鄂赣浙3省10地12株并殖吸虫基因ITS2分析

发布时间：2018-01-08 07:23

本文关键词：鄂赣浙3省10地12株并殖吸虫基因ITS2分析　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：并殖吸虫俗称肺吸虫,某些寄生于人体的虫体可引起并殖吸虫病(肺吸虫病)。并殖吸虫在分类上隶属于生物的真核总界、动物界、扁形动物门、吸虫纲、复殖目、并殖科。并殖吸虫种类命名甚多,存在同种异名和种名错定等诸多问题。并殖吸虫在东亚、东南亚、非洲和拉丁美州等国家广泛存在；中国各地也均有发现,主要在江南、西南与东北各地。从1879年Ringer首先在我国台湾发现人体病例130年以来,世界学者对并殖吸虫的特征、独立地位、地理分布和致病作用等存在分歧。主要以并殖吸虫成虫形态和生活史为依据的传统分类存在一定的局限性,如因不同地区、不同宿主、不同发育期和标本制作时个体差异所导致成虫的形态差异。而对于生活史,目前只有少数虫种的生活史被完整阐明,有一些虫种的第一和第二中间宿主尚未被证实。20世纪60-80年代,由于并殖吸虫分类研究极度活跃,出现了很多并殖吸虫新种的描述。20世纪90年代以来,分子生物学的发展为研究物种遗传变异奠定了基础。本课题基于物种DNA分析的分子生物技术较先进,具有仅需少量的材料即可进行研究、操作简单、快速、特异和敏感等特点。又基于本课题组先前研究结果显示并殖吸虫可能存在种间差异,而ITS2序列具有种株特异性。因此,本实验采用ITS2基因序列分析技术对鄂浙赣3省10地12株并殖吸虫进行遗传变异检测,以了解基因序列变化差异,为并殖吸虫分类提供依据,同时也为湖北省并殖吸虫流行区的划分提供可靠的分子生物学依据。材料与方法采集鄂赣浙3省10地：湖北省咸宁市温泉区桂花镇茅田、江西武宁县三爪、浙江绍兴兰亭乡、湖北省赤壁市赵李桥镇柘砰村、湖北省十堰市花果、湖北省五峰县、湖北鹤峰县五里镇南村、湖北鹤峰县走马镇大典、湖北随州和湖北神农架林区等10地共12株并殖吸虫。其中湖北咸宁株、湖北十堰株、江西武宁株和浙江兰亭株为成虫,湖北随州株为童虫,其余5地标本均为囊蚴。为避免混淆,本文中均以地理株命名。对12株并殖吸虫标本进行形态学观察测量,并提取4地成虫、1地童虫和5地囊蚴的基因组DNA。用3s’和BD2两对引物扩增ITS2序列,获得基因片段交由上海生工公司进行基因测序。所得12株并殖吸虫ITS2序列经Bioedit、Clustalx和MEGA4.0等软件进行遗传距离分析,并构建遗传进化树。结果1.外形观察：成虫湖北十堰株呈长梭形,湖北咸宁株、江西武宁株和浙江兰亭株等3株呈椭圆形；囊蚴5地囊蚴呈椭圆形或类圆形,囊壁2层,排泄囊内含黑色颗粒,经鉴定为并殖吸虫囊蚴。其中湖北赤壁株内壁较厚；五峰a、五峰b、走马a、走马b和五里等5株内壁较薄；湖北神农架株的囊蚴形态因故已有变形,但可以确定为并殖吸虫；童虫湖北随州株呈长梭形。2.DNA提取：DNA溶液A260/280值经测定在1.60～2.00之间,纯度符合要求。3.PCR扩增及基因测序,通过MEGA4.0分析基因序列。①各株间差异湖北咸宁株、江西武宁株、浙江兰亭株和湖北赤壁株等4株的遗传距离在0.01897～0.11278；湖北走马a、b两株,湖北五峰a、b两株,湖北十堰株,湖北五里株和湖北随州株等7株斯氏并殖吸虫在遗传树上呈现两支,最后成一大支,其7株遗传距离是0.00004-0.08401；湖北神农架发现的并殖吸虫,该株在进化树上独立成一支,与卫氏并殖吸虫遗传距离在0.01897-0.11278,与斯氏并殖吸虫遗传距离在0.01355-0.12195。②系统进化树通过MEGA4.0软件所构建的进化树中12株并殖吸虫分为三类：江西武宁株、浙江兰亭株、湖北咸宁株和湖北赤壁株等4株为卫氏并殖吸虫；湖北走马a、b两株,湖北五峰a、b两株,湖北十堰,湖北五里和湖北随州等7株为斯氏并殖吸虫；湖北神农架株独立成一类,为尚不明确的一种并殖吸虫。4.湖北省存在两个并殖吸虫流行区：以卫氏并殖吸虫为主的鄂东南和以斯氏并殖吸虫为主的鄂西两个并殖吸虫病流行区。结论1.根据基因测序分析的结果,3省10地12株并殖吸虫在基因水平上分为3类：江西武宁株、浙江兰亭株、湖北咸宁株和湖北赤壁株等4株为卫氏并殖吸虫；湖北走马a、b两株,湖北五峰a、b两株,湖北十堰,湖北五里和湖北随州等7株为斯氏并殖吸虫；湖北神农架株独立成一类,为尚不明确的一种并殖吸虫。2.湖北省存在以卫氏并殖吸虫为主的鄂东南和以斯氏为主的鄂西两个并殖吸虫病流行区。3.采用童虫、成虫和囊蚴等3个不同虫期标本,尤其是本实验首次采用童虫样本,对研究并殖吸虫遗传变异不存在影响。
