番茄斑萎病毒的分离鉴定及表达dsRNA的转基因烟草的抗病性研究
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摘要
番茄斑萎病毒(Tomato spotted wilt virus, TSWV)隶属于布尼亚病毒科(Bunuyaviridae)番茄斑萎病毒属(Tospovirus), Tospovirus是Bunuyaviridae中唯一一个可以侵染植物的属。TSWV广泛分布于世界各地,可侵染100多个科1000多种植物,常造成严重的经济损失,但在我国报道很少。近年来,随着其主要传毒介体——西花蓟马(Frankliniella occidentalis)的传播和扩散,我们发现TSWV也在逐渐蔓延扩散,分布范围日益扩大,寄主种类也在不断增加,加之TSWV难以防治,对农业生产构成了越来越严重的威胁。为了探究TSWV的扩散蔓延趋势,建立有效的TSWV防治方法,从2009～2013年,作者从云南等地采集了18份样本,分离纯化了部分TSWV株系,并对其进行了进化分析；利用RNAi,成功构建了不同片段长度的反向重复dsRNA植物表达载体,转化烟草后获得了转基因植株；并对表达TSWV dsRNA的转基因烟草的抗病性进行了初步的研究与分析。
本研究的主要内容和结果如下：
1.番茄斑萎病毒的分离及鉴定
首次从鸢尾和豇豆上分离得到TSWV。本文对采自不同地区疑似TSWV症状的样品进行了分离鉴定。从采自云南农业大学校园内的鸢尾叶片上,分离得到病原物,通过寄主症状鉴定,ELISA检测和进一步的分子生物学鉴定,确定该分离物为TSWV分离物,命名为YN-2。克隆了YN-2N基因和GN/GC基因的全长序列,经测序,YN-2N基因全长777bp (GenBank登录号：KC294570),编码258个氨基酸,GN/GC基因全长3408bp(GenBank登录号：KC294569),编码1135个氨基酸。经过与GenBank所登录的其他株系比对后,结果显示,YN-2与来自云南的番茄上(YN01)分离的TSWV的相似性最高：YN-2N基因和GN/GC基因与其相似性分别高达99.74%和99.53%；但在同源进化树分析中,TSWV N基因和GN/GC基因处于不同的分组中,由此我们推断编码GN/GC基因的MRNA与编码N基因的SRNA之间可能发生了基因重配。
另外,还从云南不同地区采集的番茄、辣椒和豇豆上分离得到了8个TSWV分离物,并对8个分离物的N基因和GN/GC基因进行了克隆与分析。8个分离物的N基因全长均为777bp,均编码258个氨基酸；GN/GC基因全长均为3408bp,均编码1135个氨基酸。8个分离物的N基因和GN/GC基因与GenBank中登录的中国的其他株系的N基因和GN/GC基因进行相似性比对后,结果显示,8个分离物的N基因和GN/GC基因与中国的其他株系的N基因和GN/GC基因相似性分别高达99.34%和99.38%。通过同源进化树分析发现,8个分离物虽然源自不同的寄主植物,但其起源是一致的。
2.转TSWV N基因dsRNA载体烟草及其抗病性研究
本研究将利用RNAi成功构建的2个不同片段长度的TSWV N基因dsRNA植物表达载体(±313-1304载体和±536-1304载体,简称313载体和536载体)通过农杆菌介导法转化烟草。并获得转313载体的三生烟阳性植株60株,转356载体的三生烟阳性植株79株,对To代和T1代转基因烟草进行了攻毒试验,攻毒试验结果表明：转536载体的T0代和T1代烟草植株的抗病效果均优于转313载体的T0代和T1代烟草植株的抗病效果；两种类型的转基因阳性植株T0代的抗病效果明显优于T1代的抗病效果。
3. TSWV GN/GC基因dsRNA载体构建及其在烟草中的表达
利用RNAi,成功构建了4个TSWV GN/GC基因不同区域及不同片段长度的反向重复dsRNA植物表达载体。根据TSWV GN/GC全长基因序列,设计含有限制性内切酶酶切位点(SwaI,AscI,SpeI,BamHI)的引物,扩增含有酶切位点的长度分别为297bp、500bp、328bp和503bp的目的片段,用于构建反向重复dsRNA植物表达载体。以最成熟的dsRNA载体pFGC1008为构建dsRNA的中间载体,其内含子是GUS基因的一段长为335bp的序列,反向重复dsRNA的植物表达载体为pCAMBIA1304,经PCR筛选、酶切和测序等方法进行验证,均表明已经成功的构建了重组载体,并且与预期相同。
将构建好的4个dsRNA植物表达载体通过农杆菌介导的方法转化烟草,目前已获得转G13-1304三生烟阳性植株20株,转G15-1304三生烟阳性植株20株,转G23-1304三生烟阳性植株15株,转G25-1304三生烟阳性植株16株。对转基因烟草的抗病性评价需要进一步的研究与分析。
The genus Tospovirus is the only phytopathogenic group in the family Bunyaviridae. Tomato spotted wilt virus (TSWV), a member of Tospovirus, is widely spread in the world, which can infect more than100genus including more than1000species of plants and cause serious losses. But it is seldom reported in China. For the past few years, with the spreading of western flower thrips (Frankliniella occidentalis), one of the main transmission vectors of TSWV, the distribution of TSWV is enlarging, the specie of its host is increasing. Since it's difficult to control, TSWV has posed very serious threat to agricultural production. In order to explore the spread trend and the control methods of TSWV, establish the detection method of TSWV, eighteen samples were collected from tomato, pepper, tobacco, cowpea, and so on in Yunnan province. Some of TSWV isolates were isolated and identified, and their phylogenetic relationship were analysed. Using the theory of RNAi, inverted-repeat dsRNA vectors with different length segments were constructed and transformed into Nicotiana tobacum cv. Samsun NN. Transgenic tobacco was achieved, and the resistance of transgenic tobacco expressed dsRNA of TSWV has been studied and analysed.
The results and main conclusions are as follows:
1. The isolation and identification of TSWV
