日本血吸虫中国大陆株28kDa GST基因在大肠杆菌中的表达

在大肠杆菌ＴＢ１中表达含日本血吸虫中国大陆株２８ｋＤａ抗原基因的重组质粒，表达产物是融合蛋白，分子量来３３ｋＤａ。采用谷胱甘肽琼脂糖亲和层析柱纯化表达产物。２，４－二硝基氯苯／谷胱甘肽分光光度测定法和琼脂糖－淀粉凝胶电泳显示重组抗原具有较高的谷胱甘肽Ｓ－转移酶活力。     

日本血吸虫中国大陆株TPI基因的克隆及其表达产物特性

磷酸丙糖异构酶（ＴＰＩ）是血吸虫病疫苗重要的候选抗原基因之一。参考日本血吸虫已发表的ＴＰＩｃＤＮＡ序列,以日本血吸虫中国大陆株成虫ｍＲＮＡ为模板,用ＲＴ－ＰＣＲ法快速克隆出一大小约８００ｂｐ的ＤＮＡ片段。ＤＮＡ序列分析证实,所扩增到的ＤＮＡ片段即为日本血吸虫中国大陆株磷酸丙糖异构酶（ＳｊＴＰＩｃ）基因。将该基因重组到表达型质粒ｐＧＥＸ－４Ｔ中,表达的ＧＳＴ融合蛋白分子量约５４ｋＤ。用谷胱甘肽琼脂糖凝胶亲和层析柱纯化的重组蛋白不仅纯度好,而且得率高,纯化产量可达３０ｍｇ／Ｌ培养物。免疫试验结果表明该重组蛋白具有良好的抗原性,是一种较为理想的抗原分子。     

日本血吸虫中国大陆株肌动蛋白编码基因的克隆和 …

根据曼氏血吸虫肌动蛋白基因ＳｍＡｃｔ２设计一对引物,以日本血吸虫中国大陆株成虫ｍＲＮＡ为模板,用ＲＴ－ＰＣＲ法扩增出一大小为１１３１ｂｐ的基因片段。测序后分析序列推断该片段为编码肌动蛋白基因的完整阅读框基因,与ＳｍＡｃｔ２碱基同源性为９２％。奖其克隆到表达载体ｐＥＴ２８ａ（＋）中,在大肠杆菌ＢＬ２１中获得了较高量的表达,融合表达产物分子量约为４７ｋＤ。利用日本血吸虫成虫粗抗原免疫大白兔所得抗血清对     

日本血吸虫（中国大陆株）谷胱甘肽转移酶基因扩增及克隆

以日本血吸虫成虫ＲＮＡ为模板逆转录合成ｃＤＮＡ链,设计合成引物,用ＰＣＲ法扩增谷胱甘肽转移酶（ＧＳＴ） 基因编码序列,将其克隆入ｐＧＥＭ—Ｔ载体,并进行初步鉴定。ｐＧＥＭ—Ｔ—ＧＳＴ克隆载体的成功构建,为进一步选择在不同表达系统中表达ＧＳＴ及其在血吸虫病免疫诊断、免疫预防中的作用研究提供了条件     

日本血吸虫中国大陆株抱雌沟蛋白编码基因的克隆和表达

根据曼氏血吸虫抱雌沟蛋白SmGcp序列和日本血吸虫编码抱雌沟蛋白保守区的基因片段jGcp1分别设计三对引物,以日本血吸虫中国大陆株成虫mRNA为模板 ,用RT－PCR法扩增出大小为1949bp的基因片段。经序列分析推断该基因片段含编码日本血吸虫抱雌沟蛋白基因的阅读框,与SmGcp碱基一致性为85％,其理论推测氨基酸组成与曼氏血吸虫抱雌沟蛋白的一致性为83．7％。将上述扩增的基因片段克隆到表达载体pET28c( )中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为80kD。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性。     

日本血吸虫(中国大陆株)谷胱甘肽转移酶编码区基因的体外扩增、克隆及在原核细胞的高效表达

为表达、获取日本血吸早谷胱甘肽转移酶（ＳｊＧＳＴ）基因工程重组蛋白,以日本血吸虫（中国大陆株）ｃＤＮＡ为模板,设计、合成特定寡核苷酸引物,ＲＴ－ＰＣＲ法扩增ＧＳＴ编码基因序列,将扩增产物连接ｐＧＥＭ－Ｔ克隆载体,再亚克隆到真、原核表达质粒ｐＢＫ－ＣＭＶ中,转染大肠杆菌ＸＬ１－ｂｌｕｅ,经ＩＰＴＧ诱导后用ＳＤＳ－ＰＡＧＥ分析表达效果。结果 ＲＴ－ＰＣＲ法特异性扩增出日本血吸虫ＧＳＴ编码区基因片段,其     

日本血吸虫中国大陆株26kD GST基因在大肠杆菌中的高效表达

用大肠杆菌高效表达日本血吸虫中国大陆株 2 6k D GST基因 (Sj2 6)并观察表达产物诱导的免疫保护效果 .将 Sj2 6基因亚克隆至 p ET2 8b(+)中构建重组表达质粒 Sj2 6/ p ET,转化大肠杆菌 BL 2 1 (DE3) .Sj2 6在诱导条件下及未诱导条件下均获得高效表达 .表达产物 (r Sj2 6GST)以包含体形式表达 ,分子量为 2 8k D左右 ,能用 6× His亲和层析柱纯化 ,纯化产量为 55～ 60 mg/ L大肠杆菌培养物 .免疫印迹及 ELSA实验证实 r Sj2 6GST具有良好抗原性 .用 r Sj2 6GST不加佐剂直接背部皮下免疫昆明系小鼠 ,获得了 1 9.6% (P<0 .0 5)的减虫率及 31 .9% (P<0 .0 5)的成熟虫体减虫率 ;且免疫组检获的虫体中未成熟虫体的比例为 32 % (66/ 2 0 6) ,明显高于对照组的 1 9.6% (56/2 85) (P<0 .0 1 ) ,说明 r Sj2 6GST免疫不仅可诱导抗日本血吸虫的部分攻击感染作用 ,而且能抑制部分虫体的发育 .     

