应用TaqMan real-time PCR研究BSV在带毒香蕉组培苗中传递

Development of TaqMan real-time PCR assay for investigating transmission of Banana streak virus in micropropagated banana

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。

The disease caused by Banana streak virus（BSV） led to great severe loss in banana production and affected the exchange of banana germplasm resources. A TaqMan real-time PCR assay was developed using the primer pair and probe based on the conserved ORF Ⅲ sequence of BSV. The assay was reproducible and could specifically detect BSV without cross reaction with Cucumber mosaic virus（CMV） and Banana bunchy top virus（BBTV）,and showed higher sensitivity than that of Endpoint PCR in detecting either DNA standard plasmid samples or field samples,which had 100 times higher than that by Endpoint PCR when detecting DNA standard plasmid samples,and 10 times higher than that by Endpoint PCR when detecting field samples. The transmission feature of BSV in the micropropagated banana samples was different when they were quantitatively detected by TaqMan real-time PCR. When comparing the differentiated banana shoots of the suckers,the concentrations of BSV in those of ‘Dajiao’of the 3th and 9th generations significantly reduced; while the concentrations of BSV inthose of ‘Fenjiao’firstly increased and then decreased in subsequent generations. However,the concentrations of BSV in those of ‘Yueyoukang 1’increased gradually in the subsequent generation. Moreover,the concentrations of BSV in ‘Dajiao’,‘Fenjiao’and ‘Yueyoukang 1’of the 10 th,11th and 12 th generations were higher in the first or（and） second upper leaves than those in the other leaves. Direct binding PCR analysis preliminarily indicated that there is no integration of BSV into the banana genomes in this study. This research elucidated the mechanisms of transmission and distribution of BSV in micropropagated banana by detecting the BSV concentrations using TaqMan real-time PCR,which would be very useful for studying the interaction between BSV and banana and detecting the BSV in the micropropagated banana.