无选择标记高含类黄酮抗晚疫病马铃薯转基因研究
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摘要
马铃薯(Solanum tuberosum)是世界上第四大粮食作物,由疫霉菌(Phytophthora infestans de Bary)引起的马铃薯晚疫病是马铃薯生产上的一种毁灭性病害。类黄酮是一类天然的植物次生代谢产物,具有抗氧化和抗病毒病等重要功能,而目前广泛种植的马铃薯品种中仅含有痕量的类黄酮。本研究通过转化马铃薯植物类黄酮调控基因AtMYB12、马铃薯野生种来源的抗晚疫病基因Rpi-vnt1.1、Rpi-mcq1.1和Rpi-mcq1.2基因,获得一系列不同组合富含黄酮醇(如芦丁等)和抗晚疫病基因的马铃薯转基因品系。有望从晚疫病抗性和营养价值上对现有品种进行改良。
利用Cre/lox特异位点重组系统载体pX6-GFP,采用高保真PCR技术,本研究通过分步克隆的策略,分别构建了pX6-patatin::AtMYB12;pX6-Rpi-mcq1.1-Rpi-mcq1.2;pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1等三个载体,并导入农杆菌工程菌AGL I中。采用茎段转化法将上述3个载体转化晚疫病感病栽培种Desiree,分别获得PCR阳性转基因植株28株、20株和20株,阳性转化效率分别达到68%、40%和45%。
利用6个不同来源的马铃薯晚疫病菌株,采用离体叶片接种的方法对携带抗病基因的转基因系进行抗病性检测。结果显示转pX6-Rpi-mcq1.1-Rpi-mcq1.2双基因植株高抗EC1,90128两个菌株;转pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1三基因植株抗所有检测的六种菌株。研究结果与预期相符,所选基因完整被转入到马铃薯品种Desiree中。
利用HPLC法对两个携带AtMYB12基因的部分转基因株系进行了类黄酮含量检测,转基因pX6-patatin::AtMYB12马铃薯中芦丁含量在0.051-3.091mg g-1之间,山奈酚-芸香糖苷的含量达到0.041-0.951mg g-1之间。转pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi- vnt1.1马铃薯中类黄酮芦丁含量在0.08-0.2 mg g-1之间。总体类黄酮含量显著提高。
基于抗病表型或类黄酮含量分析,以及外观株型性状的稳定性,每个转基因组合各选择了2个株系进行了β-雌二醇的诱导处理,经NPTII基因特异性引物分析,分别获得无抗生素筛选标记的转基因株系并重新进行了抗病性检测。结果表明β-雌二醇的诱导产生NPTII重组缺失的概率在三个转基因系统中分别为10%, 8%, 8%。
综上所述,我们通过转基因策略获得了富含类黄酮或(和)抗晚疫病的转基因新品种,有望进一步通过田间筛选并通过转基因安全评估服务生产,解决生产上的关键问题。
Potato(Solanum tuberosum) is the fourth crop in the world. Late blight, caused by Phytophthora infestans de Bary, is the most destructive disease. Flavonoids, a class of natural plant secondary metabolites, have antioxidant, anti-virus and other important functions, but only trace amounts can be detected on current widely planting potato varieties. In this study, a plant flavonoids regulating gene AtMYB12 and three new cloned late blight resistance genes (Rpi-vnt1.1, Rpi-mcq1.1 and Rpi-mcq1.2 ) were differently combinated and transformed into a cultivated potato species Desiree in order to obtain a range of flavonol-rich ( such as rutin, etc.) and potato late blight resistant trangenic lines. It is expected to improve both traits of the late blight resistance and nutritional quality.
By using the Cre/lox site-specific recombination system PX6-GFP, three transgenic constructs were made with target genes from four donors through a two-step strategy with high-fidelity PCR, which included pX6-patatin:: AtMYB12, pX6-Rpi-mcq1.1-Rpi-mcq1.2 and pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1.Three recombinant vectors were mobilised into Agrobacterium strain AGL I, and then were introduced into the susceptible cultivar Desiree via Agrobacterium-mediated stem segment transformation. In total, 28, 20, 20 positive transgenic plant individuals were obtained with 68%, 40% and 45% of transformation efficiency respectively.
Rpi gene potato were tested with six P.infestans isoltates with detached leaf method. The potato carried Rpi-mcq1.1 and Rpi-mcq1.2 were resistant only for two isolates, EC1 and 90128 . Potato carried pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1 were resistant to all six isolates fallen into the former design.
Utilizing HPLC method, We detected the content of flavonoids in potato tubes with different lines carrying AtMYB12 gene. In potato carried pX6-patatin::AtMYB12, the content of rutin is rang from 0.051 mg g-1 to 3.091 mg g-1, and the kaempferol-rutinoside is ranged from 0.041 mg g-1 to 0.951 mg g-1. In addition, flavonoid content in pX6-patatin::AtMYB12 -Rpi-mcq1.1-Rpi-vnt1.1 potato is ranged from 0.08 to 0.2 mg g-1. The result indicated the overall flavonoid content is increased significantly.