In this study, through isolating and identifying suspected TSWV samples from different areas, a TSWV strain was isolated from Iris tectorum collected in Yunnan Agricultural University. We identified it as TSWV based on the results of the host's symptoms, ELISA and molecular detection, and named it as YN-2. The complete sequences of N protein and GN/GC glycoprotein were cloned, respectively. The results showed that the complete sequences of N protein were777nt and encoded258aa (accession number:KC294570), while the GN/GC glycoprotein sequences had3408nt and1135aa (accession number:KC294569). Their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins were analyzed. The phylogenetic relationship results showed that N protein and GN/GC glycoprotein of YN-2exhibited high homology with YN01in China:99.74%and99.53%, respectively. Through analysing their phylogenetic relationships, we found that the phylogenetic comparison of N protein and GN/GC glycoprotein was in different groups. We assumed that the genome reassortment may happen between M segment that encodes GN/GC glycoprotein and S segment that encodes N protein.
Besides, eight TSWV stains were isolated from tomato, pepper and cowpea which were collected in different areas in Yunnan province, and the complete sequences of N protein and GN/GC glycoprotein of the eight TSWV stains were cloned, respectively. The complete sequences of N protein of the eight TSWV stains were777nt and encoded258aa, GN/GC glycoprotein were3408bp and encoded1135aa. And their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins in China were analyzed. Their phylogenetic relationship results showed that N protein and GN/GC glycoprotein of eight TSWV pathogens exhibited high homology with other TSWVs:99.34%and99.38%, respectively. Through analysing the phylogenetic relationships, we found that eight TSWV pathogens have the same origin, although they were isolated from different host plants.
To our knowledge, this is the first time to report that TSWV is isolated from Iris tectorum and cowpea.
2. Study on TSWV N gene dsRNA vectors being transformed Nicotiana tobacum cv. Samsun NN, and the resistance of the transgensic tobacco
TSWV N gene segment of different length dsRNA vectors (±313-1304and±536-1304,313and536for short, respectively) had been constructed using RNAi in2010, and the dsRNA vectors had been introduced into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation, respectively. After tissue culture, sixty and seventy-nine transgenic tobaccos were achieved. Through observing the phenotype of T0and T1progeny after TSWV inoculation, we found that the resistance effects of T0and T1progeny of transformed536was better than those of transformed313; and the resistance effects of To was better than its of T1in all of transgenic tobaccos.
3. Study on the construction of dsRNA vectors with TSWV different length of GN/GC gene segment, and their expression in Nicotiana tobacum cv. Samsun NN
Using RNAi, four inverted-repeat dsRNA vectors with different length segments and different regions of TSWV GN/GC gene were constructed. According to TSWV GN/GC complete gene sequences, restriction enzyme site (Ascl, Swal, BamHl, Spel) were introducted when designing primers to amplied297bp,500bp,328bp and503bp segment, respectively, to construct inverted-repeat dsRNA expression vectors. The mature dsRNA vector, pFGC1008, was used as the inverted-repeat dsRNA vector, and335bp sequences of GUS gene in pFGC1008was used as the intron, pCAMBIA1304was used as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid was consistent with expected results by PCR analysis, restriction enzyme digestion and DNA sequencing.
