日本血吸虫中间宿主湖北钉螺遗传变异及分类的研究

发布时间：2018-06-25 01:40

本文选题：日本血吸虫病 + 日本血吸虫　；参考：《复旦大学》2006年博士论文

【摘要】： 日本血吸虫病是一种严重危害人类健康的人畜共患性疾病，在流行区严重影响居民的健康和当地经济的发展。湖北钉螺是日本血吸虫的唯一中间宿主，在血吸虫病流行中起着极其重要的作用。在我国大陆，钉螺主要分布于长江以南的12个省、市、自治区。至2003年全国共有钉螺面积3，786.8Km~2。以往国内外对钉螺的形态学、地理分布、孳生环境、生理和生化特性等方面进入了较为深入的研究，显示湖北钉螺不仅在我国分布较广，而且在形态学、遗传学以及钉螺对血吸虫易感性等方面都存在着地域上的差异。国内外对钉螺遗传变异的研究中，以同工酶的研究较为多见，其次是对线粒体DNA和染色体核型等的研究，较少见对钉螺基因组遗传变异的研究。湖北钉螺的分类，多年来一直存在着争议，至今仍无定论，对钉螺种群内的遗传变异程度也存在着截然相反的结论。本次研究从钉螺形态形状和基因组DNA水平2个层次上对我国湖北钉螺的遗传变异进行系统的研究。第一部分第一节，目的探讨扩增片段长度多态性(AFLP)分子标记在钉螺遗传变异研究中应用的可能性，并进行实验条件优化和引物筛选。方法随机抽取云南大理和湖南君山阴性钉螺各1只，用异硫氰酸胍和Resin等抽取DNA，然后用64对引物对基因组DNA进行AFLP扩增，扩增产物用6％的变性聚丙烯酰胺凝胶电泳，荧光检测扩增产物。结果每对引物扩增的AFLP标记数在5—55之间，大理钉螺平均每对引物出现38.30(95％CI36.03～40.57)个标记，君山平均每对引物扩增出39.14(95％CI 36.71～41.57)个标记：每对引物扩增的多态性标记数和多态性频率分别在3～37个和28.6％～76.2％之间，分别平均为23.67(95％CI 22.12～25.22)和47.36％(95％CI 45.22％～49.50％)结论AFLP标记技术能应用于湖北钉螺的分类与遗传多样性的研究。第二节，目的探讨AFLP分子标记电泳图谱信息数量化数据的分析方法。方法随机抽取湖南君山阴性钉螺40只，进行AFLP扩增，用Glyko BandScan软件将钉螺AFLP电泳图谱信息数量化，使用不同的读带标准读带，得到相应数据集，然后对这些数据集进行遗传学统计分析与描述性总结。结果不同的标准所得到的遗传变异结果均有所差别，但随着读带标准值的增加，反映钉螺种群遗传多样性指标(如：Shannon’s信息指数)也增加，当其增加到一定水平时，又开始下降，而基因流和基因一致度则刚好相反。不同读带标准所得的遗传变异结果均呈明显的正态分布(P＞0.05)。以总灰度或以总灰度百分比划分读带标准，所得遗传变异结果的平均值均十分接近。将采集的钉螺中筛选出40只阴性钉螺，随机分为两组，平均基因一致度在总灰度百分比数据中为0.956，在总灰度数据中为0.958；两组间的平均遗传距离在总灰度百分比数据中为0.045，在总灰度数据中为0.043。结论将电泳图谱信息数量化，再以不同的读带标准去处理与分析数据的模式，是一种较为合理且准确的分析方法。第二部分第一节，目的探讨用AFLP分子标记研究湖北钉螺遗传变异的合理样本量与分子位点数。方法选取来自于湖南君山的钉螺为研究材料，用AFLP方法对钉螺基因组DNA进行扩增，然后分析钉螺样本量和分子位点数与遗传变异信息可靠性的关系。结果钉螺样本量和分子位点数与遗传多样性信息的可靠性之间存在明显的关系。当样本量低于7只时，AFLP，总位点数、多态位点数、多态位点频率、Nei's基因多样性指数和Shannon's信息指数变化很大，而当样本量超过30只时，这些指标值的变化趋于平稳。当AFLP分子位点数低于128时，多态位点频率、Nei's基因多样性指数、Shannon's信息指数以及这两个指数的标准差变化相当剧烈，当分子位点数超过338时，这些指标值的变化趋于稳定。结论在用AFLP分子标记技术研究湖北钉螺的遗传变异时，每个钉螺种群内的样本量最好不应低于30只，用于研究分析的分子位点数最好不低于338个。第二节，目的探讨湖北钉螺形态形状变异信息的可靠性与样本量的关系。方法从云南大理和湖南君山两地区随机抽取成年钉螺各60只，分别对钉螺壳形态数量性状进行测量，计算各数量性状指标的变异系数、组间变异百分比和多样性指数。结果当样本量低于30，所得到的遗传变异的结果极不可靠，随着样本量的增加遗传变异信息的可靠性也增加，当样本量超过55时，所得到的遗传变异的结果趋于稳定。结论。在湖北钉螺形态形状的遗传变异研究中，每个钉螺种群以分析不少于55个钉螺标本为好。