应用实时荧光定量PCR研究BBTV DNA1在香蕉不同组织中的含量  

Measurement the Banana Bunchy Top Virus DNA1 in Different Tissues of Banana by Real-Time Quantitation PCR Method

作　　者：周朋[1,2] 余乃通[2] 章绍延[2] 胡加谊[2] 刘志昕[2] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所农业部热带作物生物学与遗传资源利用重点实验室,海南海口571101

出　　处：《热带作物学报》2014年第7期1388-1392,共5页Chinese Journal of Tropical Crops

基　　金：2012年度海南省研究生创新科研课题项目(No.Hys2012-15);国家自然科学基金项目(No.31070131);海南省重大科技项目(No.ZDZX2013023-1)

摘　　要：为准确定量香蕉束顶病毒(Banana bunchy top virus,BBTV)DNA1组分在香蕉组织中的分布和含量,建立以SYBR Green-I荧光染料为标记的实时荧光定量PCR(Real Time Fluorescence Quantitative PCR)方法。利用B1-F/B1-R引物扩增海南BBTV DNA1组分并构建到pMD18T sample载体,再以B2-F/B2-R引物测定该质粒的标准曲线,其线性方程为Y=-3.247×LOG(X)+8.01,相关系数r2=0.997,扩增效率为103.2%,标准质粒检测灵敏度约为214 copies/μL。分别选取染病香蕉的嫩叶、叶鞘、假茎和球茎各100 mg,提取DNA并进行实时荧光定量PCR检测。结果表明:病株各部位均含BBTV病毒,但含量有明显差异,其中嫩叶(3.08×108 copies/mg)>叶鞘(2.50×108 copies/mg)>假茎(1.29×108 copies/mg)>球茎(1.67×107 copies/mg),呈依次递减。To accurately measure the distribution or amount of Banana bunchy top virus DNA1 component in banana leaf,sheath,pseudostem and corm tissues,the real-time fluorescence quantitative PCR method was used to study in this paper.A 978 bp nucleotide sequence of DNA1 was obtained by B1-F/B1-R primers and ligated to pMD18T sample vector as a plasmid standard.The plasmid standard curve was determined by the real-time fluorescence quantitative PCR by B2-F/B2-R primers with the linear equation Y=-3.247×LOG（X）＋8.01,correlation coefficient r2=0.997,and amplification efficiency 103.2％.The minimum detectable amount of the standard plasmid is approximately 214 copies/μL.The infected banana leaves,sheaths,pseudostems and corms of 4 samples were taken for DNA extraction,and real-time quantitative PCR results showed that BBTV virus was existed in these tissues.However,there are significant differences in the amount in leaf （3.08×10s copies/mg）,sheath （2.50 ×108copies/mg）,false stem（1.29×108 copies/mg） and corm（1.67×107copies/mg）,presenting in descending order.

关 键 词：香蕉束顶病毒 实时荧光定量PCR 病毒含量 

分 类 号：S668.1[农业科学—果树学]