The inverted-repeat dsRNA plant expression vectors were transformed into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation. After tissue culture, twenty transgenic tobaccos transformed G13-1304were achieved, twenty transformed G15-1304, fifteen transformed G23-1304and sixteen transformed G25-1304were also achieved. The resistance of the transgenic tobacco needs to be further analyzed.
本研究的主要内容和结果如下：
1.番茄斑萎病毒的分离及鉴定
首次从鸢尾和豇豆上分离得到TSWV。本文对采自不同地区疑似TSWV症状的样品进行了分离鉴定。从采自云南农业大学校园内的鸢尾叶片上,分离得到病原物,通过寄主症状鉴定,ELISA检测和进一步的分子生物学鉴定,确定该分离物为TSWV分离物,命名为YN-2。克隆了YN-2N基因和GN/GC基因的全长序列,经测序,YN-2N基因全长777bp (GenBank登录号：KC294570),编码258个氨基酸,GN/GC基因全长3408bp(GenBank登录号：KC294569),编码1135个氨基酸。经过与GenBank所登录的其他株系比对后,结果显示,YN-2与来自云南的番茄上(YN01)分离的TSWV的相似性最高：YN-2N基因和GN/GC基因与其相似性分别高达99.74%和99.53%；但在同源进化树分析中,TSWV N基因和GN/GC基因处于不同的分组中,由此我们推断编码GN/GC基因的MRNA与编码N基因的SRNA之间可能发生了基因重配。
另外,还从云南不同地区采集的番茄、辣椒和豇豆上分离得到了8个TSWV分离物,并对8个分离物的N基因和GN/GC基因进行了克隆与分析。8个分离物的N基因全长均为777bp,均编码258个氨基酸；GN/GC基因全长均为3408bp,均编码1135个氨基酸。8个分离物的N基因和GN/GC基因与GenBank中登录的中国的其他株系的N基因和GN/GC基因进行相似性比对后,结果显示,8个分离物的N基因和GN/GC基因与中国的其他株系的N基因和GN/GC基因相似性分别高达99.34%和99.38%。通过同源进化树分析发现,8个分离物虽然源自不同的寄主植物,但其起源是一致的。
2.转TSWV N基因dsRNA载体烟草及其抗病性研究
本研究将利用RNAi成功构建的2个不同片段长度的TSWV N基因dsRNA植物表达载体(±313-1304载体和±536-1304载体,简称313载体和536载体)通过农杆菌介导法转化烟草。并获得转313载体的三生烟阳性植株60株,转356载体的三生烟阳性植株79株,对To代和T1代转基因烟草进行了攻毒试验,攻毒试验结果表明：转536载体的T0代和T1代烟草植株的抗病效果均优于转313载体的T0代和T1代烟草植株的抗病效果；两种类型的转基因阳性植株T0代的抗病效果明显优于T1代的抗病效果。
3. TSWV GN/GC基因dsRNA载体构建及其在烟草中的表达
利用RNAi,成功构建了4个TSWV GN/GC基因不同区域及不同片段长度的反向重复dsRNA植物表达载体。根据TSWV GN/GC全长基因序列,设计含有限制性内切酶酶切位点(SwaI,AscI,SpeI,BamHI)的引物,扩增含有酶切位点的长度分别为297bp、500bp、328bp和503bp的目的片段,用于构建反向重复dsRNA植物表达载体。以最成熟的dsRNA载体pFGC1008为构建dsRNA的中间载体,其内含子是GUS基因的一段长为335bp的序列,反向重复dsRNA的植物表达载体为pCAMBIA1304,经PCR筛选、酶切和测序等方法进行验证,均表明已经成功的构建了重组载体,并且与预期相同。
将构建好的4个dsRNA植物表达载体通过农杆菌介导的方法转化烟草,目前已获得转G13-1304三生烟阳性植株20株,转G15-1304三生烟阳性植株20株,转G23-1304三生烟阳性植株15株,转G25-1304三生烟阳性植株16株。对转基因烟草的抗病性评价需要进一步的研究与分析。
The genus Tospovirus is the only phytopathogenic group in the family Bunyaviridae. Tomato spotted wilt virus (TSWV), a member of Tospovirus, is widely spread in the world, which can infect more than100genus including more than1000species of plants and cause serious losses. But it is seldom reported in China. For the past few years, with the spreading of western flower thrips (Frankliniella occidentalis), one of the main transmission vectors of TSWV, the distribution of TSWV is enlarging, the specie of its host is increasing. Since it's difficult to control, TSWV has posed very serious threat to agricultural production. In order to explore the spread trend and the control methods of TSWV, establish the detection method of TSWV, eighteen samples were collected from tomato, pepper, tobacco, cowpea, and so on in Yunnan province. Some of TSWV isolates were isolated and identified, and their phylogenetic relationship were analysed. Using the theory of RNAi, inverted-repeat dsRNA vectors with different length segments were constructed and transformed into Nicotiana tobacum cv. Samsun NN. Transgenic tobacco was achieved, and the resistance of transgenic tobacco expressed dsRNA of TSWV has been studied and analysed.
