基于RNA干扰技术的加工番茄抗CMV、ToMV的研究
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摘要
植物病毒病是近年来严重危害加工番茄的主要病害之一，这与加工番茄种植面积不断扩大、栽培时间持续和重茬地大面积增加密切相关。黄瓜花叶病毒(Cucumbermosaic virus，CMV)和番茄花叶病毒（Tomato mosaic virus，ToMV）是严重危害加工番茄的两个主要病毒。在实际生产中，这两种病毒往往复合侵染加工番茄，使加工番茄上出现严重的条斑坏死症状，果实则表现为僵小畸形，表面易形成褐色斑块。严重影响加工番茄的产量和品质。
多年来，人们一直都在努力地寻找解决病毒病对农作物的危害方法，而抗病毒育种是目前解决这一难题的主要策略，由于传统育种方法费时费力等局限性，从而大大地限制了育种工作的进程。近年来，随着植物基因工程技术的快速发展，尤其是RNAi技术的发展为解决这一难题开辟了一条新的途径。
本研究以从加工番茄上分离出来的黄瓜花叶病毒分离物NS0-4和番茄花叶病毒分离物SCS-2的全长基因序列设计干扰靶序列，利用RNAi技术构建了一条抗CMV和ToMV加工番茄的技术体系。本文的主要研究内容如下:
1. RNAi载体的构建：利用软件对CMV分离物NS0-4和ToMV分离物SCS-2全长基因序列进行分析，设计干扰片段，通过同位酶(BamHI/BglII)将两个干扰片段的序列进行拼接，同时以一段大小为266bp的核苷酸序列为内含子，构建了4个RNAi载体（pBi35STC4、pBi35STC9、pBi35STC12和pBi35STC14）。
2.烟草遗传转化的研究：分别将4个重组质粒通过电击的方法导入农杆菌GV3101中，通过菌落PCR鉴定，表明重组质粒已被成功导入到农杆菌中。然后利用农杆菌介导的叶盘法转化普通烟（Nicotiana tabacum），经过卡那霉素（50mg/L）筛选及PCR鉴定，结果表明在转pBi35STC4的56株再生烟草植株中，有32株为转基因烟草；在转pBi35STC9的44株再生烟草植株中，有28株为转基因烟草；在转pBi35STC12的58株再生烟草植株中，有35株为转基因烟草；在转pBi35STC14的50株再生烟草植株中，有30株为转基因烟草。
3.烟草上RNAi效果的初步评价：将转基因烟草、野生型烟草进行如下3种处理：第一种处理只接种CMV；第二种处理只接种ToMV；第三种处理同时接种CMV和ToMV。通过症状观察及RT-PCR检测，结果显示在接种20d野生型烟草100%均表现出了不同程度的病毒症状，在接种30d，转pBi35STC9烟草中近50%表现出了不同程度的病毒症状，而其余50%的烟草在40d后也表现出不同的病毒症状；而转pBi35STC4、pBi35STC12和pBi35STC14烟草中，有10%～30%则在接毒40d表现出了病毒症状，结果表明在T0代转基因烟草中，转pBi35STC4、pBi35STC12和pBi35STC14烟草可延迟病毒发生，抗病毒效果比转pBi35STC9烟草好。经过对转基因烟草的RNAi效果的初步评价为转化加工番茄奠定了基础。
4.加工番茄遗传转化的研究：经过烟草的初步筛选，筛选出抗性较好的3个含重组质粒（pBi35STC4、pBi35STC12和pBi35STC14）的农杆菌，并将其转化加工番茄红番3号，通过卡那霉素（50mg/L）筛选及PCR鉴定，结果显示从1200个加工番茄子叶外植体中，获得27株转pBi35STC4加工番茄再生植株，经过PCR检测，其中有14株为转基因植株，转化效率为1.16%；从1500个加工番茄子叶外植体中，获得33株转pBi35STC12加工番茄再生植株，经过PCR检测，其中有19株为转基因植株，转化效率为1.26%；从1000个加工番茄子叶外植体中，获得23株转pBi35STC14加工番茄再生植株，经过PCR检测，其中有13株为转基因植株，转化效率为1.30%。
5.加工番茄上RNAi效果的初步评价：将转基因加工番茄植株与转基因烟草一样分为3组做3种处理，通过机械摩擦接种病毒，随后观察症状和RT-PCR检测。结果显示野生型加工番茄在接毒25d时100%表现出病毒症状，而转pBi35STC4、pBi35STC12和pBi35STC14加工番茄在接毒40d仅有0%～20%表现出病毒症状。转pBi35STC12和pBi35STC14加工番茄延迟病症的发生20～25d；而转pBi35STC4加工番茄则只能延迟5～10d。总之，转pBi35STC12和pBi35STC14加工番茄对病毒的干扰效果要比转pBi35STC4加工番茄要好。这为获得抗多种病毒加工番茄奠定了基础。
In recent years, virus disease is one of the serious diseases endangering the processingtomato in xinjiang. This is due to planting area expand of processing tomato, long cultivationand continuous cropping areas to increase. Cucumber mosaic virus (CMV) and Tomatomosaic virus (ToMV) are two main viruses which causes serious damage to processing tomato.In actual production, processing tomato plants are mixed infection by two viruses, processingtomato branches appear seriously streak necrosis, while it generated brown patches and jiangsmall defects on the fruit surface.The yields and quality of the processing tomato are seriouslyaffected.
