PRSV ihpRNA干扰载体的构建及转化番木瓜的研究
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摘要
番木瓜是重要的热带水果,含有丰富的营养成分,以富含木瓜蛋白酶而著称。番木瓜产业发展过程中遇到很多问题,其中番木瓜环斑病毒(Papaya ringspot virus, PRSV)病严重影响番木瓜生产。并且,这些问题依赖常规育种方法难以得到解决,必须依靠生物技术的方法。为研究番木瓜广谱抗病毒机制,培育适合我国南方地区栽培的番木瓜新品系,本研究利用RNAi介导的抗病机理,通过花粉管通道法将几个不同ihpRNA植物表达载体遗传转化番木瓜,为我国番木瓜生物技术研究提供理论及实践依据。具体研究结果如下：
1、基于PRSV-HN株系CP基因序列及其保守序列,利用非对称重叠延伸PCR (Asymmetric Overlap Extension PCR),建立了快速构建植物ihpRNA结构的方法。该方法只需在一个PCR管中进行一步反应法即可获得ihpRNA结构,具有简单、快速、成本低等优点。利用本方法,成功构建了hpRNA-S1(9351-9705bp). hpRNA-S2(9406-9705bp)、 hpRNA-S3(9535-9705bp)、hpRNA-S4(9590-9705bp)、 hpRNA-S5(9655-9705bp)、 hpRNA-S6(9720-10073bp)、 hpRNA-S8(9940-10073bp)、hpRNA-S9(9974-10073bp)、hpRNA-S13(9590-10073bp)、 hpRNA-S14(9655-10073bp)10个PRSV-CP基因不同位置和不同大小靶片段的ihpRNA结构。
2、利用两轮重叠延伸PCR (Overlap Extension PCR, OE-PCR)技术,建立了快速构建嵌合型植物ihpRNA结构的方法。该方法可以同时靶向多个基因或一个基因的多个位点,且构建过程简单、快速。基于PRSV-HN株系CP和VPg基因的保守序列,利用本方法,成功构建了1个嵌合型植物ihpRNA结构hpRNA-CV,该结构同时靶向PRSV上CP基因和VPg基因,有望增强RNA干扰效果,提高番木瓜对PRSV的持久抗性和广谱抗性。
3、利用植物表达载体pB1121和pCambia3300,构建了hpRNA-S5、 hpRNA-S6、hpRNA-S8以及嵌合型的hpRNA-CV结构的植物表达载体,分别为pCambia-hpRNA-S5、pCambia-hpRNA-S6、pCambia-hpRNA-S8和pCambia-hpRNA-CV。
4、通过花粉管通道法,将pCambia-hpRNA-S5、pCambia-hpRNA-S6、 pCambia-hpRNA-S8、pCambia-hpRNA-CV质粒DNA导入番木瓜,进行番木瓜遗传转化,转化后收获的转化番木瓜种子依次进行了除草剂(PPT)筛选、PCR检测、及RT-PCR验证试验,最终共获得了24株转基因阳性的番木瓜种子苗,其中：转pCambia-hpRNA-S5植株5株；转pCambia-hpRNA-S6植株6株；转pCambia-hpRNA-S8植株8株；转pCambia-hpRNA-CV植株5株。RT-PCR检测结果表明：筛选获得的转基因植株中目标片段获得了稳定表达。
5、综合PRSV攻毒实验后供试番木瓜苗的生长发病情况和ELISA检测结果,初步认为共获得了10个抗性的转基因番木瓜株系。转pCambia-hpRNA-S5基因获得2个抗性株系；转pCambia-hpRNA-S8基因获得4个抗性株系；转pCambia-hpRNA-S6基因获得2个抗性株系；转pCambia-hpRNA-CV基因获得2个抗性株系。且初步证明转基因番木瓜抗病植株病毒积累量显著降低,病毒累积量与植株抗性性呈负相关性。
6、Northern blot结果表明,当ihpRNA载体转化番木瓜后,能够较好地沉默病毒PRSV-CP基因,使转基因植株具有明显的抗PRSV病毒活性。Northern blot产生的杂交信号,随着抗病性的减弱,PRSV-CP基因RNA的积累增多,杂交信号逐渐增强。抗病植株内PRSV-CP基因RNA的积累量明显低于感病株系,即转基因植株内PRSV-CP基因RNA的积累量与植株抗病性呈负相关性。
Papaya (Carica papaya L.) is an important fruit in tropical and subtropical regions, which is famous for containing rich nutrients and papaya protease. Many factors affect the development of papaya industry, and Papaya ringspot virus (PRSV), is a major disease. PRSV causes significant yield losses in papaya. These problems are much harder to solve rely on conventional breeding methods, and it must depend on the method of biological technology. In order to study the broad-spectrum antiviral mechanism in papaya and develop transgenic papaya with durable PRSV resistance in south China, we used hpRNA anti-virus technique and genetic transformed the four different ihpRNA expression vectors into papaya plants throught pollen tube channel transformation technology, to provided a basis of theory and practice for papaya biotechnology research in China.
