苹果锈果类病毒内蒙古金红分离物的基因组序列分析

Complete genome sequence analysis on apple scar skin viroid separated from infected Malus pumila Jinhong in Inner Mongolia

【目的】研究苹果锈果类病毒内蒙古金红分离物的基因组序列,为苹果锈果类病毒的有效防控提供参考。【方法】鉴定内蒙古自治区园艺研究院实验农场果实表现花脸的金红苹果叶片是否被苹果锈果类病毒(apple scar skin viroid,ASSVd)侵染。提取植物总RNA并以获得的总RNA为模板合成cDNA第一链,通过RT-PCR技术检测样品中的ASSVd,并经克隆测序获得ASSVd内蒙古金红分离物全基因组序列。采用MEGA 6.0、DNAMAN软件对不同寄主和不同地区来源的ASSVd分离物核苷酸序列进行分析,利用线上工具RNA Folding Form预测ASSVd内蒙古金红分离物的RNA二级结构。【结果】在疑似感病内蒙古金红苹果叶片样品中检测到ASSVd。ASSVd内蒙古金红分离物的序列长度为330 nt(GenBank登录号MZ476527),与ASSVd新疆梨分离物(JX861258)的亲缘关系最近,核苷酸序列一致性也最高,为97.9%;其次与ASSVd内蒙古塞外红苹果分离物(MN598205)的亲缘关系较近,序列一致性为97.0%;与ASSVd内蒙古塞外红苹果分离物(MN598204)的核苷酸一致性最低,为87.5%。系统发育分析显示,ASSVd的遗传关系与寄主和地理来源相关性不大。多重对比分析结果表明,ASSVd的变异主要集中在左末端区、致病区和中央保守区。RNA Folding Form预测ASSVd内蒙古金红分离物RNA基因组具有杆状二级结构,自由能为-499.49 kJ/mol。【结论】内蒙古金红苹果样品被ASSVd侵染,获取了其基因组序列相关信息。

【Objective】The full-length sequence of apple scar skin viroid(ASSVd)from infected Malus pumila Jinhong in Inner Mongolia was analyzed to provide basis for effective disease control.【Method】The occurrence of ASSVd on Malus pumila Jinhong with dapple fruits in the experimental farm of the Inner Mongolia Horticulture Research Institute was identified.Total RNA of plants was extracted and used as template to synthesize the first strand of cDNA.The ASSVd isolate from Inner Mongolia Jinhong was amplified using reverse transcription-polymerase chain reaction.The complete genome nucleotide sequence of ASSVd was aligned with other isolates detected in different hosts and regions using MEGA 6.0 and DNAMAN software.The secondary structure of ASSVd genome was predicted using the online tool of RNA folding form.【Result】The presence of ASSVd was confirmed in the Jinhong with dapple disease.The genome of the ASSVd isolate from Jinhong consisted of 330 nucleotides with GenBank of MZ476527.It had the closest genetic relationship with Xinjiang pear isolate JX861258 and the highest nucleotide sequence identity was 97.9%.It was also closely related to the isolate MN598205 from Inner Mongolia with the sequence identity of 97.0%.The nucleotide identity of 87.5%with MN598204 was the lowest.The phylogenetic tree showed that the genetic diversity of ASSVd isolates was unrelated to either geographical distribution or host plant species.The multiple alignment result indicated that variations of ASSVd genomes were mainly distributed in TL,P and C domains.RNA folding form predicted that RNA genome of ASSVd isolate from Jinhong had rod-shaped secondary structure with free energy of-499.49 kJ/mol.【Conclusion】The infection of Jinhong apple with dappling fruits by ASSVd was confirmed and key genome information was obtained.