Over the years, it has been difficult to find a solution to control crop virus diseaseeffectively, and anti-virus breeding becomes the main strategy to solve this problem. Beacusethe traditional breeding method takes long time, the progress of breeding work was limitedgreatly. In recent years, with rapid development of plant gene engineering technology,especially the development of RNAi technology, it provides a new way to solve this difficultproblem.
Interefence target fragments were designed based on the full-length sequences of CMVNS0-4and ToMV SCS-2isolated from processing tomato and technology system for breedingprocessing tomato against CMV and ToMV were constructed by RNAi.
1. The construction of RNAi expression vectors: The full-length sequence of the isolateof CMV NS0-4and ToMV SCS-2were analysised using the software, respectively. Twointerfence fragments were designed and joined with enzyme (BamHI/BglII), using a266bpnucleotide for introns. Four RNAi vectors (pBi35STC4、pBi35STC9、pBi35STC12andpBi35STC14) were constructed. Through a series of enzyme digestion and sequencing, theRNAi vectors were successfully constructed.
2. The research of tobacco genetic transformation: The four recombinant plasmids weretransformed into Agrobacterium GV3101by electric shock respectively and successfullyintroduced by PCR identification, then transformed into Nicotiana tabacum by leaf discmethod. Through kanamycin (50mg/L) screening and PCR detection, the results showed that32plants were transgenic tobacco plants with pBi35STC4in56regenerated tobacco plants,28plants were transgenic tobacco plants with pBi35STC9in44regenerated tobacco plants,35plants were transgenic tobacco plants with pBi35STC12in58regenerated tobacco plantsand30plants were transgenic tobacco plants with pBi35STC14in50regenerated tobaccoplants. A further sign of purpose fragments successfully were introduced to the genome ofNicotiana tabacum.
3. The preliminary evaluation of RNAi effects on tobacco: Transgenic tobacco and wildtype tobacco were processed3kinds of treatments which is inoculation by CMV only, byToMV only and by CMV and ToMV at the same time. The results showed that100%of wildtype tobacco showed a different degree of symptoms of the virus20d after inoculation, nearly50%of transgenic tobacco with pBi35STC9showed different levels of the symptoms30dafter inoculation, while the remaining50%of the tobacco also showed different symptoms40d after inoculation.10%~30%of the transgenic tobacco plants with pBi35STC4,pBi35STC12and pBi35STC14showed symptoms40d after inoculation. This suggests thatamong T0generation of transgenic tobacco with pBi35STC4, pBi35STC12and pBi35STC14can delay the symptoms and antiviral effect is better than pBi35STC9. After a preliminaryevaluation of the effect of RNAi transgenic tobacco has paved the way to transformprocessing tomato.
4. The research of processing tomato genetic transformation: Through the preliminaryscreening of tobacco, three recombinant plasmids (pBi35STC4, pBi35STC12andpBi35STC14) of high resistance were introduced to processing tomato Hongfan3byAgrobacterium-mediated transformation. The results showed that27regenerated plants wereobtained from1200callus of processing tomato,14of them were identified to be thetransgenic plants by PCR and the transformational rate was1.16%.33regenerated plantswere obtained from1500callus of processing tomato,19of them were identified to be thetransgenic plants by PCR and the transformational rate was1.26%.23regenerated plantswere obtained from1000callus of processing tomato,13of them were identified to be thetransgenic plants by PCR and the transformational rate was1.30%. A further sign of purposefragments were successfully introduced to the genome of the processing tomato.
5. The preliminary evaluation of RNAi effects on processing tomato: The transgenictomato plants and wild type tomato plants were divided into3groups and they wereincoulated by mechanical friction. Then by observation of the symptom and RT-PCRdetection,the results showed that100%of wild type processing tomato plants showed adifferent degree of symptoms25d after inoculation, while0%~20%of the transgenicprocessing tomato plants with pBi35STC4, pBi35STC12and pBi35STC14showed symptoms40d after inoculation.The results showed transgenic processing tomato plants withpBi35STC12and pBi35STC14could delay the happening of the disease20~25d, Whiletransgenic processing tomato plants with pBi35STC4could only delay5~10d. Anyhow, theeffect pBi35STC12and pBi35STC14on virus interference is better than pBi35STC4. Thismethod laid a solid foundation for processing tomato to improve resistance against viruses.