第三部分第一节，目的探讨湖北钉螺种群内的遗传变异及其程度。方法采用扩增片段长度多态性(AFLP)分子标记技术对9省(云南、四川、广西、福建、湖南、湖北、江西、安徽、江苏)13个钉螺种群基因组DNA进行扩增，分析钉螺种群内的遗传变异。结果13个钉螺种群AFLP扩增片段数在403～472之间，江西星子钉螺种群内遗传多样性较高，多态位点频率、Nei's基因多样性指数和Shannon's信息指数分别为93.22％、0.345和0.510，而广西宜州钉螺种群内遗传多样性较低，以上3指标分别为55.80％、0.191和0.287；广西宜州钉螺种群内的相似性较大，相似系数(中位数)为0.904，而江苏丹徒钉螺种群内的相似性较低，相似系数(中位数)为0.748；13个钉螺种群内的遗传变异差异显著(P＜0.01)，5个光壳钉螺种群内的相似系数普遍高于8个肋壳钉螺种群内的相似系数。结论我国大陆广泛分布的钉螺，种群内存在一定程度的遗传变异。不同地区钉螺种群内遗传变异程度不同，有的相差较大。第二节，目的探讨湖北钉螺种群内的形态形状变异及其程度。方法在中国大陆血吸虫病流行的7个省中，采集不同环境类型的21个钉螺种群，每个种群各测量60只湖北钉螺的11个形态性状。采用形态性状的变异系数、种群内个体间的欧氏距离与多样性指数、及主成分分析3种方法，分析不同湖北钉螺种群内形态性状的变异程度。结果在种群内个体间的欧氏距离和形态性状多样性方面，都以江西都昌的变异最大，平均欧氏距离为2.88，方差为2.33，极差为9.05，多样性指数为1.56，个体间平均距离以四川西昌湖北钉螺种群的最小，仅为1.37，而多样性指数以四川丹棱湖北钉螺种群的最小，为0.91。在不同光壳钉螺种群中，以云南大理钉螺个体间的平均距离最大，为1.90，但方差和极差以江苏宜兴的最大，分别为0.55和4.46。结论不同湖北钉螺种群内形态性状变异程度存在较大的差异，肋壳钉螺种群内的形态性状变异程度普遍高于光壳钉螺种群内的变异程度。第四部分第一节，目的探讨湖北钉螺种群间的遗传变异及其程度。方法采用AFLP分子标记技术对来自中国大陆10省的25个种群钉螺基因组DNA样品池进行扩增，分析钉螺各种群间的遗传变异并对钉螺种群进行聚类分析。结果25个钉螺种群间的相似系数GS_(DICE)在0.694～0.831之间，Nei无偏遗传一致性在0.635～0.799之间，遗传距离D在0.169～0.306之间，Nei无偏遗传距离在0.225～0.452之间，指名亚种(包括或不包括广西钉螺)钉螺种群间的遗传变异程度明显高于滇川亚种钉螺种群间的遗传变异程度(P＜0.01)，光壳钉螺种群间的遗传变异程度明显高于肋壳钉螺种群间的遗传变异程度(P＜0.01)。25个钉螺种群被聚成3类，A类包括来自福建福清和广西宜州的光壳钉螺种群；B类包括来自四川西昌、普格、丹棱、蒲江、广汉和云南大理的光壳钉螺种群；C类则由其它来自长江中下游地区的17个钉螺种群组成。结论在我国分布的湖北钉螺已发生较大的遗传变异，基因组水平上的钉螺种群聚类结果和其地理分布基本一致。第二节，目的探讨湖北钉螺种群间的形态形状变异及其程度。方法采用数量分类法对27个钉螺种群的11个螺壳形态形状指标进行了聚类和主成分分析。结果27个钉螺种群中，以湖南华容钉螺种群与四川丹棱钉螺种群间的形态形状变异最大，其欧氏距离达10.29，而安徽贵池与枞阳钉螺种群间的形态形状变异最小，其欧氏距离仅为0.62。无论指名亚种包括还是不包括广西钉螺，滇川亚种钉螺种群间欧氏距离均明显低于指名亚种种群间的(P＜0.05)：11个光壳钉螺种群间的欧氏距离明显低于16个肋壳钉螺种群间的欧氏距离(P＜0.05)。主成分分析将27个钉螺种群分为三组，第一组包括江陵、贵池、枞阳、丹徒、铜陵1、江宁的钉螺。第二组包括南昌、华容、都昌、汉川、阳新、蔡甸、南县、星子、君山、江山的钉螺。第三组包括宜兴、西昌、广汉、蒲江、丹棱、大理、福清、石门、铜陵2、宜州、普格的钉螺。结论指名亚种钉螺种群间的形态形状变异大于滇川亚种的，肋壳钉螺种群间的形态形状变异大于光壳钉螺的。主成分分析的分类结果与环境类型基本一致。第五部分第一节，目的探讨湖北钉螺的空间遗传结构。方法分析25个钉螺种群间的遗传距离与地理距离的相关性。结果25个钉螺种群间的遗传距离D和Nei无偏遗传距离，，都与其地理距离存在明显的正相关性(P＜0.001)，相关系数分别为0.5234和0.5622；湖北钉螺指名亚种种群间的遗传距离与地理距离也存在正相关(P＜0.001)，遗传距离D的相关系数为0.5276，Nei无偏遗传距离的为0.5770；无论是肋壳钉螺还是光壳钥一螺，钉螺种群间的遗传距离都与地理距离存在正相关((P＜0.001)，肋壳钉螺种群间的遗传距离D和Nei无偏遗传距离与地理距离的相关系数分别为0.3612和0.3916，光壳钉螺的相关系数分别为0.7535和0.7500。