RNA干涉技术在烟草抗TMV和PVY病毒育种中的应用
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摘要
烟草普通花叶病和烟草马铃薯Y病毒病是世界各烟草产区普遍发生的一类主要病害,也是我国北方烟区的主要病毒病害,尤其对我国黄淮烟区和东北烟区的影响较为严重,他们严重影响烟叶的产量和品质,而且在烟株上经常复合侵染,加大病害的防治难度。选育抗病品种是生产中防治病害的主要手段,但存在着抗性资源狭窄、抗病品种选育时间慢等诸多局限,不能有效地控制复合侵染对烟草生产带来的危害。随着分子生物学的发展,RNA干涉技术为病毒病害的防治提供了一条新途径。为了解决TMV和PVY复合侵染对烟草生产带来的危害,本试验以RNAi机制为依据,设计了双基因融合的RNAi靶序列,并成功构建植物双元表达载体系统。农杆菌侵染叶盘,最终获得9株转基因烟草苗。经PCR检测、TMV与PVY病毒验证,3株转基因烟草对TMV和PVY具有免疫级抗性。证实了以RNAi为基础的双基因融合策略在增强植物抵抗两种病毒侵染方面的效果。本试验主要内容如下:
1.TMV和PVY目的序列的克隆
分别对TMV和PVY外壳蛋白基因序列进行Blast序列比对,找出TMV、PVY外壳蛋白基因的保守序列,针对各自的保守序列确定进行融合的目的序列,设计TMV与PVY目的序列引物TMV-m/TMV-n;PVY-m/PVY-n。通过重新接种鉴定TMV与PVY病毒致病性后,分别提取TMV和PVY的总RNA,经RT-PCR反应获得TMV与PVY外壳蛋白基因的cDNA序列,应用TMV与PVY目的序列引物进行PCR扩增,产物连接进入pMD18-T Simple Vector,转化到大肠杆菌中扩繁,获得进行融合的TMV、PVY目的序列。
2.目的基因的融合
利用限制性内切酶对含有TMV片段的T载体质粒DNA和含有PVY片段的T载体质粒DNA进行双酶切;分别回收含TMV片段的pMD18-T载体质粒大片段和PVY小片段。然后两个DNA片段经T4 DNA Ligase连接,产物转化到大肠杆菌DH5α感受态细胞中扩繁,获得双基因融合的RNAi靶序列。
3.构建RNAi双元载体系统
采用酶切后连接方法,根据RNAi载体多克隆位点及目的序列边缘的相同酶切位点,构建含双基因融合序列(TMV-PVY-r)正反向重复结构的中间载体(pUCCRNAi-TMV-PVY-2)和表达载体(pC2300-35S-OCS-TMV-PVY-2)。分别以TMV-m,TMV-n/PVY-m,PVY-n;35S-F/35S-R超强启动子引物进行PCR扩增验证、限制性内切酶酶切验证,证实连接正确后经大肠杆菌扩繁,提取质粒转化农杆菌LBA4404感受态细胞。完成RNAi双元载体系统构的构建。
4.农杆菌介导法转化烟草
利用叶盘转化法,将试验获得的RNAi工程农杆菌侵染无菌烟草叶圆片,成功筛选出抗卡那霉素烟草个体86株并将其移栽至温室培养。
5.分子生物学检测转基因烟草及转基因烟草的抗性验证
利用改良CTAB法提取转基因烟草DNA,分别以特异引物(TMV-m/TMV-n;PVY-m/PVY-n)与超强启动子引物(35S-F/35S-R)对卡那霉素抗性材料进行PCR鉴定,确定9株转基因烟草苗为阳性。初步证实目的基因已经插入烟草基因组DNA中,利用人工接种TMV和PVY进行转基因烟草的抗病性鉴定。结果表明:转TMV和PVY外壳蛋白基因阳性个体的抗病性明显高于阴性的非转基因烟草,并且有3株达到了免疫级水平。
6.荧光定量PCR分析双基因融合RNAi效果
选择免疫级抗性的转基因烟草3株,田间未感染TMV与PVY的正常烟草1株,田间感染TMV与PVY的烟草1株,提取各自总RNA,经荧光定量PCR反应分析证实:三株免疫级抗性转基因烟草的TMV与PVY病毒外壳蛋白基因的mRNA拷贝数均低于接种的对照植株,说明RNAi确实引起了TMV与PVY外壳蛋白基因转录产物mRNA的降解,双基因融合干涉成功。为RNAi在抵抗多种病毒侵染方面提供了理论基础。
7.得到T0代转基因烟草并获得了T1代转基因材料。
Tobacco mosaic virus and Potato virus Y are two main diseases in the world tobacco production areas and they are also affect he north of our country.The virus affect the jield and quality seriously. It is difficult to control the two virus jointly affect the tobacco. Breeding for disease resistance is used as the important measure in agriculture.But the existing varieties have a narrow resource of resistance and a long time selection.As development of the molecular biology. RNAi technology provides a new way to improve plant resistance. To solution the dangerous of TMV and PVY affect tobacco jointly, the research follow the RNAi medicaments. IT connected the TMV and PVY CP gene as the target and constructed the RNAi expression vector containing 35s promoter.Finally, the expression vector was transformed into Agrobacterium tumefaciens LBA4404. With leaf dish transformation, we got nine trans-formants. They were approved with PCR reaction and virus identification. Three of which are immne resistance.The effect of RNA interference of double gene in improving the plant’s antivirotic capacity were verified primarily in the experiment. The main contents are as follows:
