摘要
【目的】比较分析野生柑橘与栽培柑橘上衰退病毒(CTV)分离株间的分子遗传特征,为深入解析CTV的遗传进化提供相关依据。【方法】运用RT-PCR对我国云南、广西、四川、湖南、江西等省(区)的11个野生柑橘上的CTV分离株和4个国内栽培甜橙及柚上的CTV强弱毒代表株分离株的p23基因进行扩增、测序,所获序列与Gen Bank收录的具有代表性的国外CTV分离株的相应基因序列进行比对分析。【结果】野生柑橘与国内外栽培柑橘上CTV分离株的p23基因序列相似率为87.7%~99.3%;密码子中碱基含量GC%<AT%,密码子转换数多于颠换数,第3位的碱基替换数最多,其次为第1位,最少是第2位;非同义突变与同义突变的比值(dN/dS)小于1,表明p23基因在进化过程中承受着净化选择。系统聚类分析表明,来自于野生柑橘上的11个CTV分离株在构建的系统发育树中分属不同的簇支,与不同来源地具有较高相似性和较低遗传距离的栽培柑橘上的CTV分离株构成同一组群。【结论】野生柑橘与栽培柑橘上的CTV分离株在碱基含量、密码子转换和颠换率及非同义突变与同义突变比值(dN/dS)等遗传特征方面均表现出一致性,系统发育分析表明,野生柑橘上CTV分离株基于p23基因序列的聚类关系与其地理来源之间无明显相关性。
Abstract： [Objective] This study focused on comparative analysis of the genetic evolution characteristics of the Citrus tristeza virus （CTV） isolates between wild citrus and cultivated citrus, which provided the theoretical basis for further study on the genetic evolution of CTV. [Methods] A total of 11 CTV isolates in this study were collected from naturally wild citrus in Yunnan （isolates CT-W1, CT-W2, CT-W3, CT- W4, CT-W5）, Sichuan （isolates CT-W6, CT-W7）, Hunan （isolates CT-W8, CT-W9）, Jiangxi （isolate CT- W10）, Guangxi （isolate CT-Wll） provinces of China. The severe isolate CT3 and the mild isolate CT9 from cultivated pummelos, together with the severe isolate CT4 and the mild isolate CT21 from cultivated sweet oranges were kindly p^ovided by the Citrus Research Institute, Chinese Academy of Agricultural Sciences as being representative of CTV isolates from cultivated citrus in China. The nucleic acids of the samples with 11 CTV isolates from wild citrus and 4 CTV isolates from cultivated citrus in China were each extracted. The primer pairs of p23f and p23r were used as a specific primer for amplifying the p23 gene, the sense primer was p23f： 5＇ -ATGGATAATACTAGCGCACA-3＇, and the anti-sense primer was p23r： 5＇-TCAGATGAAGTGGTGTTCAC-3＇. The cDNA of CTV was amplified and performed in a 25 μ L reaction volume. PCR products were separated by electrophoresis in a 1.2% agarose gel and detected by ethidium bromide staining, and the amplified products of approximately 630 bp for the complete p23 gene were gel purified and the nucleotide sequences were determined in both directions by means of an ABI Prism DNA sequencer 377 （Sangon Biological Engineering ＆ Technology and Service, Shanghai, China）. Other CTV nucleotide sequences used in this study were obtained from the indicated GenBank entries： American isolates T36, T30 and SY568, Israel isolate VT, Spain isolates T385 and T318A, Japanese iso- late NuagA, Egypt isolate Qaha, Mexico isolate Mexic-ctv. Multiple alignments of the nucleotide sequenc- es were realized using the BioEdit program. The version 6.0 of the MEGA program was used to estimate the base content, the transition and transversion, and their ratio （Ts/Tv）, the rates of non-synonymous（dN） and synonymous（ds） substitutions and their ratios were determined by the Nei ＆ Gojobori methods, the sta- tistic for differentiation using the Z-test （P〈0.05）. Based on the neighbor-joining method, phylogenetic re- lationships were inferred taking the homologous sequences of Beet yellow virus （BYV） as the outgroup, and the reliability of the clusters was evaluated by using the bootstrap analysis base on 1 000 repetitions. [ Resuits ]The sequences of p23 genes of the 11 wild CTV isolates were found having a similarity ranging from 88.1 to 99.8%, the identity of the sequence ranged from 87.7 to 97.9% and 88.0 to 99.3% when compared with 4 Chinese CTV isolates of cultivated citrus and 9 exotic cultivated citrus, respectively. In this investi- gation, the GC base contents of the p23 gene were less than the AT base contents, the base A content was the highest and the base C was the lowest among the four bases in the p23 genes in the 11 CTV isolates from wild type citrus, which was the same as that from cultivated citrus. More transitions were found in the p23 genes than transversions with the transitions/transversions ratio ranging from 3.4 to 5.3 among the spe- cies; the substitution frequency was the highest at the third codon, followed by the first and the second co- don. The significant difference was found among the rates of non-synonymous mutations（dN） and of synony- mous mutations（ds） in the p23 genes （P=0.000〈0.05）. The ratio of non-synonymous （dN） to synonymous（ds） substitutions is far lower than 1, suggesting that the p23 gene might have experienced a purifying selection in the CTV evolution process. Phylogenetic analysis revealed that the 11 CTV isolates from wild citrus be- longed to different phylogenetic clusters regardless of their geographic origins, and shared higher homolo- gy and lower genetic distance with other CTV isolates of cultivated citrus from different geographic origins, for example, isolates CT-W1, CT-W2, CT-W3, CT-W4, CT-W5 were from Yunnan province in China, but dropped into different clusters, the same as the isolates CT-W6, CT-W7 from Sichuan province and the isolates CT-W8, CT-W9 from Hunan province. [Conclusion] Comparative analysis found that the genetic characteristics of the base content, transition and tranversion, non-synonymous and synonymous substitutions of CTV isolates from wild citrus were similar to those of CTV isolates from cultivated citrus. Phylogenetic analysis showed that there was no significant correlation between the geographical origins of Citrus tristeza virus isolates from wild citrus and its clustering relationship of p23 gene sequences.
出处
《果树学报》
CAS
CSCD
北大核心
2017年第7期828-834,共7页
Journal of Fruit Science
基金
国家自然科学基金(30900977)
江西省自然科学基金(20142BAB204010)
江西省高等学校科技落地项目(KJLD13079)
江西省重点研发项目(20161BBF60070)
关键词
野生柑橘
栽培柑橘
柑橘衰退病毒
p23基因
遗传特征
Wild citrus
Cultivated citrus
Citrus tristeza virus
p23 gene
Genetic characterization
