论文标题:柑橘速衰病毒的遗传多样性研究
The Genetic Diversity of Citrus Tristeza Virus
论文作者
论文导师 王国平,论文学位 博士,论文专业 果树学
论文单位 华中农业大学,点击次数 103,论文页数 80页File Size3502K
2006-06-01论文网 http://www.lw23.com/lunwen_544191007/
Citrus tristeza virus; CP gene; p23 gene; RdRp gene; SSCP; molecular variability; co-infection; phylogenetic analysis; molecular marker; strain identification
由柑橘速衰病毒(Citrus tristeza virus，CTV)引发的柑桔速衰病是全世界柑桔生产中破坏力最强、经济意义最为重要的病害之一。它主要危害以酸橙作砧木的柑桔，也能引起葡萄柚和某些甜橙品种的茎陷和果实变小等症状。本研究应用RT-PCR、单链构象多态性(Single-strand conformation polymorphism，SSCP)、DNA测序和系统发育分析等方法，目的是要调查我国柑橘CTV感染的基本情况，比较不同分离物的序列特征，探索CTV的系统进化关系，最终为开发分子标记鉴定我国CTV株系提供理论指导。结果如下： 1．以随机采样的方式，从湖北、湖南和江西3省10县的柑橘产区收集242份温州蜜柑(Citrus unshiu Marc．)、橘(C．reticulata Blanc．)、甜橙(C．sinensis Osb．))和柚(C．grandis Osb．)样品。对PAS-ELISA法检测呈阳性的样品进行CTV的CP(Coating protein，CP)基因的RT-PCR扩增，结果来自14个样品采集地的120份样品感染了CTV，单个采样点的感染率为20.0％-71.4％，总感染率达49.6％，表明CTV在这些地区已经广泛分布。SSCP分析显示，120份CP基因的RT-PCR扩增产物共产生120种SSCP谱型，每一种谱型含有2-7条不等的单链DNA带，暗示这些样品采集地区存在广泛的CTV混合感染。 以CTAB-LiCl法制备的总RNA为模板，可以对柑橘进行CTV的RT-PCR检测。该法制备总RNA的过程简单，成本低廉。 2．以柑橘叶片总RNA为模板，用RT-PCR扩增CP基因，扩增产物经克隆与测序证实，来自湖南道县和江西崇义3份橘的实牛苗样品(HN、JX-1和JX-2)都感染了CTV。通过扩增产物克隆前和克隆后的PCR-SSCP分析，发现来自HN和JX-1的CP基因各自只含有1种独特的序列，来自JX-2的序列则含有2种主要的序列。这表明来自3份样品的CTV的CP基因不仅存在序列变异，而且样品JX-2的CTV群体可能因为混合感染而呈异质性。 通过双向测序和序列分析，4个CP基因序列都没有碱基插入、缺失或终止密码子插入，含有完整的翻译区。核苷酸序列比对分析表明，4个克隆之间的相似性为93％-98％；克隆HN-K与葡萄牙分离物28C、以色列茎陷分离物VT的相似性都达到98％；克隆JX-1-1与印度茎馅分离物Pune和Bangalore的相似性都在98％以上；克隆JX-2-7、JX-2-17与日本苗黄分离物NUagA和美国加利福尼亚严重茎陷分离物SY568的相似性也在97％-98％之间。4个克隆与佛罗里达速衰分离物T36、弱毒分离物T30和西班牙弱毒分离物T385的相似性仅有91％-93％。系统树分析显示，4个中国克隆分属3个不同的分支，每个分支由中国克隆和地理来源不同的茎陷或苗黄分离物组成。 3．以SSCP分析显示CTV混合感染程度较轻的江西省于都县的1份甜橙样品为材
Citrus tristeza disease caused by Citrus tristeza virus (CTV) is one of the most destructive and economically important diseases of commercial citrus worldwide. It mainly causes losses of sweet orange trees on sour orange rootstock, and also causes stem pitting syndrome and reduces fruit size of grapefruits and some sweet orange varieties. In this study, RT-PCR, SSCP, DNA sequencing and phylogenetic analysis are used to investigate the CTV infection of citrus in China, to compare sequence characteristics of different isolates, to explore their phylogenetic relationships, and give theoretical guides in developing molecular markers to finally identify the CTV strains in China. The results are described as following:1. 242 samples of satsuma mandarin (Citrus unshiu Marc), tangerine (C. reticulata Blanc), sweet orange (C. sinensis Osb.) and pummelo (C. grandis Osb.) were randomly collected from ten counties of Hubei, Hunan, and Jiangxi. CP (Coating protein) genes of CTV from the samples preliminarily screened by PAS-EL1SA were amplified by RT-PCR. The amplification results showed that 120 samples from fourteen sampling sites were infected with CTV, the infecting rate of single site ranged from 20.0% to 71.4%, and the total rate was up to 49.6%. Thus it indicated that CTV extensively existed in these regions. Single-strand conformation polymorphism (SSCP) analysis showed that 120 amplified products of CP gene made up a profile of 120 kinds of SSCP patterns and each pattern consisted of two to seven single-strand DNA bands. This suggested that a great mixed infection of CTV existed among these sampling regions.The total RNA extracted by a method of CTAB-LiCl could always work as efficient templates in RT-PCR detection of CTV in citrus, and its preparation was simple and not expensive.2. CP genes of CTV were amplified by RT-PCR with total RNA as templates extracted from three tangerine seedling samples (HN, JX-1 and JX-2) collected from Daoxian, Hunan and Chongyi, Jiangxi. The results of cloning and sequencing of amplified products demonstrated that three samples were infected with CTV. The SSCP analysis results before and after cloning the amplified products showed that the CP genes from HN and JX-1 only contained a type of unique sequence, but JX-2 had a population of sequences, two of which were predominant. This indicated that genetic variation existed among the CP genes of CTV from three samples, and JX-2 was heterogeneous probably due to the mixed infection.Bi-directional sequencing and sequence analysis showed that four CP gene sequences had full-length translating regions without any base insertion, deletion, or stop code insertion. Nucleotide sequence alignment results indicated that four clones shared 93%-99% of nucleotide sequence similarity. Clone HN-K had 98% of similarity with Portuguese isolate 28C and Israel stem pitting isolate VT, clone JX-1-1 had 98-99% with Indian isolate Pune and Bangalore, and clone JX-2-7 and JX-2-17 had over 97% with Japanese seedling yellows
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