用草本植物番茄鉴定五种柑橘类病毒

doi:10.3864/j.issn.0578-1752.2016.04.017

摘要/Abstract

摘要： 【目的】通过嫁接木本指示植物进行柑橘类病毒鉴定需要合适的季节和较长的显症时间，并且柑橘木本指示植物的遗传转化和基因功能验证体系尚不成熟，研究其与寄主间的互作较为困难。因此，寻找合适的草本鉴定和实验寄主对于更好地进行柑橘类病毒的鉴定及研究其与寄主间的互作具有重要意义。论文旨在明确中国报道的5种主要柑橘类病毒，包括柑橘裂皮类病毒（Citrus exocortis viroid，CEVd）、柑橘曲叶类病毒（Citrus bent leaf viroid，CBLVd）、啤酒花矮化类病毒（Hop stunt viroid, HSVd）、柑橘矮化类病毒（Citrus dwarfing viroid，CDVd）和柑橘树皮裂纹类病毒（Citrus bark cracking viroid，CBCVd）对番茄（Solanum lycopersicum）的侵染能力，进而明确其可否作为5种类病毒的鉴别寄主或实验寄主。【方法】将5种柑橘类病毒的野生型分子变种（分别为CEVd.188，长370 nt；CBLVd.032，长328 nt；HSVd.cit106，长296 nt；CVd-III.072，长295 nt；CVd IV Ca，长285 nt）二聚体cDNA克隆质粒（克隆于pGEM-T载体），采用限制性内切酶Nde I在37℃条件下进行酶切线性化处理，经琼脂糖凝胶电泳分析鉴定酶切成功后，将该含有目标片段的线性化重组质粒采用T7 RNA多聚酶在37℃条件下进行体外转录，获得类病毒RNA二聚体溶解于1% K2HPO4接种缓冲液中，机械摩擦接种Alisa Craig和 Rutgers番茄（S. lycopersicum var. Alisa Craig和 var. Rutgers）叶片，置于24℃温室条件下培养。接种60 d后，采集新生叶片釆用CTAB法提取番茄叶片总RNA，通过RT-PCR对接种的类病毒进行检测。将PCR产物经琼脂糖凝胶电泳分析后，切胶回收目标片段，与pMD18-T载体连接，热击转化大肠杆菌DH5α感受态细胞。经PCR鉴定后挑取阳性克隆进行序列测定，采用DNAMAN version 6.0软件对获得的cDNA序列进行相似性分析。【结果】线性化质粒体外转录后经琼脂糖凝胶电泳分析显示，每个质粒转录产物均可检测到目标大小的RNA条带。接种后，RT-PCR检测显示5种类病毒侵染率表现出显著差异，CEVd、CDVd和HSVd可以侵染大部分接种植株（侵染率分别为7/8、5/8和7/8）。从接种成功的植株样品中对所接种类病毒后代的cDNA克隆和序列测定显示，随机测定的5—10个克隆中均能找到与接种类病毒cDNA一致的核苷酸序列，后代序列与接种序列间的相似性在99.7%以上；后代分子变异分析显示变异位点小于10个，变异率小于0.3%；而 CBLVd和CBCVd不能侵染或侵染率很低（0或1/8）。【结论】CEVd、CDVd和HSVd能够侵染Rutgers和Alisa Craig番茄，2种番茄均可以作为3种类病毒的草本鉴定和实验寄主；而 CBLVd和CBCVd 难于侵染Rutgers和Alisa Craig番茄。在接种的Alisa Craig番茄品种上，各类病毒接种植株均比接种缓冲液对照表现显著的矮化效果；在Rutgers植株上，仅有接种了CEVd的植株表现较对照的矮化效果。

关键词: 柑橘类病毒, 番茄, 草本指示植物, 侵染性RNA

Abstract: 【Objective】 A suitable season and a long time are needed for the symptom visualization by grafting onto woody indicator plant to identify the viroids infecting citrus. Moreover, the system is not well developed for genetic transformation and function identification in citrus indicator plants, thus, it is difficult to study the interaction between viroids and their host in such plant. Therefore, it will be important to find suitable herbaceous plants as indicator and experimental host for viroid idenfication and study on the interaction between viroids and their host. In this study, five viroids including Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) have been reported to be the major ones infecting citrus trees in China. The objective of this study is to identify whether the five viroids infect tomato (Solanum lycopersicum), and then to clarify whether the tomato can be adopted as the herbraceous and experimental host plant for the five viroids.【Method】 After enzyme-digested with Nde I at 37℃ into linearized form, the pGEM-T plasmids containing the dimerized cDNA of each wild type of five citrus viroids (CEVd.188, 370 nt in length; CBLVd.032, 328 nt in length; HSVd.cit106, 296 nt in length; CVd-III.072, 295 nt in length; CVd IV Ca, 285 nt in length) were in vitro transcribed with T7 RNA polymerase at 37℃, the obtained dimeric RNAs were inoculated into tomato leaves of S. lycopersicum vars. Alisa Craig and Rutgers by mechicanlly rubbling manners, and incubated in a greenhouse at 24℃. New developed tomato leaves were collected and extracted their total RNAs after 60 days post inoculation (dpi), and subjected to detection of the inoculated viroids by RT-PCR. The resulted RT-PCR products were analyzed on agarose gel, purified by excising the target bands from the gel, ligated with pMD18-T vector, and transformed into competent cells of Escherichia coli DH5α. After idetification by PCR, positive clones were choicely sequenced and their cDNA sequences were aligned using software DNAMAN version 6.0. 【Result】After transcribed based on the lineralized plasmids, analyses on agarose gel revealed the target RNA bands with expect sizes for all the plasmids. After inoculation, RT-PCR identification of the inoculated viroids revealed that the infectivity significantly varied among the five viroids. Of which, CEVd, CDVd and HSVd infected most inoculated seedlings, with total infectivity of 7/8, 5/8 and 7/8, respectively. Sequencing five to ten clones chosen in random showed that each parental sequence could be found in the progeny sequences, and their progeny sequences had similarities more than 99.7% with the parental sequences, with varibilities less than 0.3% (less than 10 mutations). While CBLVd and CBCVd could not infect the inoculated seedlings or had a vey low infectivity (1/8) for the latter. 【Conclusion】 CEVd, CDVd and HSVd can infect S. lycopersicum vars. Alisa Craig and Rutgers, which could be acted as herbaceous indicator and experimental plants. Whereas it is difficult to infect vars. Rutgers and Alisa Craig for CBLVd and CBCVd. For the inoculated seedlings of var. Alisa Craig, they all were significantly dwarfed than the control seedlings inoculated with buffer for all these five viroids; whereas for the inoculated seedlings of var. Rutgers, only the ones inoculated with CEVd showed the dwarfed effect compared with the controls.

Key words: citrus viroids, tomato, herbraceous indicator, infecious RNA

董艳娜，郑银英，徐文兴. 用草本植物番茄鉴定五种柑橘类病毒[J]. 中国农业科学, 2016, 49(4): 784-790.

DONG Yan-na, ZHENG Yin-ying, XU Wen-xing. Identification of Five Viroids Infecting Citrus with Herbaceous Plant Tomato[J]. Scientia Agricultura Sinica, 2016, 49(4): 784-790.

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