结论在我国大陆广泛分布的湖北钉螺种群间具有明显的空间遗传结构。第二节目的探讨湖北钉螺种群间的形态形状变异的空间相关性。方法分析27个钉螺种群间形态形状变异及其与地理距离的相关性。结果27个钉螺种群间形态形状变异与其地理距离存在明显的正相关(P＜0.001)，相关系数r为0.2791；这种显著的相关性在指名亚种和肋壳钉螺种群中仍然存在，但在滇川亚种和光壳钉螺种群中却未发现((P＞0.05)，指名亚种包括广西钉螺种群时相关系数为0.2655(P＜0.01))，不包括时相关系数为0.2567(P＜0.01)，肋壳钉螺种群的相关系数r为0.3121(P＜0.001)。结论在我国大陆分布的肋壳钉螺种群间的表型变异具有明显的空间结构。
[Abstract]:Schistosomiasis japonica is a zoonotic disease which seriously endangers human health. It seriously affects the health of residents and the development of local economy in the epidemic area. Oncomelania Snail is the only intermediate host of Schistosoma japonicum in Hubei. It plays an extremely important role in the epidemic of schistosomiasis. Oncomelania Snail is mainly distributed in the south of the Yangtze River in the mainland of China, 12 The total area of Oncomelania snails in the province, city and autonomous region in 2003 was 3786.8Km~2. in the past countries and abroad on the morphology, geographical distribution, breeding environment, physiological and biochemical characteristics, which showed that Oncomelania hupensis in Hubei is not only widely distributed in China, but also in morphology, genetics and Oncomelania susceptibility to Schistosoma japonicum. There are regional differences in all aspects. In the study of the genetic variation of Oncomelania hupensis, the research of isozymes is more common, the next is the study of mitochondrial DNA and chromosome karyotype, and the study on the genetic variation of Oncomelania snails is rare. The classification of Oncomelania snails in Hubei has been disputed for many years, and it is still undecided. The genetic variation in the snail population also has the opposite conclusion. This study systematically studies the genetic variation of Oncomelania hupensis in Hubei, Hubei, from the shape of Oncomelania snails and the level of genomic DNA.
