摘要： 为获得兼抗多种病毒（类病毒）的马铃薯植株，分别克隆了靶向PVX、PVY和PSTVd蛋白的P25、HC-Pro和Virp1基因，以拟南芥pre-miR159a为骨架设计了针对P25、HC-Pro和Virp1基因的amiRNA序列，通过Overlapping PCR将3个amiRNA片段连接，并导入植物双元表达载体pCAMBIA1300-221，构建植物表达载体p1300-221-pre-amiR-P25-HCPro-Virp1，经PCR及酶切验证，表达载体构建正确。农杆菌介导法将含目的基因质粒菌液侵染马铃薯品种费乌瑞它脱毒微型薯片，经再生、压力筛选，抗性植株分化及植株壮苗共获得转化株系15株，PCR检测结果表明，其中10株检测到与目的基因大小一致的片段（729 bp）。qRT-PCR结果显示，外源amiR -P25-HCPro-Virp1基因在10个转化植株中均有表达，相对表达量在7.68~21.37。对T₀阳性转化植株的攻毒试验，将PVX、PVY和PSTVd病毒等量混合液摩擦接种至6~8叶期的植株叶片，20 d后观察发现，对照植株感病严重，出现植株矮小、叶片斑驳等症状，而转化植株未表现出感病症状，生长正常。RT-PCR进行病毒特异性检测，结果显示，转化植株中也未检测到病毒序列，这表明转amiR-P25-HCPro-Virp在转化植株中能稳定表达，转化植株能够兼抗PVX、PVY和PVSTd 3种病毒（类病毒）。得到了同时兼抗PVX、PVY和PVSTd转化植株，且抗性显著，为马铃薯抗病毒育种提供了新的遗传资源。

关键词: 马铃薯, amiRNA, PVX, PVY, PSTVd

Abstract: In order to obtain potato plants which could be resistant to multiple viruses (viroid) simultaneously, P25, HC-Pro, and Virp1 gene targeting PVX, PVY, and PSTVd proteins were cloned, respectively. Three types of amiRNAs targeting sequences encoding the silencing suppressor P25, HC-Pro and Virp1 were designed by using Arabidopsis thaliana miR159a as backbone. These three amiRNA sequences were connected by Overlapping PCR. The synthetic of P25, HC-Pro and Virp1 gene was inserted into the expression vector pCAMBIA1300-221 to form p1300-221-preamiR-P25-HCPro-Virp1, and the vector was verified by PCR and restriction enzyme digestion. Pre-amiR-P25-HCPro-Virp1 was transformed into minituber of potato cultivar Favorita by Agrobacterium tumefaciens infection. 15 transformed plants were obtained through regenerating, pressure screening and differentiation.PCR results showed that 10 of them were detected the same size fragment (729 bp) as the target gene. Further qRT-PCR testing confirmed that amiR-P25-HCPro-Virp1 gene was expressed in 10 transformed plants, and the relative expression was between 7.68-21.37. Inoculated the T₀ transgenic plants with the mixture virus of PVX, PVY and PSTVd by friction into the leaves of the plants at the 6-8 leaf stage. The plant growth was observed after 20 days and the results showed that the control plants were severely susceptible, with symptoms such as short plants and mottled leaves, while the transformed plants did not show infection symptoms, and growth normally. There was no virus detected by RT-PCR either. This indicated that the transfected amiR-P25-HCPro-Virp could be stably expressed in the transformed plants, and the transformed plants were resistant to PVX, PVY and PVSTd viruses (viroid). Transformed plants resistant to PVX, PVY and PVSTd were obtained, and the resistance was significant, which provided new genetic resources for potato virus-resistant breeding.

Key words: Potato, amiRNA, PVX, PVY, PSTVd

中图分类号: 

• S532