多年来，人们一直都在努力地寻找解决病毒病对农作物的危害方法，而抗病毒育种是目前解决这一难题的主要策略，由于传统育种方法费时费力等局限性，从而大大地限制了育种工作的进程。近年来，随着植物基因工程技术的快速发展，尤其是RNAi技术的发展为解决这一难题开辟了一条新的途径。
本研究以从加工番茄上分离出来的黄瓜花叶病毒分离物NS0-4和番茄花叶病毒分离物SCS-2的全长基因序列设计干扰靶序列，利用RNAi技术构建了一条抗CMV和ToMV加工番茄的技术体系。本文的主要研究内容如下:
1. RNAi载体的构建：利用软件对CMV分离物NS0-4和ToMV分离物SCS-2全长基因序列进行分析，设计干扰片段，通过同位酶(BamHI/BglII)将两个干扰片段的序列进行拼接，同时以一段大小为266bp的核苷酸序列为内含子，构建了4个RNAi载体（pBi35STC4、pBi35STC9、pBi35STC12和pBi35STC14）。
2.烟草遗传转化的研究：分别将4个重组质粒通过电击的方法导入农杆菌GV3101中，通过菌落PCR鉴定，表明重组质粒已被成功导入到农杆菌中。然后利用农杆菌介导的叶盘法转化普通烟（Nicotiana tabacum），经过卡那霉素（50mg/L）筛选及PCR鉴定，结果表明在转pBi35STC4的56株再生烟草植株中，有32株为转基因烟草；在转pBi35STC9的44株再生烟草植株中，有28株为转基因烟草；在转pBi35STC12的58株再生烟草植株中，有35株为转基因烟草；在转pBi35STC14的50株再生烟草植株中，有30株为转基因烟草。
3.烟草上RNAi效果的初步评价：将转基因烟草、野生型烟草进行如下3种处理：第一种处理只接种CMV；第二种处理只接种ToMV；第三种处理同时接种CMV和ToMV。通过症状观察及RT-PCR检测，结果显示在接种20d野生型烟草100%均表现出了不同程度的病毒症状，在接种30d，转pBi35STC9烟草中近50%表现出了不同程度的病毒症状，而其余50%的烟草在40d后也表现出不同的病毒症状；而转pBi35STC4、pBi35STC12和pBi35STC14烟草中，有10%～30%则在接毒40d表现出了病毒症状，结果表明在T0代转基因烟草中，转pBi35STC4、pBi35STC12和pBi35STC14烟草可延迟病毒发生，抗病毒效果比转pBi35STC9烟草好。经过对转基因烟草的RNAi效果的初步评价为转化加工番茄奠定了基础。
4.加工番茄遗传转化的研究：经过烟草的初步筛选，筛选出抗性较好的3个含重组质粒（pBi35STC4、pBi35STC12和pBi35STC14）的农杆菌，并将其转化加工番茄红番3号，通过卡那霉素（50mg/L）筛选及PCR鉴定，结果显示从1200个加工番茄子叶外植体中，获得27株转pBi35STC4加工番茄再生植株，经过PCR检测，其中有14株为转基因植株，转化效率为1.16%；从1500个加工番茄子叶外植体中，获得33株转pBi35STC12加工番茄再生植株，经过PCR检测，其中有19株为转基因植株，转化效率为1.26%；从1000个加工番茄子叶外植体中，获得23株转pBi35STC14加工番茄再生植株，经过PCR检测，其中有13株为转基因植株，转化效率为1.30%。
5.加工番茄上RNAi效果的初步评价：将转基因加工番茄植株与转基因烟草一样分为3组做3种处理，通过机械摩擦接种病毒，随后观察症状和RT-PCR检测。结果显示野生型加工番茄在接毒25d时100%表现出病毒症状，而转pBi35STC4、pBi35STC12和pBi35STC14加工番茄在接毒40d仅有0%～20%表现出病毒症状。转pBi35STC12和pBi35STC14加工番茄延迟病症的发生20～25d；而转pBi35STC4加工番茄则只能延迟5～10d。总之，转pBi35STC12和pBi35STC14加工番茄对病毒的干扰效果要比转pBi35STC4加工番茄要好。这为获得抗多种病毒加工番茄奠定了基础。
In recent years, virus disease is one of the serious diseases endangering the processingtomato in xinjiang. This is due to planting area expand of processing tomato, long cultivationand continuous cropping areas to increase. Cucumber mosaic virus (CMV) and Tomatomosaic virus (ToMV) are two main viruses which causes serious damage to processing tomato.In actual production, processing tomato plants are mixed infection by two viruses, processingtomato branches appear seriously streak necrosis, while it generated brown patches and jiangsmall defects on the fruit surface.The yields and quality of the processing tomato are seriouslyaffected.
