目的 明确中缅边境地区间日疟原虫几丁质酶(Plasmodium vivax chitinase,PvCHT1)基因的遗传多样性,探究PvCHT1基因的地理差异,为我国间日疟疫苗的设计提供参考.方法 收集中缅边境地区(云南腾冲)、中国内陆地区(安徽合肥、河南郑州)PvCHT1基因序列,同时从美国国家生物技术信息中心(National Center for Bio-technology Information,NCBI)下载获取缅甸、柬埔寨、泰国、越南、印度尼西亚、马来西亚、巴布亚新几内亚、巴西、哥伦比亚、秘鲁和墨西哥等11个国家的PvCHT1基因序列.采用MEGA、DnaSP、KaKs_Calculator、Arlequin、STRUCTURE和 NETWORK软件,分别对所有基因序列的遗传多样性、基因进化、遗传分化、种群结构和单倍型网络进行分析.结果 共获得中缅边境地区(6条)、中国内陆地区(10条)PvCHT1基因序列16条,从NCBI下载其他11个国家的PvCHT1基因序列551条.遗传多样性分析显示,中缅边境地区的PvCHT1基因有25个多态位点,其中16个为非同义突变,最常见的突变位点是E617D(占31.57%)和I272M(占31.04%);中缅边境地区的PvCHT1基因核苷酸多样性(π=0.000 79)略高于中国内陆地区(π=0.000 71)和缅甸(π=0.000 75).基因进化分析显示,中缅边境PvCHT1基因的中性检验(Tajima's D)值<0,非同义替换与同义替换之比(Ka/Ks)>1.遗传分化分析显示,中缅边境地区PvCHT1基因与中国内陆之间的近交系数(FST)为0.31,与缅甸的FST为-0.05,与柬埔寨、泰国、越南、印度尼西亚、马来西亚之间的范围为0.04～0.15,与巴布亚新几内亚、巴西、哥伦比亚、秘鲁、墨西哥之间的FST范围为0.24～0.56.种群结构分析显示,所有种群结构的最佳组数为7,其中,中缅边境种群由K1～K6组分构成,以K5为主.单倍型网络分析显示,存在4个单倍型地理集群,除中国内陆和墨西哥外,其他国家均共享单倍型H5.结论 中缅边境地区PvCHT1基因高度保守,提示其可作为传播阻断疫苗候选靶标;PvCHT1基因在不同种群中存在明显的地理差异,因此在设计疫苗时应更具针对性.
Analysis of the genetic characteristics of chitinase gene of Plasmodium vivax along the China-Myanmar border
Objective To understand the genetic diversity of the Plasmodium vivax chitinase(PvCHT1)gene along the China-Myanmar border,and explore its geographical variations for evidence to invent the malaria vaccines against P.vivax in China.Methods PvCHT1 gene sequences were collected from regions along the China-Myanmar border(Tengchong,Yunnan Province)and inland China(Hefei,Anhui Province;Zhengzhou,Henan Province),as well as from 11 countries,including Myanmar,Cambodia,Thailand,Vietnam,Indonesia,Malaysia,Papua New Guinea,Brazil,Co-lombia,Peru and Mexico through the National Center for Biotechnology Information(NCBI)of the United States.Mega,DnaSP,KaKs_Calculator,Arlequin,STRUCTURE and NETWORK software were used to analyze the genetic di-versity,gene evolution,genetic differentiation,population STRUCTURE and haplotype NETWORK of all the harvested gene sequences.Results A total of 16 PvCHT1 gene sequences were obtained,including 6 from the China-Myanmar border region and 10 from inland China.Besides,another 551 PvCHT1 gene sequences were harvested from 11 other countries via NCBI.Genetic diversity analysis revealed 25 polymorphic sites in the PvCHT1 gene,of which 16 were non-synonymous mutations.The most common mutation sites were E617D(31.57%)and I272M(31.04%).The nucleo-tide diversity of the PvCHT1 gene in the China-Myanmar border region(π=0.000 79)was slightly higher than that in in-land China(π=0.000 71)and Myanmar(π=0.000 75).Gene evolution analysis indicated that the neutral test(Tajima's D)value of the PvCHT1 gene in the China-Myanmar border region was less than 0,with a ratio of non-synonymous to synonymous mutations(Ka/Ks)greater than 1.Genetic differentiation analysis showed that the fixation(FST)of the PvCHT1 gene in the China-Myanmar border region was 0.31 with inland China,-0.05 with Myanmar,and it ranged from 0.04 to 0.15 with Cambodia,Thailand,Vietnam,Indonesia and Malaysia.Additionally,it ranged from 0.24 to 0.56 with Papua New Guinea,Brazil,Colombia,Peru and Mexico.Population structure analysis indicated that the optimal number of clusters for all populations was 7,with the population at the China-Myanmar border comprised primarily of groups K1 to K6,dominated by K5.Haplotype network analysis showed strong geographical differentiation in the PvCHT1 gene,with four haplotype geographical clusters identified.Haplotype H5 was shared in all countries except in-land China and Mexico.Conclusion PvCHT1 gene showing high conservation in the China-Myanmar border,which suggests that this gene can be a potential candidate target for transmission-blocking vaccines.Furthermore,significant geographical variation exists in the PvCHT1 gene among different populations.The findings indicate that design of the vaccine should be more targeted.
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