Part one
The first section, objective to explore the possibility of the application of amplified fragment length polymorphism (AFLP) molecular markers in the study of Oncomelania snails genetic variation, and to optimize the experimental conditions and primer screening. The method was used to randomly select 1 snail negative Oncomelania snails in Dali and Hunan, Yunnan, Hunan and Junshan, and use guanidine thiocyanate and Resin to extract DNA, and then 64 pairs of primers were used for genomic DNA. The amplified products were amplified by 6% denatured polyacrylamide gel electrophoresis with 6% AFLP markers per pair of primers and 38.30 (95%CI36.03 to 40.57) markers per pair of Oncomelania Oncomelania in Dali, and 39.14 (95%CI 36.71 to 41.57) markers per pair of primers in Junshan. The polymorphic markers and polymorphic frequencies per pair of primers were 3~37 and 28.6% to 76.2%, respectively, 23.67 (95%CI 22.12 ~ 25.22) and 47.36% (95%CI 45.22% ~ 49.50%), respectively. The AFLP marker technique could be applied to the study of the classification and genetic diversity of Oncomelania hupensis in Hubei.
The second section is to discuss the quantitative data analysis method of AFLP molecular marker electrophoresis map information. Methods 40 Hunan Junshan negative Oncomelania snails were randomly selected for AFLP amplification. The data of the AFLP electrophoresis Atlas of Oncomelania snails were quantified by Glyko BandScan software, and the corresponding data sets were obtained by using different reading band standard reading bands, and then the data were collected. The results of genetic variation obtained by different criteria were different, but as the standard value of the reading band increased, the index of genetic diversity of the Oncomelania Snail population (such as the Shannon 's information index) also increased, and when it increased to a certain level, it began to decline, and the gene flow and gene were consistent. The results of the genetic variation obtained from the standard of different reading bands were all positive in normal distribution (P > 0.05). The average value of the results of the genetic variation was very close. 40 negative Oncomelania snails were selected from the snails collected and divided into two groups, and the average gene consistency was found. It is 0.956 in the total gray scale percentage data and 0.958 in the total gray scale data; the average genetic distance between the two groups is 0.045 in the total gray percentage data. It is more reasonable and accurate to use the 0.043. conclusion to quantify the information of the electrophoretic atlas in the total gray level data and then to deal with and analyze the data by different reading band standards. The method of analysis.
The second part
In the first section, objective to study the rational sample size and number of molecular points of the genetic variation of Oncomelania hupensis in Hubei by AFLP molecular markers. Methods selected Oncomelania snails from Junshan, Hunan as the research materials, amplified the Oncomelania snails genome DNA by AFLP method, and analyzed the relationship between the number of Oncomelania snails and the number of molecular bits and the reliability of genetic variation information. Results there was a significant relationship between the number of Oncomelania snails and the number of molecular sites and the reliability of genetic diversity. When the sample size was less than 7, AFLP, the total number of points, the number of polymorphic sites, the frequency of polymorphic loci, the Nei's gene diversity index and the Shannon's information index changed greatly, and the changes of these index values when the sample size exceeded 30 When the number of AFLP points is less than 128, the frequency of polymorphic loci, the Nei's gene diversity index, the Shannon's information index and the standard deviation of the two indices are very intense. When the number of points is more than 338, the changes tend to be stable. Conclusion the genetic variation of Oncomelania Snail in Hubei is studied by AFLP molecular marker technique. At the same time, the sample size of each snail population should not be less than 30. The number of molecular sites used for research and analysis is best not less than 338.