The main results were as follows:
1Based on the conservative sequence of CP gene of PRSV-HN strain, we developed a novel method to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently throught asymmetric Overlap Extension PCR systerm. This method is a simple, rapid and economic (without any expensive reagent) technique for ihpRNA structure constructing. It is a one-step PCR in one tube without any intermediate purification. Based on the conservative CP gene (coat protein) sequence from hainan PRSV (PRSV-HN), we successfully generated10ihpRNA structure which target on difference sites of PRSV-CP gene, for respectively:hpRNA-Sl (9351-9705bp), hpRNA-S2(9406-9705bp), hpRNA-S3(9535-9705bp), hpRNA-S4(9590-9705bp), hpRNA-S5(9655-9705bp), hpRNA-S6(9720-10073bp), hpRNA-S8(9940-10073bp), hpRNA-S9(9974-10073bp), hpRNA-S13(9590-10073bp), hpRNA-S14(9655-10073bp).
2Based on two rounds of Gene Splicing by Overlap Extension PCR (OE-PCR) technology, we developed a method to obtain mosaic ihpRNA structure. Its advantage is that it can target at several genes or multiple parts of one gene. Based on CP gene and VPg gene of PRSV-HN, we built one mosaic ihpRNA structure, named as hpRNA-CV. It targets on the two difference site of PRSV, which is promising to obtain broad-spectrum resistance and enhance the resistance to PRSV in papaya.
3Using the pBI121and pCambia3300as background, we obtained four plant expression vector: pCambia-hpRNA-S5, pCambia-hpRNA-S8, pCambia-hpRNA-S6and pCambia-hpRNA-CV, which based on the structure of hpRNA-S5, hpRNA-S8, hpRNA-S6and hpRNA-CV.
4The plant expression vectors with the ihpRNA structure were transformed into papaya plants (Sui zhong hong) through the pollen tube channel technology. The plasmid DNA was used as material in pollen tube channel technology platform. Through herbicide selection、 PCR detection, we got24plants of transfermation papaya, they are5plants of pCambia-hpRNA-S5,8plants of pCambia-hpRNA-S8,6plants of pCambia-hpRNA-S6and5plants of pCambia-hpRNA-CV. RT-PCR test results showed that the interest genes were stable expressed in transgenic plants.
5Throught resistance test of PRSV virus poison attack and ELISA test, we got10resistance transgenic papaya plants. In detail:there are2transgenic plants of pCambia-hpRNA-S5,4transgenic plants of pCambia-hpRNA-S8,2transgenic plants of pCambia-hpRNA-S6,2transgenic plants of pCambia-hpRNA-CV. The tests show that the cumulant of virus decreased significantly than the control plants. That is to say that the cumulant of virus and plant resistance to PRSV have a negative correlation.
