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用草本植物番茄鉴定五种柑橘类病毒 被引量:4

Identification of Five Viroids Infecting Citrus with Herbaceous Plant Tomato
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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c DNA克隆质粒(克隆于p GEM-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p MD18-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c DNA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c DNA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c DNA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。 [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 identifcation 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 eDNA 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 ofEscherichia coli DH5a. After idetification by PCR, positive clones were choicely sequenced and their eDNA 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.
出处 《中国农业科学》 CAS CSCD 北大核心 2016年第4期784-790,共7页 Scientia Agricultura Sinica
基金 国家自然科学基金(31201488)
关键词 柑橘类病毒 番茄 草本指示植物 侵染性RNA citrus viroids tomato herbraceous indicator infecious RNA
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参考文献20

  • 1Duran-Vila N, Roistacher C N, Rivera-Bustamante R, Semancik J S. A definition of citrus viroid groups and their relationship to the exocortis disease. Journal of General Virology, 1988, 69(12): 3069-3080.
  • 2Ito T, Ieki H, Ozaki K, Ito T. Characterization of a new citrus viroid species tentatively termed Citrus viroid OS. Archives of Virology, 2001, 146(5): 975-982.
  • 3Serra P, Barbosa C, Daròs J, Flores R, Duran-Vila N. Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid. Virology, 2008, 370(1): 102-112.
  • 4Flores R, Hernández C, Martínez de Alba A E, Daròs J A, Di Serio F. Viroids and viroid-host interaction. Annual Review of Phytopathology, 2005, 43: 117-139.
  • 5Ashulin L, Lachman O, Hadas R, Bar-Joseph M. Nucleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Research, 1991, 19(17): 4767.
  • 6Semancik J S, Roistacher C N, Rivera-Bustamante R, Duran-Vila N. Citrus cachexia viroid, a new viroid of citrus: Relationship to viroids of the exocortis disease complex. Journal of General Virology, 1988, 69: 3059-3068.
  • 7Zhang B L, Liu G Y, Liu C Q, Wu Z J, Jiang D M, Li S F. Characterisation of Hop stunt viroid (HSVd) isolates from jujube trees (Ziziphus jujuba). European Journal of Plant Pathology, 2009, 125: 665-669.
  • 8杨元爱,李世访,成卓敏,王红清.杏和李树啤酒花矮化类病毒的检测与序列分析[J].园艺学报,2006,33(6):1193-1198. 被引量:8
  • 9周莹,李世访,成卓敏,赵剑波,姜全.桃树上啤酒花矮化类病毒(Hop stunt viroid)的检测及序列分析[J].植物病理学报,2006,36(6):501-507. 被引量:10
  • 10Sano T, Hataya T, Terai Y, Shikata E. Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan. Journal of General Virology, 1989, 70(6): 1311-1319.

二级参考文献34

  • 1唐科志,彭军,王雪峰,周彦,周常勇,刘科宏,刘英,杨方云.利用一步法RT-PCR检测柑橘裂皮病类病毒[J].园艺学报,2005,32(3):408-413. 被引量:14
  • 2欧阳立,赵学源,陈炜,田波,游标,胡坤元.柑桔裂皮病类病毒(CEV)的双向聚丙烯酰胺凝胶电泳检测[J].西南农业大学学报(自然科学版),1989,11(3):258-260. 被引量:5
  • 3彭军,周常勇,唐科志,谭万忠.柑橘裂皮病类病毒的一步RT-PCR法检测及其在甜橙体内的分布[J].果树学报,2005,22(6):744-747. 被引量:8
  • 4Yamamoto H,Kagami Y,Kurokawa M,Nishimura S,Kubo S,Inoue M,Murayama D.Studies on hop stunt disease.Memoirs of the Faculty of Agriculture,Hokkaido University,1970,7:491~512 (in Japanese)
  • 5Shikata E.New viroids from Japan.Seminars in Virology,1990,1:107~115
  • 6Astruc N,Marcos J F,Macquaire G,Candresse T,Pallas V.Studies on the diagnosis of Hop stunt viroid in fruit trees:identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents.European Journal of Plant Pathology,1996,102:837~846
  • 7Canizares M,Marcos J,Pallas V.Molecular characterization of an almond isolate of Hop stunt viroid (HSVd) and conditions for eliminating spurious hybridization in its diagnostics in almond samples.European Journal of Plant Pathology,1999,105:553~558
  • 8Polivka H,Staub U,Gross H J.Variation of viroid profiles in individual grapevine plants:novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I.Journal of General Virology,1996,77:155~161
  • 9Sano T,Hataya T,Terai Y,Shikata E.Hop stunt viroid strains from dapple fruit disease of plum and peach in Japan.Journal of General Virology,1989,70 (6):1311~1319
  • 10Diener T,Smith D,Hammond R,Albanese G,La Rosa R,Davino M.Citrus B viroid identified as a strain of Hop stunt viroid.Plant Disease,1988,72:691~693

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