苹果锈果类病毒火焰海棠分离物全基因组克隆及序列分析

Cloning and genome sequence analysis of apple scar skin viroid from Malus ’Flame’

【目的】检测从山东青岛采集的有“花脸”症状的火焰海棠(Malus‘Flame’)果实是否带有苹果锈果类病毒(Apple scar skin viroid,ASSVd)。【方法】提取带有“花脸”症状的火焰海棠果皮总RNA,设计ASSVd特异性引物,利用RTPCR方法进行检测。设计基因特异性引物扩增ASSVd基因组全长,并进行克隆、测序,利用CLC RNAWorkbench4预测其二级结构。用DANMAN软件比对来自中国不同地区、不同寄主的苹果锈果类病毒分离物基因序列。用MEGA-7软件分析山东火焰海棠ASSVd分离物与来自不同国家、不同寄主ASSVd分离物的遗传关系。【结果】从山东青岛采集的表现“花脸”症状的火焰海棠果皮中检测到ASSVd,推测火焰海棠果皮的“花脸”症状可能由ASSVd引起。利用基因特异性引物克隆4条ASSVd基因组全长序列,其长度为333~334 bp,在GenBank的登录号依次为MK102981-MK102984。ASSVd序列比对结果显示,来源于不同寄主的分离物存在差异。系统进化树显示ASSVd不存在明显的地区专化性,进一步对ASSVd二级结构分析发现不同寄主的ASSVd致病区(P)存在明显的差异。【结论】从山东青岛采集的带有“花脸”症状的火焰海棠果实带有ASSVd。本研究从火焰海棠检测到ASSVd,明确其为自然寄主之一,且火焰海棠果实的“花脸”症状形成可能与ASSVd侵染有关。

【Objective】Apple scar skin viroid(ASSVd)is the pathogen of the apple scar skin disease,which is one of the major diseases in apple(Malus domestica).ASSVd induces different symptoms on different apple cultivars.For instance,it induces scar skin symptoms on the apple fruits of‘Ralls Janet’and‘Golden Delicious’,while it induces dapple symptoms on red-skinned cultivars,such as‘Red Gold’‘Red Fuji’and‘Jonathan’.These viroid-induced symptoms severely affect the market value of fresh apples,and as a result,they lead to great economic loss for the farmers.To date,ASSVd has been detected from apple,pear,wild pear,peach,apricot and sweet cherry trees.However,Malus‘Flame’has not been reported to be infected with ASSVd.In the orchard of Qingdao,Shandong Province,we found the fruits of Malus‘Flame’showed dapple symptoms,which was very similar with the apple dapple symptoms induced by ASSVd.Thus,to detect whether or not dapple fruits of Malus‘Flame’collected from Qingdao,Shandong Province were infected with ASSVd,we used reverse-transcript PCR to perform this experiment.We also intended to obtain the full length of the ASSVd genome and perform the sequence analysis.【Methods】We first obtained the peels of the dapple fruits of Malus‘Flame’,and extracted the total RNA using the RNA extraction kit.Then the first strand cDNA was synthesized by reverse transcriptase,and the products were stored at-20℃.Then specific primers were designed to detect ASSVd by RT-PCR.To obtain the full length of ASSVd genome sequence,a new pair of primers was designed according to the obtained ASSVd sequences and the full length of ASSVd genome was amplified with two rounds RT-PCR.The secondary structure of ASSVd was predicted with CLC RNAWorkbench4 to test whether these isolates had the same domains with reported ASSVd sequences in the secondary structure.Then,the ASSVd isolates obtained from Shandong Province were aligned with those from different regions of China,and also from different fruit tree species by using DNAMAN software to analyze the sequence similarity.The phylogenetic tree of ASSVd isolates from Malus‘Flame’with those from other countries and plant species was generated with MEGA-7 to analyze the transgenic relationship among these isolates.【Results】We first extracted the total RNA and synthesized the first strand cDNA.By using the cDNA as a template and the specific primers,the RT-PCR results indicated that the dapple fruits of Malus‘Flame’were indeed infected with ASSVd,and this suggested that the dapple symptom was mostly likely induced by ASSVd.To obtain the full length of ASSVd genome sequences,we designed a new pair of primers according to the previous obtained sequences,and through two sets of RT-PCR assays,four ASSVd isolates were obtained and the length of the genome ranged from 333-334 nt,and the GenBank accession number of these four sequences are MK102981,MK102982,MK102983,and MK102984,respectively.The secondary structure analysis showed that it had 31 loops,which have been reported to play key roles in the replication and transportation in the case of other viroids,such as potato spindle tuber viroid,(PSTVd).We also found that the“rod”structure of these four ASSVd isolates had five domains,the left terminal domain,the pathogenicity domain,the central domain,the various domain and the right terminal domain.And this was very similar with the secondary structure of ASSVd isolates that have been reported.The alignment with ASSVd isolates from different fruit tree species of China indicated that the four isolates obtained from Shandong Province were in very high similarity,however,the pathogenicity domain(30-50 nt and 250-270 nt region)of all the aligned sequences were different,and this indicated that the pathogenicity domain may determine the host preferences since they varied significantly among different isolates.The phylogenetic tree showed that the ASSVd isolates from Malus‘Flame’had a closest relationship with ASSVd from Malus domestica(Iran,HQ326090.1),and then from the Malus domestica(Xinjiang,China,KC110859.1;Shanxi,China,KU507023.1).This may suggest that the ASSVd we isolated from Malus‘Flame’dapple fruits from Qingdao,Shandong Province was most likely transported from other places,such as Xinjiang or Shanxi Province,and both are the major production areas of apple in China,but not evolved from the local species.【Conclusion】The Malus‘Flame’dapple fruits we collected from Qingdao,Shandong province were infected with ASSVd,and this study confirms that Malus‘Flame’is a natural host for ASSVd in China.In addition,our study also implies that the dapple symptom of Malus‘Flame’fruits is most likely induced by ASSVd.