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由香蕉线条病毒(Banana streak virus,BSV)引起的香蕉线条病,严重影响了香蕉产量及种质资源交流。本研究根据BSV ORF Ⅲ基因保守序列设计引物和探针,建立了BSV的实时荧光定量PCR检测方法。该法检测BSV时,与黄瓜花叶病毒(Cucumber mosaic virus,CMV)和香蕉束顶病毒(Banana bunchy top virus,BBTV)无交叉反应;不论是检测质粒DNA还是香蕉病株总DNA,其灵敏度都比普通PCR高,检测质粒DNA的灵敏度比普通PCR高100倍,检测香蕉病株总DNA的灵敏度比普通PCR高10倍,且具有良好的重复性。利用建立的实时荧光定量PCR方法检测我国主栽的不同香蕉品种‘大蕉’、‘粉蕉’和‘粤优抗一号’香蕉组培苗中BSV的浓度,发现不同品种BSV传递规律不同。‘大蕉’第3代、第9代组培分化芽中BSV含量较原代显著减少;‘粉蕉’中BSV含量随继代数增加表现为先增加后减少;‘粤优抗一号’中BSV含量随继代数增加呈增加趋势。BSV在‘粉蕉’、‘大蕉’和‘粤优抗一号’第10代到第12代组培苗中以顶部第1片或(及)第2片叶中含量最高。直接结合PCR分析初步表明不同品种在原代和组培至第12代,其植株中的BSV均为游离状态。本文通过对带毒香蕉组培苗中BSV的含量监测,明确了BSV在不同品种带毒香蕉组培苗中的传递和分布规律,为研究BSV与寄主互作及BSV检测提供了理论依据。
Banana streak virus (Banana streak virus, BSV) caused by banana streak disease, severely affected the banana yield and germplasm exchange. In this study, primers and probes were designed based on the conserved sequence of ORF Ⅲ gene in BSV, and a real-time fluorescence quantitative PCR assay was established for BSV. The method for detecting BSV showed no cross-reaction with Cucumber mosaic virus (CMV) and banana bunchy top virus (BBTV). The sensitivity of both BSA Compared with ordinary PCR, the sensitivity of detecting plasmid DNA is 100 times higher than that of ordinary PCR. The sensitivity of detecting total DNA of banana plant is 10 times higher than that of ordinary PCR, and has good repeatability. The established real-time fluorescence quantitative PCR method was used to detect the BSV concentrations in different plant species of banana cultivars, such as ’Banana’, ’Banjia’ and ’Yueyoukai 1’, and found that the BSV transmission of different varieties was different. The BSV content in the third and the ninth generation of plantains decreased significantly compared with that of the first generation. The content of BSV in ’Banana’ increased first and then decreased with the increase of subsequent generations. The content of BSV increased with the following generations. The highest content of BSV was found in the top 1 or (or) 2 leaves of BSA, ’plantain’ and ’Yueyou AN 1’ from the 10th to 12th generation. Directly combined with PCR analysis showed that different varieties in the primary and tissue culture to 12 generations, the BSV in their plants are free state. In this paper, the BSV content in banana tissue culture seedlings was monitored, the transmission and distribution regularity of BSV in banana tissue culture seedlings were determined, which provided a theoretical basis for the study of BSV interaction with host and BSV detection.