[Abstract]:Paragonimus commonly known as paragonimiasis, some parasitic in the human body can cause paragonimiasis (paragonimiasis). In the classification of Paragonimus belonging to the eukaryotic organisms total animal kingdom, animal world, flat door, Trematoda, Digenea, paragonimidae Paragonimus species named. So, there are many problems and new synonyms. The wrong set of Paragonimus in East Asia, Southeast Asia, Africa and Latin America, widely exists in other countries; China around were also found, mainly in the south, southwest and northeast country. From 1879 Ringer first in Taiwan in China discovered the human disease cases 130 years since independence the world of Paragonimus, differences in characteristics, geographical distribution and pathogenicity. Mainly Paragonimus adult morphology and life history on the basis of the traditional classification has certain limitation, as a result of different regions, different hosts and different developmental stages and making samples The morphological differences between adults in individual differences. For life history, only a few species life history was completely elucidated, some species of the first and second intermediate host.20 century 60-80 has not yet been confirmed, because of Paragonimus taxonomy is extremely active, since there are a lot of description of the new species of Paragonimus.20 in 90s, the development of molecular biology has laid the foundation for the study of species genetic variation. This project is based on the analysis of the species DNA molecular biology technology is more advanced, has studied only a small amount of material can be simple, rapid, specific and sensitive features. Based on the previous research results show that the research group of Paragonimus may there are differences between species, and the ITS2 sequence has a strain specificity. Therefore, this experiment used ITS2 gene sequence analysis technology of Hubei Province Zhejiang Jiangxi 3 10 12 strains of Paragonimus were genetic variation In order to provide evidence for classification of paragonimiasis and provide a reliable molecular biological basis for the division of paragonimiasis endemic areas in Hubei province.