6Northern blot results revealed that PRSV-CP gene was silenced in ihpRNA transgenic papaya plants, which enable the transgenic plants have obvious resistance to PRSV. The signals of Northern blot hybridization increased following the disease-resistant's weaken of transgenic plants. The RNA accumulation of PRSV-CP gene in PRSV-resistant plants is significantly lower than the sensitive plants. That is to say the RNA accumulation of PRSV-CP gene in the transgenic plant showed a negative correlation with virus resistance.
1、基于PRSV-HN株系CP基因序列及其保守序列,利用非对称重叠延伸PCR (Asymmetric Overlap Extension PCR),建立了快速构建植物ihpRNA结构的方法。该方法只需在一个PCR管中进行一步反应法即可获得ihpRNA结构,具有简单、快速、成本低等优点。利用本方法,成功构建了hpRNA-S1(9351-9705bp). hpRNA-S2(9406-9705bp)、 hpRNA-S3(9535-9705bp)、hpRNA-S4(9590-9705bp)、 hpRNA-S5(9655-9705bp)、 hpRNA-S6(9720-10073bp)、 hpRNA-S8(9940-10073bp)、hpRNA-S9(9974-10073bp)、hpRNA-S13(9590-10073bp)、 hpRNA-S14(9655-10073bp)10个PRSV-CP基因不同位置和不同大小靶片段的ihpRNA结构。
2、利用两轮重叠延伸PCR (Overlap Extension PCR, OE-PCR)技术,建立了快速构建嵌合型植物ihpRNA结构的方法。该方法可以同时靶向多个基因或一个基因的多个位点,且构建过程简单、快速。基于PRSV-HN株系CP和VPg基因的保守序列,利用本方法,成功构建了1个嵌合型植物ihpRNA结构hpRNA-CV,该结构同时靶向PRSV上CP基因和VPg基因,有望增强RNA干扰效果,提高番木瓜对PRSV的持久抗性和广谱抗性。
3、利用植物表达载体pB1121和pCambia3300,构建了hpRNA-S5、 hpRNA-S6、hpRNA-S8以及嵌合型的hpRNA-CV结构的植物表达载体,分别为pCambia-hpRNA-S5、pCambia-hpRNA-S6、pCambia-hpRNA-S8和pCambia-hpRNA-CV。
4、通过花粉管通道法,将pCambia-hpRNA-S5、pCambia-hpRNA-S6、 pCambia-hpRNA-S8、pCambia-hpRNA-CV质粒DNA导入番木瓜,进行番木瓜遗传转化,转化后收获的转化番木瓜种子依次进行了除草剂(PPT)筛选、PCR检测、及RT-PCR验证试验,最终共获得了24株转基因阳性的番木瓜种子苗,其中：转pCambia-hpRNA-S5植株5株；转pCambia-hpRNA-S6植株6株；转pCambia-hpRNA-S8植株8株；转pCambia-hpRNA-CV植株5株。RT-PCR检测结果表明：筛选获得的转基因植株中目标片段获得了稳定表达。
5、综合PRSV攻毒实验后供试番木瓜苗的生长发病情况和ELISA检测结果,初步认为共获得了10个抗性的转基因番木瓜株系。转pCambia-hpRNA-S5基因获得2个抗性株系；转pCambia-hpRNA-S8基因获得4个抗性株系；转pCambia-hpRNA-S6基因获得2个抗性株系；转pCambia-hpRNA-CV基因获得2个抗性株系。且初步证明转基因番木瓜抗病植株病毒积累量显著降低,病毒累积量与植株抗性性呈负相关性。
6、Northern blot结果表明,当ihpRNA载体转化番木瓜后,能够较好地沉默病毒PRSV-CP基因,使转基因植株具有明显的抗PRSV病毒活性。Northern blot产生的杂交信号,随着抗病性的减弱,PRSV-CP基因RNA的积累增多,杂交信号逐渐增强。抗病植株内PRSV-CP基因RNA的积累量明显低于感病株系,即转基因植株内PRSV-CP基因RNA的积累量与植株抗病性呈负相关性。
Papaya (Carica papaya L.) is an important fruit in tropical and subtropical regions, which is famous for containing rich nutrients and papaya protease. Many factors affect the development of papaya industry, and Papaya ringspot virus (PRSV), is a major disease. PRSV causes significant yield losses in papaya. These problems are much harder to solve rely on conventional breeding methods, and it must depend on the method of biological technology. In order to study the broad-spectrum antiviral mechanism in papaya and develop transgenic papaya with durable PRSV resistance in south China, we used hpRNA anti-virus technique and genetic transformed the four different ihpRNA expression vectors into papaya plants throught pollen tube channel transformation technology, to provided a basis of theory and practice for papaya biotechnology research in China.