1. Cloning the target sequence of TMV-coat protein gene and PVY-coat protein gene
TMV-coat protein gene and PVY-coat protein gene were multiple sequence alignmented by BlastP on NCBI website respectively.The TMV and PVY’high homologous sequences were connected as the target sequence for RNA interference, according to which the primer pairs(TMV-m/TMV-n;PVY-m/PVY-n)of the target sequence were designed. After the TMV and PVY total RNA were extracted, the cDNA were gotten by the RT-PCR reaction. The TMV and PVY target sequences were obtained by PCR reaction using the cDNA as template with the primer pair (TMV-m/TMV-n;PVY-m/PVY-n). PCR products were linked to pMD18-T Vector respectively and replicated in E. f DH 5αrespectively.
2. Connected TMV coat protein and PVY coat protein
The pMD18-T-TMV and pMD18-T-PVY vector were digested restrictively by double-endonuclease respectively.Then we connected the PVY sequence and pMD18-T vector which contains TMV sequence. Finally the target gene was digested restrictively by double-endonuclease .So far the fusion gene was constructed.
3. Constructing RNA interference double-plasmid expression vector
After purifying the digested product, the double gene was inserted into pUCCRNAi vector by the counter-repeat way. Then the pUCCRNAi vector was inserted into expression vector. Finally,the recombination plasmid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid. With restriction endonuclease digestion, the double-plasmid expression vector was constructed successfully.
4. Tissue -cultured tobacco was infected by engineering-Agrobacterium tumefaciens.
The tissue-cultured tobacco was infected by Agrobacterium tumefaciens with leaf-dish transformation.Based on tissue–culturing in kanamycin selective medium, the kanamycin resistance tobaccos were planted in greenhouse.
5. Molecular biological examination and diseases resistance identification for transgenic tobacco
After the transgenic tobacco’RNA were extracted, We selecteded the kanamycin resistance plants with PCR reaction of TMV-m/TMV-n ; PVY-m/PVY-n and 35S-F/35S-R primers respectively. All the transgenic tobaccos were investigated and compare with the negative plants through manual vaccinating TMV and PVY. Three of the transgenic tobacco reached the immune resistance. Comparing with negative plant, the antiviral property positive of tobacco was increased significantly in transgenic tobacco.
