccos.
A transformation system was established for the tomato line B-l, The optimum medium and ratio of hormone (MS+Zeatin 0.5mg/ml+BA 3.0mg/ml+IAA0.2mg/ml) to regenerate buds have been founded. The rate of shoot differentiation has reached 500% (the number of buds /per disc).
The transformed tomato plants with 2a replicase gene were also obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tomatos showed CMV RP gene could be amplified from 7 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tomato. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed two samples had hybridization signal with CMV Pl-RP gene as probe, and no hybridization signal was observed in control. This result indicated the exogenous CMV Fny RNA2 replicase gene has been transcribed in the transformed tomatos.
The transgenic tobaccos with CMV Pl-RP gene and tomatos with CMV Fny RNA2 2a replicase gene were inoculated with the strain of CMV PI and CMV RB respectively. Some of the transgenic plants were found susceptible to virus infection, delayed symptoms appearance were found in several transgenic plants and a few transgenic plants showed resistance to CMV. Further experiment is still going on.
The transgenic tobaccos with movement proteins of ToMV, TMV and r
酶（Replicase）基因的特异引物，以CMV Fny RNA2 cDNA的克隆pFny209为模板，
进行PCR扩增，得到全长2.5kb的2a复制酶基因扩增产物（RP）。对此产物进行纯化，
并用NcoⅠ和BspHⅠ进行双酶切，得到三个片段。将不含有GDD保守区的两个片段用
T4 DNA连接酶连接，并对连接产物进行PCR扩增，得到2.2kb左右的缺失GDD保守
区的CMV 2a复制酶基因扩增产物（RP△GDD）。以CMV P1 RNA2基因组为模板，
根据CMV Fny RNA2基因组的发表序列设计引物，进行PCR扩增，得到5'端及3'端
分别缺失的2a复制酶基因（P1-RP）。将三个不同长度的2a复制酶基因片断克隆到
pGEM-T Easy Vector上，筛选获得重组质粒pTEVRP、pTEVRP△GDD和pTEVP1-RP。
对重组质粒进行序列测定，结果表明序列正确。
将三个复制酶基因重组于植物表达载体中，构建植物基因转化载体系统。用SmaⅠ
和BamHⅠ对重组质粒pTEVRP和pTEVRP△GDD双酶切，获得全长的（RP）及缺失
GDD保守区的2a复制酶基因片段（RP△GDD）；用SmaⅠ和XbaⅠ对重组质粒
PTEVP1-RP双酶切获得5'端及3'端分别缺失的复制酶基因片段（P1-RP）。将这些目
的基因片段定向克隆到植物表达载体pBI121中，PCR及酶切鉴定重组质粒正确。由此
获得了不同长度的2a复制酶基因的植物表达载体pBIRP、pBIRP△GDD和pBIP1-RP。
通过三亲交配法分别将三个植物表达载休导入根癌农杆菌EHA105中，PCR及酶
切鉴定，证实质粒均已导入农杆菌菌株。
通过叶盘转化法，获得了转化CMV P1-RP基因的烟草植株。对10株转基因烟草
进行PCR检测，9株能够扩增出目的基因。取PCR鉴定为阳性的3个植株，进行Southern
blot印迹分析，其杂交结果均为阳性，该结果表明CMV P1-RP基因已整合于烟草基
因组DNA中。对3株烟草的Northern blot印迹杂交结果也为阳性，表明转入烟草的CMV
P1-RP基因已在受体植物中转录表达。
建立了番茄亲本B-1的遗传转化体系，找到了适宜番茄B-1分化的培养基及最佳
l 激素比率，芽的分化率（叶盘上形成的芽数与叶盘的比）可达500％以上。
l 通过叶盘转化法，同样获得了转化CMV Fnv全长的 Za复制酶基因的番茄植株。
l 对 10株转基因番茄进行 PCR检测，有 7株能够扩增出目的基因。取 3株 PCR鉴定为
l 阳性的植株，进行 S。uthm hi仇印迹分析，其杂交结果均为阳性，而且其中 1株存在
l 多拷贝，表明 CMV Ffiy Za复制酶基因已整合于番茄基因组 DNA中。对 3株番茄的
INorthern blot印迹分析发现2株杂交信号为阳性，表明转入番茄的CMV Ffly Za复制
D 酶基因已在部分受体植物中得到表达。
l 用 CMV PI株系和 CMV RB株系接种转基因植物，通过症状观察和 ELISA检测，
l 转基因植株有三种不同的抗性反应。即症状严重型；症状延迟型；无症状型。ELISA
l 测定结果也表明，一部分转基因植物对 CMV PI株系和 CMV RB株系没有抗性；一部
D 分转基因植物具有延迟发病作用；少部分转基因植物具有抗病作用。有关抗性机理的
0 研究正在进行中。
D 导入TOMV、TMV移动蛋白基因的烟草和导入CMV PI－RP复制酶基因的烟草，分
【别经组织培养扩繁并移栽于温室中。4－5叶期分别接种CMVRB株系、PVX、PVY、l
ITMV，并以非转化普通烟为对照。症状观察表明，病毒在转基因植株上的症状与对照植l
D 株上的症状无明显差异，ELISA测定也表明转化植株体内病毒的含量与对照相近。
将导入ToMV移动蛋白基因的烟草接种TMV，一个月后取其系统叶接种心叶烟D
和三生烟（NN基因型），并以TMV接种心叶烟和三生烟为对照。症状观察结果表明l
两者接种的JL’叶烟和三生烟病斑大小相近、病斑颜色相同。随后10个月中定期取系统D
叶接种心叶烟和三生烟，观察病斑大小的变化，没有发现所转基因与接种病毒之间的D
重组。将导入CMV PI．RP复制酶基因的烟草接种CMV RB株系，一个月后取其系统l
叶接种蚕豆，并以CMV RB株系接种蚕豆为对照。结果表明两者接种的蚕豆病斑大小 l
相同。随后6个月中定期取系统叶接种蚕豆，观察病斑大小的变化，也没有发现所转0
基因与接种病毒之间的重组。D