The second section, objective to investigate the relationship between the reliability of the morphological variation information of Oncomelania snails in Hubei and the relationship between the sample size and the reliability of the snails. Methods 60 adult snails were randomly selected from the two regions of Dali and Junshan in Hunan, Yunnan, and the quantitative traits of the nail shells were measured to calculate the variation coefficients of the quantitative traits, the percentage of variation and diversity among the groups. Results when the sample size is less than 30, the results of the genetic variation are extremely unreliable. The reliability of the genetic variation is also increased with the increase of sample size. The results of the genetic variation tend to be stable when the sample size exceeds 55. Conclusion. In the study of the genetic variation of the shape and shape of Oncomelania snails in Hubei, each snails population is analyzed. No less than 55 Oncomelania Snail specimens are good.
The third part
In the first section, objective to investigate the genetic variation and its degree in the population of Oncomelania hupensis in Hubei. Methods the amplified fragment length polymorphism (AFLP) molecular marker technique was used to amplify the gene group DNA of 13 Oncomelania snails population in 9 provinces (Yunnan, Sichuan, Guangxi, Fujian, Hunan, Hubei, Jiangxi, Anhui, Jiangsu), and analyzed the genetic variation in the Oncomelania snails population. The results were 13 nails. The number of AFLP amplified fragments in the snail population was 403~472, and the genetic diversity of the Oncomelania snails population in Jiangxi stars was higher, the frequency of polymorphic loci, the Nei's gene diversity index and the Shannon's information index were 93.22%, 0.345 and 0.510 respectively, while the genetic diversity of Oncomelania Snail population in Guangxi Yizhou was lower, and the above 3 indexes were 55.80%, 0.191 and 0.287, respectively. The similarity of Oncomelania hupensis population in West Yizhou was larger, the similarity coefficient (median) was 0.904, but the similarity of Oncomelania Snail population in Jiangsu Dantu was lower and the similarity coefficient (median) was 0.748, and the difference of genetic variation within 13 snails population was significant (P < 0.01). The similarity coefficient of 5 light shell snails was generally higher than that in the 8 ribbed snails population. Similarity coefficient. Conclusion the population of Oncomelania Snail in China is widely distributed in a certain degree of genetic variation. The genetic variation of Oncomelania Snail populations in different regions is different, and some are different.
The second section was to investigate the morphological and shape variation and its degree in the population of Oncomelania hupensis in Hubei. Methods 21 Oncomelania Snail populations of different environmental types were collected in 7 provinces of China's schistosomiasis epidemic. Each population measured 11 morphological characters of Oncomelania hupensis in 60 Hubei, using morphological variation coefficient and Euclidean distance among individuals in the population. 3 methods of diversity index and principal component analysis were used to analyze the variation degree of morphological characters in different Hubei Oncomelania Snail population. The variation of Euclidean distance and morphological traits among individuals in the population was the largest in Jiangxi Duchang, with the average Euclidean distance of 2.88, the variance of 2.33, the range of 9.05, and the diversity index of 1.56, respectively. The average distance between the snail population in Xichang and Hubei, Sichuan, is the smallest, only 1.37, and the diversity index is the smallest of Hubei Oncomelania Snail population in Sichuan. It is 0.91. in the different light shell snails population, the average distance between the Oncomelania snails is the largest and 1.90 in Yunnan Dali, but the variance and maximum difference is the largest of Yixing in Jiangsu, respectively 0.55 and 4.46. knot, respectively. The variation degree of morphological characters in the population of Oncomelania hupensis in different Hubei was different, and the variation degree of morphological characters in the snail population was higher than that of the Oncomelania snails population.