6.Analyze the effect of double gene RNA interference by Fluorescent Quantitative PCR reaction
We extracted the DNA of three transgenic tobacco which reached the immune resistance、one normal tobacco and one plant that was infected TMV and PVY.With Fluorescent quantitative PCR reaction, we got the real-time PCR curve of samples and standard curre (TMV and PVY).The result indicated that the mRNA copies of three transgenic tobaccos were less than the infected plant.These demonstrated that the RNAi cause the decrease of viral mRNA.The RNA interference of TMC-cp and PVY-cp double gene has succeeded.The approach provide a foundation in multiple infect with RNAi.
7. Gotten the tobacco transformants of T0 and transgenic plant of T1.
1.TMV和PVY目的序列的克隆
分别对TMV和PVY外壳蛋白基因序列进行Blast序列比对,找出TMV、PVY外壳蛋白基因的保守序列,针对各自的保守序列确定进行融合的目的序列,设计TMV与PVY目的序列引物TMV-m/TMV-n;PVY-m/PVY-n。通过重新接种鉴定TMV与PVY病毒致病性后,分别提取TMV和PVY的总RNA,经RT-PCR反应获得TMV与PVY外壳蛋白基因的cDNA序列,应用TMV与PVY目的序列引物进行PCR扩增,产物连接进入pMD18-T Simple Vector,转化到大肠杆菌中扩繁,获得进行融合的TMV、PVY目的序列。
2.目的基因的融合
利用限制性内切酶对含有TMV片段的T载体质粒DNA和含有PVY片段的T载体质粒DNA进行双酶切;分别回收含TMV片段的pMD18-T载体质粒大片段和PVY小片段。然后两个DNA片段经T4 DNA Ligase连接,产物转化到大肠杆菌DH5α感受态细胞中扩繁,获得双基因融合的RNAi靶序列。
3.构建RNAi双元载体系统
采用酶切后连接方法,根据RNAi载体多克隆位点及目的序列边缘的相同酶切位点,构建含双基因融合序列(TMV-PVY-r)正反向重复结构的中间载体(pUCCRNAi-TMV-PVY-2)和表达载体(pC2300-35S-OCS-TMV-PVY-2)。分别以TMV-m,TMV-n/PVY-m,PVY-n;35S-F/35S-R超强启动子引物进行PCR扩增验证、限制性内切酶酶切验证,证实连接正确后经大肠杆菌扩繁,提取质粒转化农杆菌LBA4404感受态细胞。完成RNAi双元载体系统构的构建。
4.农杆菌介导法转化烟草
利用叶盘转化法,将试验获得的RNAi工程农杆菌侵染无菌烟草叶圆片,成功筛选出抗卡那霉素烟草个体86株并将其移栽至温室培养。
5.分子生物学检测转基因烟草及转基因烟草的抗性验证
利用改良CTAB法提取转基因烟草DNA,分别以特异引物(TMV-m/TMV-n;PVY-m/PVY-n)与超强启动子引物(35S-F/35S-R)对卡那霉素抗性材料进行PCR鉴定,确定9株转基因烟草苗为阳性。初步证实目的基因已经插入烟草基因组DNA中,利用人工接种TMV和PVY进行转基因烟草的抗病性鉴定。结果表明:转TMV和PVY外壳蛋白基因阳性个体的抗病性明显高于阴性的非转基因烟草,并且有3株达到了免疫级水平。
6.荧光定量PCR分析双基因融合RNAi效果
选择免疫级抗性的转基因烟草3株,田间未感染TMV与PVY的正常烟草1株,田间感染TMV与PVY的烟草1株,提取各自总RNA,经荧光定量PCR反应分析证实:三株免疫级抗性转基因烟草的TMV与PVY病毒外壳蛋白基因的mRNA拷贝数均低于接种的对照植株,说明RNAi确实引起了TMV与PVY外壳蛋白基因转录产物mRNA的降解,双基因融合干涉成功。为RNAi在抵抗多种病毒侵染方面提供了理论基础。
7.得到T0代转基因烟草并获得了T1代转基因材料。
Tobacco mosaic virus and Potato virus Y are two main diseases in the world tobacco production areas and they are also affect he north of our country.The virus affect the jield and quality seriously. It is difficult to control the two virus jointly affect the tobacco. Breeding for disease resistance is used as the important measure in agriculture.But the existing varieties have a narrow resource of resistance and a long time selection.As development of the molecular biology. RNAi technology provides a new way to improve plant resistance. To solution the dangerous of TMV and PVY affect tobacco jointly, the research follow the RNAi medicaments. IT connected the TMV and PVY CP gene as the target and constructed the RNAi expression vector containing 35s promoter.Finally, the expression vector was transformed into Agrobacterium tumefaciens LBA4404. With leaf dish transformation, we got nine trans-formants. They were approved with PCR reaction and virus identification. Three of which are immne resistance.The effect of RNA interference of double gene in improving the plant’s antivirotic capacity were verified primarily in the experiment. The main contents are as follows:
