本文關(guān)鍵詞：鄂贛浙3省10地12株并殖吸蟲(chóng)基因ITS2分析　出處：《華中科技大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】：并殖吸蟲(chóng)俗稱肺吸蟲(chóng),某些寄生于人體的蟲(chóng)體可引起并殖吸蟲(chóng)病(肺吸蟲(chóng)病)。并殖吸蟲(chóng)在分類上隸屬于生物的真核總界、動(dòng)物界、扁形動(dòng)物門、吸蟲(chóng)綱、復(fù)殖目、并殖科。并殖吸蟲(chóng)種類命名甚多,存在同種異名和種名錯(cuò)定等諸多問(wèn)題。并殖吸蟲(chóng)在東亞、東南亞、非洲和拉丁美州等國(guó)家廣泛存在；中國(guó)各地也均有發(fā)現(xiàn),主要在江南、西南與東北各地。從1879年Ringer首先在我國(guó)臺(tái)灣發(fā)現(xiàn)人體病例130年以來(lái),世界學(xué)者對(duì)并殖吸蟲(chóng)的特征、獨(dú)立地位、地理分布和致病作用等存在分歧。主要以并殖吸蟲(chóng)成蟲(chóng)形態(tài)和生活史為依據(jù)的傳統(tǒng)分類存在一定的局限性,如因不同地區(qū)、不同宿主、不同發(fā)育期和標(biāo)本制作時(shí)個(gè)體差異所導(dǎo)致成蟲(chóng)的形態(tài)差異。而對(duì)于生活史,目前只有少數(shù)蟲(chóng)種的生活史被完整闡明,有一些蟲(chóng)種的第一和第二中間宿主尚未被證實(shí)。20世紀(jì)60-80年代,由于并殖吸蟲(chóng)分類研究極度活躍,出現(xiàn)了很多并殖吸蟲(chóng)新種的描述。20世紀(jì)90年代以來(lái),分子生物學(xué)的發(fā)展為研究物種遺傳變異奠定了基礎(chǔ)。本課題基于物種DNA分析的分子生物技術(shù)較先進(jìn),具有僅需少量的材料即可進(jìn)行研究、操作簡(jiǎn)單、快速、特異和敏感等特點(diǎn)。又基于本課題組先前研究結(jié)果顯示并殖吸蟲(chóng)可能存在種間差異,而ITS2序列具有種株特異性。因此,本實(shí)驗(yàn)采用ITS2基因序列分析技術(shù)對(duì)鄂浙贛3省10地12株并殖吸蟲(chóng)進(jìn)行遺傳變異檢測(cè),以了解基因序列變化差異,為并殖吸蟲(chóng)分類提供依據(jù),同時(shí)也為湖北省并殖吸蟲(chóng)流行區(qū)的劃分提供可靠的分子生物學(xué)依據(jù)。 材料與方法采集鄂贛浙3省10地：湖北省咸寧市溫泉區(qū)桂花鎮(zhèn)茅田、江西武寧縣三爪、浙江紹興蘭亭鄉(xiāng)、湖北省赤壁市趙李橋鎮(zhèn)柘砰村、湖北省十堰市花果、湖北省五峰縣、湖北鶴峰縣五里鎮(zhèn)南村、湖北鶴峰縣走馬鎮(zhèn)大典、湖北隨州和湖北神農(nóng)架林區(qū)等10地共12株并殖吸蟲(chóng)。其中湖北咸寧株、湖北十堰株、江西武寧株和浙江蘭亭株為成蟲(chóng),湖北隨州株為童蟲(chóng),其余5地標(biāo)本均為囊蚴。為避免混淆,本文中均以地理株命名。對(duì)12株并殖吸蟲(chóng)標(biāo)本進(jìn)行形態(tài)學(xué)觀察測(cè)量,并提取4地成蟲(chóng)、1地童蟲(chóng)和5地囊蚴的基因組DNA。用3s’和BD2兩對(duì)引物擴(kuò)增ITS2序列,獲得基因片段交由上海生工公司進(jìn)行基因測(cè)序。