Over the years, it has been difficult to find a solution to control crop virus diseaseeffectively, and anti-virus breeding becomes the main strategy to solve this problem. Beacusethe traditional breeding method takes long time, the progress of breeding work was limitedgreatly. In recent years, with rapid development of plant gene engineering technology,especially the development of RNAi technology, it provides a new way to solve this difficultproblem.
Interefence target fragments were designed based on the full-length sequences of CMVNS0-4and ToMV SCS-2isolated from processing tomato and technology system for breedingprocessing tomato against CMV and ToMV were constructed by RNAi.
1. The construction of RNAi expression vectors: The full-length sequence of the isolateof CMV NS0-4and ToMV SCS-2were analysised using the software, respectively. Twointerfence fragments were designed and joined with enzyme (BamHI/BglII), using a266bpnucleotide for introns. Four RNAi vectors (pBi35STC4、pBi35STC9、pBi35STC12andpBi35STC14) were constructed. Through a series of enzyme digestion and sequencing, theRNAi vectors were successfully constructed.
2. The research of tobacco genetic transformation: The four recombinant plasmids weretransformed into Agrobacterium GV3101by electric shock respectively and successfullyintroduced by PCR identification, then transformed into Nicotiana tabacum by leaf discmethod. Through kanamycin (50mg/L) screening and PCR detection, the results showed that32plants were transgenic tobacco plants with pBi35STC4in56regenerated tobacco plants,28plants were transgenic tobacco plants with pBi35STC9in44regenerated tobacco plants,35plants were transgenic tobacco plants with pBi35STC12in58regenerated tobacco plantsand30plants were transgenic tobacco plants with pBi35STC14in50regenerated tobaccoplants. A further sign of purpose fragments successfully were introduced to the genome ofNicotiana tabacum.
3. The preliminary evaluation of RNAi effects on tobacco: Transgenic tobacco and wildtype tobacco were processed3kinds of treatments which is inoculation by CMV only, byToMV only and by CMV and ToMV at the same time. The results showed that100%of wildtype tobacco showed a different degree of symptoms of the virus20d after inoculation, nearly50%of transgenic tobacco with pBi35STC9showed different levels of the symptoms30dafter inoculation, while the remaining50%of the tobacco also showed different symptoms40d after inoculation.10%~30%of the transgenic tobacco plants with pBi35STC4,pBi35STC12and pBi35STC14showed symptoms40d after inoculation. This suggests thatamong T0generation of transgenic tobacco with pBi35STC4, pBi35STC12and pBi35STC14can delay the symptoms and antiviral effect is better than pBi35STC9. After a preliminaryevaluation of the effect of RNAi transgenic tobacco has paved the way to transformprocessing tomato.
4. The research of processing tomato genetic transformation: Through the preliminaryscreening of tobacco, three recombinant plasmids (pBi35STC4, pBi35STC12andpBi35STC14) of high resistance were introduced to processing tomato Hongfan3byAgrobacterium-mediated transformation. The results showed that27regenerated plants wereobtained from1200callus of processing tomato,14of them were identified to be thetransgenic plants by PCR and the transformational rate was1.16%.33regenerated plantswere obtained from1500callus of processing tomato,19of them were identified to be thetransgenic plants by PCR and the transformational rate was1.26%.23regenerated plantswere obtained from1000callus of processing tomato,13of them were identified to be thetransgenic plants by PCR and the transformational rate was1.30%. A further sign of purposefragments were successfully introduced to the genome of the processing tomato.
5. The preliminary evaluation of RNAi effects on processing tomato: The transgenictomato plants and wild type tomato plants were divided into3groups and they wereincoulated by mechanical friction. Then by observation of the symptom and RT-PCRdetection,the results showed that100%of wild type processing tomato plants showed adifferent degree of symptoms25d after inoculation, while0%~20%of the transgenicprocessing tomato plants with pBi35STC4, pBi35STC12and pBi35STC14showed symptoms40d after inoculation.The results showed transgenic processing tomato plants withpBi35STC12and pBi35STC14could delay the happening of the disease20~25d, Whiletransgenic processing tomato plants with pBi35STC4could only delay5~10d. Anyhow, theeffect pBi35STC12and pBi35STC14on virus interference is better than pBi35STC4. Thismethod laid a solid foundation for processing tomato to improve resistance against viruses.
引文
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