1. Cloning the target sequence of TMV-coat protein gene and PVY-coat protein gene
TMV-coat protein gene and PVY-coat protein gene were multiple sequence alignmented by BlastP on NCBI website respectively.The TMV and PVY’high homologous sequences were connected as the target sequence for RNA interference, according to which the primer pairs(TMV-m/TMV-n;PVY-m/PVY-n)of the target sequence were designed. After the TMV and PVY total RNA were extracted, the cDNA were gotten by the RT-PCR reaction. The TMV and PVY target sequences were obtained by PCR reaction using the cDNA as template with the primer pair (TMV-m/TMV-n;PVY-m/PVY-n). PCR products were linked to pMD18-T Vector respectively and replicated in E. f DH 5αrespectively.
2. Connected TMV coat protein and PVY coat protein
The pMD18-T-TMV and pMD18-T-PVY vector were digested restrictively by double-endonuclease respectively.Then we connected the PVY sequence and pMD18-T vector which contains TMV sequence. Finally the target gene was digested restrictively by double-endonuclease .So far the fusion gene was constructed.
3. Constructing RNA interference double-plasmid expression vector
After purifying the digested product, the double gene was inserted into pUCCRNAi vector by the counter-repeat way. Then the pUCCRNAi vector was inserted into expression vector. Finally,the recombination plasmid was transferred into Agrobacterium tumefaciens which only contained assistant plasmid. With restriction endonuclease digestion, the double-plasmid expression vector was constructed successfully.
4. Tissue -cultured tobacco was infected by engineering-Agrobacterium tumefaciens.
The tissue-cultured tobacco was infected by Agrobacterium tumefaciens with leaf-dish transformation.Based on tissue–culturing in kanamycin selective medium, the kanamycin resistance tobaccos were planted in greenhouse.
5. Molecular biological examination and diseases resistance identification for transgenic tobacco
After the transgenic tobacco’RNA were extracted, We selecteded the kanamycin resistance plants with PCR reaction of TMV-m/TMV-n ; PVY-m/PVY-n and 35S-F/35S-R primers respectively. All the transgenic tobaccos were investigated and compare with the negative plants through manual vaccinating TMV and PVY. Three of the transgenic tobacco reached the immune resistance. Comparing with negative plant, the antiviral property positive of tobacco was increased significantly in transgenic tobacco.
6.Analyze the effect of double gene RNA interference by Fluorescent Quantitative PCR reaction
We extracted the DNA of three transgenic tobacco which reached the immune resistance、one normal tobacco and one plant that was infected TMV and PVY.With Fluorescent quantitative PCR reaction, we got the real-time PCR curve of samples and standard curre (TMV and PVY).The result indicated that the mRNA copies of three transgenic tobaccos were less than the infected plant.These demonstrated that the RNAi cause the decrease of viral mRNA.The RNA interference of TMC-cp and PVY-cp double gene has succeeded.The approach provide a foundation in multiple infect with RNAi.
7. Gotten the tobacco transformants of T0 and transgenic plant of T1.
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