The main results were as follows:
1Based on the conservative sequence of CP gene of PRSV-HN strain, we developed a novel method to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently throught asymmetric Overlap Extension PCR systerm. This method is a simple, rapid and economic (without any expensive reagent) technique for ihpRNA structure constructing. It is a one-step PCR in one tube without any intermediate purification. Based on the conservative CP gene (coat protein) sequence from hainan PRSV (PRSV-HN), we successfully generated10ihpRNA structure which target on difference sites of PRSV-CP gene, for respectively:hpRNA-Sl (9351-9705bp), hpRNA-S2(9406-9705bp), hpRNA-S3(9535-9705bp), hpRNA-S4(9590-9705bp), hpRNA-S5(9655-9705bp), hpRNA-S6(9720-10073bp), hpRNA-S8(9940-10073bp), hpRNA-S9(9974-10073bp), hpRNA-S13(9590-10073bp), hpRNA-S14(9655-10073bp).
2Based on two rounds of Gene Splicing by Overlap Extension PCR (OE-PCR) technology, we developed a method to obtain mosaic ihpRNA structure. Its advantage is that it can target at several genes or multiple parts of one gene. Based on CP gene and VPg gene of PRSV-HN, we built one mosaic ihpRNA structure, named as hpRNA-CV. It targets on the two difference site of PRSV, which is promising to obtain broad-spectrum resistance and enhance the resistance to PRSV in papaya.
3Using the pBI121and pCambia3300as background, we obtained four plant expression vector: pCambia-hpRNA-S5, pCambia-hpRNA-S8, pCambia-hpRNA-S6and pCambia-hpRNA-CV, which based on the structure of hpRNA-S5, hpRNA-S8, hpRNA-S6and hpRNA-CV.
4The plant expression vectors with the ihpRNA structure were transformed into papaya plants (Sui zhong hong) through the pollen tube channel technology. The plasmid DNA was used as material in pollen tube channel technology platform. Through herbicide selection、 PCR detection, we got24plants of transfermation papaya, they are5plants of pCambia-hpRNA-S5,8plants of pCambia-hpRNA-S8,6plants of pCambia-hpRNA-S6and5plants of pCambia-hpRNA-CV. RT-PCR test results showed that the interest genes were stable expressed in transgenic plants.
5Throught resistance test of PRSV virus poison attack and ELISA test, we got10resistance transgenic papaya plants. In detail:there are2transgenic plants of pCambia-hpRNA-S5,4transgenic plants of pCambia-hpRNA-S8,2transgenic plants of pCambia-hpRNA-S6,2transgenic plants of pCambia-hpRNA-CV. The tests show that the cumulant of virus decreased significantly than the control plants. That is to say that the cumulant of virus and plant resistance to PRSV have a negative correlation.
6Northern blot results revealed that PRSV-CP gene was silenced in ihpRNA transgenic papaya plants, which enable the transgenic plants have obvious resistance to PRSV. The signals of Northern blot hybridization increased following the disease-resistant's weaken of transgenic plants. The RNA accumulation of PRSV-CP gene in PRSV-resistant plants is significantly lower than the sensitive plants. That is to say the RNA accumulation of PRSV-CP gene in the transgenic plant showed a negative correlation with virus resistance.
引文
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