According to the published RNA2 sequence of Cucumber mosaic virus ( CMV ) Fny, the specific primers were designed. The entire CMV Fny 2a replicase gene (RP) with length of 2.5kb was amplified by polymerase chain reaction (PCR). This PCR product was digested with Nco I and BspH I, and three fragments were obtained. Two fragments which couldn't encode GDD motif were ligated and amplified by PCR. And a 2.2kb product with deletion of GDD motif was obtained. The specific primers were also designed for amplification of a 530bp fragment containing partial replicase gene with GDD motif using CMV PI as template. These three replicase genes of CMV were cloned into pGEM-T Easy Vector. DNA sequence analyses confirmed that the ORFs of the amplicated replicase gene were correct.
In order to construct expression vectors, the 2a replicase gene and the 2a replicase gene
without GDD motif were cleavaged from pGEM-T Easy Vector with Sma I and BamR I. The 530bp partial CMV PI 2a replicase gene fragment was also cleavaged from pGEM-T Easy Vector with Sma I and Xba I. These replicase genes were cloned into plant expression vector pBI121. PCR and double digestion confirmed that the recombination plasmids contain the target genes.
The three genes in plant expression vectors were transferred into Agrobacterium tumefaciens EHA105 by tri-parental mating method. PCR and double digestion also confirmed that the three of target genes were transferred into Agrobacterium tumefaciens.
The transformed tobacco plants with CMV Pl-RP gene were obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tobaccos showed CMV Pl-RP gene could be amplified from 9 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tobacco. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed all the samples had hybridization signal with CMV Pl-RP gene as probe, and no
hybridization signal was observed in control. This result indicated the exogenous CMV Pl-RP gene has been transcribed hi the transformed tobaccos.
A transformation system was established for the tomato line B-l, The optimum medium and ratio of hormone (MS+Zeatin 0.5mg/ml+BA 3.0mg/ml+IAA0.2mg/ml) to regenerate buds have been founded. The rate of shoot differentiation has reached 500% (the number of buds /per disc).
The transformed tomato plants with 2a replicase gene were also obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tomatos showed CMV RP gene could be amplified from 7 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tomato. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed two samples had hybridization signal with CMV Pl-RP gene as probe, and no hybridization signal was observed in control. This result indicated the exogenous CMV Fny RNA2 replicase gene has been transcribed in the transformed tomatos.
The transgenic tobaccos with CMV Pl-RP gene and tomatos with CMV Fny RNA2 2a replicase gene were inoculated with the strain of CMV PI and CMV RB respectively. Some of the transgenic plants were found susceptible to virus infection, delayed symptoms appearance were found in several transgenic plants and a few transgenic plants showed resistance to CMV. Further experiment is still going on.
The transgenic tobaccos with movement proteins of ToMV, TMV and r
引文
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