所得12株并殖吸蟲(chóng)ITS2序列經(jīng)Bioedit、Clustalx和MEGA4.0等軟件進(jìn)行遺傳距離分析,并構(gòu)建遺傳進(jìn)化樹(shù)。 結(jié)果1.外形觀察：成蟲(chóng)湖北十堰株呈長(zhǎng)梭形,湖北咸寧株、江西武寧株和浙江蘭亭株等3株呈橢圓形；囊蚴5地囊蚴呈橢圓形或類圓形,囊壁2層,排泄囊內(nèi)含黑色顆粒,經(jīng)鑒定為并殖吸蟲(chóng)囊蚴。其中湖北赤壁株內(nèi)壁較厚；五峰a、五峰b、走馬a、走馬b和五里等5株內(nèi)壁較��；湖北神農(nóng)架株的囊蚴形態(tài)因故已有變形,但可以確定為并殖吸蟲(chóng)；童蟲(chóng)湖北隨州株呈長(zhǎng)梭形。2.DNA提�。篋NA溶液A260/280值經(jīng)測(cè)定在1.60～2.00之間,純度符合要求。3.PCR擴(kuò)增及基因測(cè)序,通過(guò)MEGA4.0分析基因序列。①各株間差異湖北咸寧株、江西武寧株、浙江蘭亭株和湖北赤壁株等4株的遺傳距離在0.01897～0.11278；湖北走馬a、b兩株,湖北五峰a、b兩株,湖北十堰株,湖北五里株和湖北隨州株等7株斯氏并殖吸蟲(chóng)在遺傳樹(shù)上呈現(xiàn)兩支,最后成一大支,其7株遺傳距離是0.00004-0.08401；湖北神農(nóng)架發(fā)現(xiàn)的并殖吸蟲(chóng),該株在進(jìn)化樹(shù)上獨(dú)立成一支,與衛(wèi)氏并殖吸蟲(chóng)遺傳距離在0.01897-0.11278,與斯氏并殖吸蟲(chóng)遺傳距離在0.01355-0.12195。②系統(tǒng)進(jìn)化樹(shù)通過(guò)MEGA4.0軟件所構(gòu)建的進(jìn)化樹(shù)中12株并殖吸蟲(chóng)分為三類：江西武寧株、浙江蘭亭株、湖北咸寧株和湖北赤壁株等4株為衛(wèi)氏并殖吸蟲(chóng)；湖北走馬a、b兩株,湖北五峰a、b兩株,湖北十堰,湖北五里和湖北隨州等7株為斯氏并殖吸蟲(chóng)；湖北神農(nóng)架株獨(dú)立成一類,為尚不明確的一種并殖吸蟲(chóng)。4.湖北省存在兩個(gè)并殖吸蟲(chóng)流行區(qū)：以衛(wèi)氏并殖吸蟲(chóng)為主的鄂東南和以斯氏并殖吸蟲(chóng)為主的鄂西兩個(gè)并殖吸蟲(chóng)病流行區(qū)。 結(jié)論1.根據(jù)基因測(cè)序分析的結(jié)果,3省10地12株并殖吸蟲(chóng)在基因水平上分為3類：江西武寧株、浙江蘭亭株、湖北咸寧株和湖北赤壁株等4株為衛(wèi)氏并殖吸蟲(chóng)；湖北走馬a、b兩株,湖北五峰a、b兩株,湖北十堰,湖北五里和湖北隨州等7株為斯氏并殖吸蟲(chóng)；湖北神農(nóng)架株獨(dú)立成一類,為尚不明確的一種并殖吸蟲(chóng)。2.湖北省存在以衛(wèi)氏并殖吸蟲(chóng)為主的鄂東南和以斯氏為主的鄂西兩個(gè)并殖吸蟲(chóng)病流行區(qū)。3.采用童蟲(chóng)、成蟲(chóng)和囊蚴等3個(gè)不同蟲(chóng)期標(biāo)本,尤其是本實(shí)驗(yàn)首次采用童蟲(chóng)樣本,對(duì)研究并殖吸蟲(chóng)遺傳變異不存在影響。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R383.2

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本文編號(hào)：1396155