Materials and methods collected in Hubei and Jiangxi Zhejiang 3 province 10: osmanthus Zhen Mao Tian in Hubei province Xianning city hot spring area, three claw Jiangxi Wuning County, Zhejiang Shaoxing Lanting Pavilion Township, Zhao Li Qiao Zhen zhe Bang village in Hubei Province, Chibi City, Hubei province and Shiyan City, five peaks county of Hubei Province, five in Hefeng County town of Hubei village Hubei County, Hefeng zouma town Hubei Suizhou Hubei Shennongjia forest region ceremony, and in 10 a total of 12 strains of Paragonimus in Hubei. The Xianning Hubei line, Shiyan line, Jiangxi Wuning line and Zhejiang Lanting Pavilion were adults, Hubei Suizhou strains of schistosomula, the remaining 5 samples were metacercariae. To avoid confusion, this paper are the geographical strains named. Morphological observation and measurement of 12 strains of Paragonimus were extracted and 4 adults, 1 to 5 metacercariae of schistosomula and genomic DNA. by 3S and BD2 two primers amplified ITS2 gene fragment sequence, obtained by Shanghai SANGON company for sequencing. The 12 The genetic distance of Paragonimus strain ITS2 was analyzed by Bioedit, Clustalx and MEGA4.0 software, and the genetic evolution tree was constructed.
Results 1. shape observation: adult Hubei Shiyan strain fusiform, Hubei Xianning strain, Jiangxi strain and Zhejiang strain of Wuning Lanting Pavilion and other 3 strains were oval; metacercariae of 5 metacercariae were oval or round, cystic wall 2, excretory sac containing black granules were identified as Paragonimus metacercaria. Hubei Chibi plant wall thick; five peaks of a, five peaks of B, a B and five horse, horse in 5 strains of thin wall; Hubei Shennongjia strains of metacercariae form for existing deformation, but can be identified as Paragonimus; schistosomula Hubei Suizhou strain fusiform.2.DNA extraction: DNA solution A260/280 the value measured at 1.60 ~ 2, the purity of compliance with the requirements of.3.PCR amplification and gene sequencing, gene sequence analysis by MEGA4.0. The difference between strains of Hubei Xianning strain, Jiangxi strain in Wuning, Zhejiang and Hubei Chibi Lanting Pavilion strain strains and 4 strains of genetic distance ranged from 0.01897 to 0.11278; Hubei Ma a, two strains of b, Hubei five peak a, two strains of B, the Hubei Shiyan line, Hubei line and Hubei Suizhou in five strains and 7 strains of Paragonimus skrjabini in genetic tree has two branches, and finally into a large, 0.00004-0.08401 is the genetic distance of 7 strains of Hubei; Shennongjia found Paragonimus, the strains in the evolutionary tree on an independent branch, and the genetic distance of Paragonimus westermani in 0.01897-0.11278, and Paragonimus skrjabini genetic distance between 0.01355-0.12195. and the phylogenetic tree by the phylogenetic tree constructed by MEGA4.0 software in 12 strains of Paragonimus were divided into three categories: Jiangxi Wuning strain, Zhejiang strain and Xianning strain of Hubei Lanting Pavilion, Hubei Chibi strains of 4 strains of Paragonimus westermani; Hubei zouma a, two were B, five peaks of Hubei a, two strains of B, Hubei Shiyan, Hubei and Hubei five in Suizhou and other 7 strains of Paragonimus skrjabini; Hubei Shennongjia strain of an independent class, as it is not clear a kind of Paragonimus.4. in Hubei Province There are two paragonimiasis endemic areas: the two paragonimiasis endemic areas in Western Hubei, Southeast Hubei and Paragonimus, dominated by Paragonimus weir.
Conclusion: 1. according to the sequencing results, 3, 10 and 12 strains of Paragonimus in gene level is divided into 3 categories: Jiangxi Wuning strain, Zhejiang strain and Xianning strain of Hubei Lanting Pavilion, Hubei Chibi strains 4 strains of Paragonimus westermani; Hubei zouma a, two were B, five peaks of Hubei a B, two strains, Hubei Shiyan, Hubei and Hubei five in Suizhou and other 7 strains of Paragonimus skrjabini; Hubei Shennongjia strain of an independent class, with Paragonimus westermani mainly in Southeastern Hubei and Steinernema based Hubei two paragonimiasis flow zone by.3. schistosomula it is not clear for a species of Paragonimus in Hubei province.2., adult and metacercaria 3 specimens of different stages, especially for the first time, the schistosomula samples of Paragonimus genetic variation has no effect.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R383.2

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