山楂属植物中苹果褪绿叶斑病毒的RT-PCR检测及病毒脱除方法  

Detection of Apple chlorotic leaf spot virus in hawthorn and comparison of virus elimination methods

作　　者：郑文燕[1] 郭巍[1] 马跃[1] 李晓明[1] 代红艳[1] 

机构地区：[1]沈阳农业大学园艺学院,沈阳110866

出　　处：《果树学报》2016年第12期1576-1583,共8页Journal of Fruit Science

基　　金：国家自然科学基金(31470678)

摘　　要：【目的】建立优化的山楂属植物苹果褪绿叶斑病毒(ACLSV)的RT-PCR检测体系,获得以山楂组培苗为试材的高效ACLSV脱除方法。【方法】基于转录组高通量测序解析的山楂属植物ACLSV基因组序列设计病毒检测引物,比较c DNA模板浓度、Taq DNA聚合酶种类及采样部位对病毒RT-PCR检测效果的影响。通过组培苗热处理和培养基中添加三氮唑核苷来脱除山楂植株中的ACLSV。【结果】基于转录组高通量测序获得的2个ACLSV山楂分离物的全基因组序列设计并筛选出适合于山楂属植物ACLSV的RT-PCR检测引物。建立了优化的山楂属植物ACLSV的RT-PCR检测体系。对80份山楂样品进行病毒检测,其中有18个样本检测出ACLSV,带毒率为22.5%。比较了热处理(37℃、30d)、化学处理(培养基中添加20 mg·L-1的三氮唑核苷,处理40 d)、热处理与化学处理结合方法(处理30 d)3种方法对山楂组培苗中ACLSV的脱除效率,表明热处理或热处理与化学处理结合的方法能够完全脱除山楂中的ACLSV,而单独化学处理的脱毒率不能达到100%。【结论】建立了优化的山楂属植物ACLSV的RT-PCR检测体系,表明组培苗热处理是脱除山楂植株中ACLSV的有效方法。【Objective】Virus disease in fruit tree was first reported in 1923 by American scholars. More than 600 kinds of virus diseases have been found in fruit trees around the world by 2003. Apple chlorotic leaf spot virus（ACLSV）, as one of the important latent recessive virus in fruit trees, was widely distributed in a variety of plants, including apple, pear, peach, plum and cherry. In 2015, ACLSV was found in hawthorn（Crataegus pinnatifida） in China. The virus can cause a decline in hawthorn trees and the quality of fruits, and even bring devastating disasters in fruit yield. Propagation of the virus-free hawthorn plants by tissue culture is the main method to prevent and cure virus disease, while, it demands a rapid, accurate and specific virus detection method. In this study, we established an optimized RT-PCR detection system for detection of ACLSV in hawthorn plants. At the same time, we studied the technology for ACLSV elimination from hawthorn with tissue culture.【Methods】Young plant leaves and fruits of the accessions of Crataegus spp. in this study were collected from the National Hawthorn Germplasm Repository in Shenyang Agricultural University. Total RNA of hawthorn was extracted from leaves or young fruits by magnetic bead RNA extraction machine produced by Thermo Fisher Scientific. The sample was 100 mg, and the amount of buffer was 30 μL. Agarose gel electrophoresis was applied to analyze RNA quality and integri-ty, and Nano Drop 2000 ultraviolet spectrophotometer was used to test the concentration and purity of RNA. Primers were designed based on the genomic sequence of ACLSV hawthorn isolates. Reverse transcription reactions were performed at 37 ℃ for 30 min, then 85 ℃ for 5 s with Prime Script？RT reagent kit（Ta Ka Ra, Dalian） according to the manufacturer＇s instructions. PCR reactions were carried out in 20 μL total volumes with reaction mixtures that contained 1 μL c DNA, 1.6 μL of each d NTP（2.5 mmol·L-1）, 2.0μL 10× PCR buffer, 1.0 μL Mg Cl2（25 mmol·L-1）,

关 键 词：山楂 苹果褪绿叶斑病毒 RT-PCR 组培 脱毒 

分 类 号：S661.5[农业科学—果树学]