The results and main conclusions are as follows:
1. The isolation and identification of TSWV
In this study, through isolating and identifying suspected TSWV samples from different areas, a TSWV strain was isolated from Iris tectorum collected in Yunnan Agricultural University. We identified it as TSWV based on the results of the host's symptoms, ELISA and molecular detection, and named it as YN-2. The complete sequences of N protein and GN/GC glycoprotein were cloned, respectively. The results showed that the complete sequences of N protein were777nt and encoded258aa (accession number:KC294570), while the GN/GC glycoprotein sequences had3408nt and1135aa (accession number:KC294569). Their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins were analyzed. The phylogenetic relationship results showed that N protein and GN/GC glycoprotein of YN-2exhibited high homology with YN01in China:99.74%and99.53%, respectively. Through analysing their phylogenetic relationships, we found that the phylogenetic comparison of N protein and GN/GC glycoprotein was in different groups. We assumed that the genome reassortment may happen between M segment that encodes GN/GC glycoprotein and S segment that encodes N protein.
Besides, eight TSWV stains were isolated from tomato, pepper and cowpea which were collected in different areas in Yunnan province, and the complete sequences of N protein and GN/GC glycoprotein of the eight TSWV stains were cloned, respectively. The complete sequences of N protein of the eight TSWV stains were777nt and encoded258aa, GN/GC glycoprotein were3408bp and encoded1135aa. And their phylogenetic relationships with other TSWVs isolated from different hosts in different geographic origins in China were analyzed. Their phylogenetic relationship results showed that N protein and GN/GC glycoprotein of eight TSWV pathogens exhibited high homology with other TSWVs:99.34%and99.38%, respectively. Through analysing the phylogenetic relationships, we found that eight TSWV pathogens have the same origin, although they were isolated from different host plants.
To our knowledge, this is the first time to report that TSWV is isolated from Iris tectorum and cowpea.
2. Study on TSWV N gene dsRNA vectors being transformed Nicotiana tobacum cv. Samsun NN, and the resistance of the transgensic tobacco
TSWV N gene segment of different length dsRNA vectors (±313-1304and±536-1304,313and536for short, respectively) had been constructed using RNAi in2010, and the dsRNA vectors had been introduced into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation, respectively. After tissue culture, sixty and seventy-nine transgenic tobaccos were achieved. Through observing the phenotype of T0and T1progeny after TSWV inoculation, we found that the resistance effects of T0and T1progeny of transformed536was better than those of transformed313; and the resistance effects of To was better than its of T1in all of transgenic tobaccos.
3. Study on the construction of dsRNA vectors with TSWV different length of GN/GC gene segment, and their expression in Nicotiana tobacum cv. Samsun NN
Using RNAi, four inverted-repeat dsRNA vectors with different length segments and different regions of TSWV GN/GC gene were constructed. According to TSWV GN/GC complete gene sequences, restriction enzyme site (Ascl, Swal, BamHl, Spel) were introducted when designing primers to amplied297bp,500bp,328bp and503bp segment, respectively, to construct inverted-repeat dsRNA expression vectors. The mature dsRNA vector, pFGC1008, was used as the inverted-repeat dsRNA vector, and335bp sequences of GUS gene in pFGC1008was used as the intron, pCAMBIA1304was used as plant expression vector. The recombinant inverted-repeat dsRNA vector plasmid was consistent with expected results by PCR analysis, restriction enzyme digestion and DNA sequencing.
The inverted-repeat dsRNA plant expression vectors were transformed into Nicotiana tobacum cv. Samsun NN via Agrobacterium tumefaciens-mediated transformation. After tissue culture, twenty transgenic tobaccos transformed G13-1304were achieved, twenty transformed G15-1304, fifteen transformed G23-1304and sixteen transformed G25-1304were also achieved. The resistance of the transgenic tobacco needs to be further analyzed.
引文
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