Based on resistance phenotype, flavonoid content analysis and the stability of mor- phological traits, 2 lines from each selected transgenic combination were treated withβ-estradiol, and were tested by PCR to obtain NPTII marker-free plants, whose resistance were analysed again. The result indicated that the percentage of marker-free in 3 transgenic combinations is 10%, 8%, 8% respectively. In summary, we have obtained new flavonoid-rich or (and) late blight resistance varieties by transgenic strategy, which is expected to be further screened in the field and to pass the safety assessment, and to address the key problem in production.
利用Cre/lox特异位点重组系统载体pX6-GFP,采用高保真PCR技术,本研究通过分步克隆的策略,分别构建了pX6-patatin::AtMYB12;pX6-Rpi-mcq1.1-Rpi-mcq1.2;pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1等三个载体,并导入农杆菌工程菌AGL I中。采用茎段转化法将上述3个载体转化晚疫病感病栽培种Desiree,分别获得PCR阳性转基因植株28株、20株和20株,阳性转化效率分别达到68%、40%和45%。
利用6个不同来源的马铃薯晚疫病菌株,采用离体叶片接种的方法对携带抗病基因的转基因系进行抗病性检测。结果显示转pX6-Rpi-mcq1.1-Rpi-mcq1.2双基因植株高抗EC1,90128两个菌株;转pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1三基因植株抗所有检测的六种菌株。研究结果与预期相符,所选基因完整被转入到马铃薯品种Desiree中。
利用HPLC法对两个携带AtMYB12基因的部分转基因株系进行了类黄酮含量检测,转基因pX6-patatin::AtMYB12马铃薯中芦丁含量在0.051-3.091mg g-1之间,山奈酚-芸香糖苷的含量达到0.041-0.951mg g-1之间。转pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi- vnt1.1马铃薯中类黄酮芦丁含量在0.08-0.2 mg g-1之间。总体类黄酮含量显著提高。
基于抗病表型或类黄酮含量分析,以及外观株型性状的稳定性,每个转基因组合各选择了2个株系进行了β-雌二醇的诱导处理,经NPTII基因特异性引物分析,分别获得无抗生素筛选标记的转基因株系并重新进行了抗病性检测。结果表明β-雌二醇的诱导产生NPTII重组缺失的概率在三个转基因系统中分别为10%, 8%, 8%。
综上所述,我们通过转基因策略获得了富含类黄酮或(和)抗晚疫病的转基因新品种,有望进一步通过田间筛选并通过转基因安全评估服务生产,解决生产上的关键问题。
Potato(Solanum tuberosum) is the fourth crop in the world. Late blight, caused by Phytophthora infestans de Bary, is the most destructive disease. Flavonoids, a class of natural plant secondary metabolites, have antioxidant, anti-virus and other important functions, but only trace amounts can be detected on current widely planting potato varieties. In this study, a plant flavonoids regulating gene AtMYB12 and three new cloned late blight resistance genes (Rpi-vnt1.1, Rpi-mcq1.1 and Rpi-mcq1.2 ) were differently combinated and transformed into a cultivated potato species Desiree in order to obtain a range of flavonol-rich ( such as rutin, etc.) and potato late blight resistant trangenic lines. It is expected to improve both traits of the late blight resistance and nutritional quality.
By using the Cre/lox site-specific recombination system PX6-GFP, three transgenic constructs were made with target genes from four donors through a two-step strategy with high-fidelity PCR, which included pX6-patatin:: AtMYB12, pX6-Rpi-mcq1.1-Rpi-mcq1.2 and pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1.Three recombinant vectors were mobilised into Agrobacterium strain AGL I, and then were introduced into the susceptible cultivar Desiree via Agrobacterium-mediated stem segment transformation. In total, 28, 20, 20 positive transgenic plant individuals were obtained with 68%, 40% and 45% of transformation efficiency respectively.
Rpi gene potato were tested with six P.infestans isoltates with detached leaf method. The potato carried Rpi-mcq1.1 and Rpi-mcq1.2 were resistant only for two isolates, EC1 and 90128 . Potato carried pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1 were resistant to all six isolates fallen into the former design.
Utilizing HPLC method, We detected the content of flavonoids in potato tubes with different lines carrying AtMYB12 gene. In potato carried pX6-patatin::AtMYB12, the content of rutin is rang from 0.051 mg g-1 to 3.091 mg g-1, and the kaempferol-rutinoside is ranged from 0.041 mg g-1 to 0.951 mg g-1. In addition, flavonoid content in pX6-patatin::AtMYB12 -Rpi-mcq1.1-Rpi-vnt1.1 potato is ranged from 0.08 to 0.2 mg g-1. The result indicated the overall flavonoid content is increased significantly.
Based on resistance phenotype, flavonoid content analysis and the stability of mor- phological traits, 2 lines from each selected transgenic combination were treated withβ-estradiol, and were tested by PCR to obtain NPTII marker-free plants, whose resistance were analysed again. The result indicated that the percentage of marker-free in 3 transgenic combinations is 10%, 8%, 8% respectively. In summary, we have obtained new flavonoid-rich or (and) late blight resistance varieties by transgenic strategy, which is expected to be further screened in the field and to pass the safety assessment, and to address the key problem in production.
引文
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