The fourth part
In the first section, objective to explore the genetic variation and its degree among Oncomelania Snail populations in Hubei. Method AFLP molecular marker technique was used to amplify the DNA sample pool of Oncomelania snails from 25 populations of 10 provinces of China. Genetic variation among Oncomelania snails was analyzed and the cluster analysis of Oncomelania snails population was analyzed. The results of the similarity system among 25 Oncomelania snails population The number of GS_ (DICE) was between 0.694 and 0.831, Nei unbiased genetic consistency was between 0.635 and 0.799, genetic distance D was between 0.169 and 0.306, Nei unbiased genetic distance was 0.225 to 0.452. The genetic variation of Oncomelania snails among subspecies (including or excluding Oncomelania Guangxi) was significantly higher than that between Oncomelania Oncomelania populations in Yunnan and Sichuan Degree (P < 0.01), the genetic variation among Oncomelania hupensis populations was significantly higher than that between Oncomelania Snail populations (P < 0.01) and.25 snail populations were grouped into 3 groups. A group included light shell snail populations from Fuqing, Fujian and Yizhou of Guangxi, and B included light shells from Sichuan Xichang, Pug, Dan prism, Pujiang, Guanghan and Yunnan. The population of Oncomelania snails and C are composed of 17 Oncomelania Snail populations from the middle and lower reaches of the Yangtze River. The conclusion is that the Oncomelania snails in Hubei have been genetically altered, and the cluster results of Oncomelania Snail populations at the genome level are basically the same as their geographical distribution.
In the second section, the morphological and shape variation and the degree of the population of Oncomelania Snail population in Hubei were studied. Methods the quantitative classification method was used to cluster and principal component analysis on the morphological and shape indexes of 11 spiral shells of 27 snail populations. The results showed that among the 27 Oncomelania hupensis population, the shape and shape variation between Oncomelania and Oncomelania snails population in Huarong, Hunan and Sichuan was the most. The Euclidean distance between Anhui Guichi and Zongyang snail population is the smallest, and the Euclidean distance is only 0.62., the Euclidean distance is only 0.62. or not in Guangxi snail. The Euclidean distance between the snail population in the subspecies of Yunnan and Sichuan is significantly lower than that among the subspecies (P < 0.05):11 light shell snail populations. The distance is significantly lower than the Euclidean distance between 16 ribbed snail populations (P < 0.05). Principal component analysis divides 27 Oncomelania Snail populations into three groups. The first group includes Jiangling, Guichi, Zongyang, Dantu, Tongling 1, and Oncomelania Jiangning. The second groups include Nanchang, Huarong, Duchang, Hanchuan, Yangxin, Caidian, Nanxian, stars, stars, and Oncomelania Snail. Xichang, Guanghan, Pujiang, Pujiang, Daneng, Dali, Fuqing, Shimen, Tongling 2, Yizhou and Pug. Conclusion the morphological and shape variation among the snail populations of the subspecies is greater than that of the subspecies in Yunnan and Sichuan, and the morphological and shape variation among the Oncomelania Snail population is larger than that of the light shell snails. The classification results of principal component analysis are basically the same as the environmental types.
The fifth part
The first section was to investigate the spatial genetic structure of Oncomelania hupensis in Hubei. Methods the correlation between genetic distance and geographical distance between 25 Oncomelania Snail populations was analyzed. The results showed that the genetic distance between 25 Oncomelania Snail populations, D and Nei, had significant positive correlation with their geographical distance (P < 0.001), and the correlation coefficients were 0.5234 and 0.5622, respectively. There was a positive correlation between the genetic distance and geographical distance between the subgroups of Oncomelania snails in Hubei (P < 0.001), the correlation coefficient of the genetic distance D was 0.5276, and the Nei unbiased genetic distance was 0.5770. The genetic distance between the Oncomelania Snail and the light shell was positively correlated with the geographical distance (P < 0.001), and the ribbed snails. Genetic distance between populations and the correlation between D and Nei unbiased genetic distance and geographical distance
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R184

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