日本血吸虫中国大陆株21.7kD蛋白编码基因的克隆和表达

根据日本血吸虫菲律宾株编码21.7kD蛋白的基因设计引物,以日本血吸虫中国大陆株成虫mRNA为模板,用RT-PCR法扩增出大小为558bp的基因片段。经序列分析推断该基因片段为编码日本血吸虫中国大陆株21.7kD蛋白基因的完整阅读框,与菲律宾株该基因的碱基序列同源性为98%。将其克隆到表达载体pET28a(+)中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为25.4kD。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该基因的表达产物具有抗原性。     

中国大陆五个地区日本血吸虫人工感染恒河猴的研究

本文报告了具有分布区代表性的中国大陆5地日本血吸虫人工感染恒河猴的实验结果。在相同宿主、相同感染度和相同感染期的条件下,恒河猴感染云南、广西两地日本血吸虫后的平均虫卵开放前期、两性虫体体长及性腺发育以及肝组织中单个虫卵引起增生期肉芽肿的平均体积与感染安徽、湖北、四川三地虫体的有所不同。这表明恒河猴宿主机体对感染前两地虫体的应答情况与对感染后三地的有异,并提示中国大陆境内日本血吸虫可能存在不同的品系。     

The genetic structure of the steppe plant Iris aphylla L. at the northern limit of its geographical range

Genetic diversity of Iris aphylla was examined in seven Polish populations located at the northern limit of the geographical range of the species. Using RAPD analysis, 491 DNA fragments were amplified, all of which were polymorphic. The peripheral populations were characterized by a low number of unique bands (one to three per population) and moderate values of genetic diversity parameters (percent of polymorphic loci = 30.6%, Shannon's index = 0.149 and Nei's gene diversity = 0.097, on average), which can be explained as being due to the recent expansion of the European biota and the extremely low frequency or absence of seed set and of juveniles, as well as limited pollen and seed dispersal. There was also no significant correlation between ecological properties (population size; number of flowering ramets and fruits) and genetic diversity parameters. Despite this fact, more fixed loci were noted in the smallest populations than were in the largest ones. All populations were significantly differentiated and geographically structured. Analysis of molecular variance between regions showed a low and nonsignificant value, but relatively high molecular variation was partitioned among and within populations. Only one group of south-eastern populations was structured according to isolation by distance pattern and neighbour-joining cluster analysis. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 245–255.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract

Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated F_ST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.     

中国特有种四合木种群遗传多样性的RAPD分析

采用随机扩增多态性 DNA(RAPD)技术检测了西鄂尔多斯高原特有种四合木种群 5个斑块的遗传多样性。2 2个扩增引物产生 1 1 6条带 ,Shannon信息指数和 Nei指数对 RAPD数据的分析表明 :四合木种群存在较高的遗传多样性 ,其中千里山斑块的遗传多样性和多态位点比例较高 ,石嘴山斑块的最低。遗传多样性的 86.5 %存在于斑块内 ,斑块间的遗传变异为 1 3 .5 %。遗传距离与地理距离无直接相关关系。这些结果说明 ,遗传多样性反映了四合木种群基因组 DNA存在较高的变异性 ;同时各斑块间存在一定的基因流 ,四合木各斑块可看成是处于同一种群的半隔离状态 (meta-种群 ) ,对四合木应注意保护遗传多样性丰富的 meta-种群。     

Genetic diversity analysis of Hepatacodium miconioides at different altitude in Tiantal Mountain,Zhejiang Province,China and its relationship with environmental factors

The genetic diversity within and among populations of Hepatacodium miconioides collected at three different altitudes in Tiantai Mountain,Zhejiang Province and its relationships to environmental factors were analyzed by random amplified polymorphic DNA(RAPD)technique.Amplification using 12 random primers of 60 plants and 122 repetitive loci were produced.The percentage of polymorphic loci of three populations ranged from 18.85% to 23.77% with an average of 21.86%,indicating the relatively low genetic diversity of H.miconioides.The average Shannon index of phenotypic diversity(0.1329)and Nei index(0.0925)within populations were relatively low.A distinct genetic differentiation existed among populations Of H miconioides in spite of the relatively small geographical distribufion.The average genetic diversity within populations of H.miconioides accounted for 33.58% of the total genetic diversity while the genetic diversity among populations accounted for 66.42% as estimated by the Shannon index of phenotypic diversity,The genetic differentiation among populations of H.miconioides was 0.6546,as estimated by Nei index.The gene flow estimated from Gsr was only 0.2656 and it indicated that gene flow among populations of H.miconioides was relatively low.The mean value of the genetic identity among populations of H.miconioides was 0.7126 and the average of genetic distance of H.miconioides was 0.3412.The genetic identity between populations at the elevation of 990m and at the elevation of 780 m was the highest.The genetic identity between population at the elevation 500 m and other two populations was relatively low.The correlation analysis showed that the genetic diversity within populations was significantly related with the soil total nitrogen.     

Genetic diversity analysis of Hepatacodium miconioides at different altitude in Tiantai Mountain, Zhejiang Province, China and its relationship with environmental factors

The genetic diversity within and among populations of Hepatacodium miconioides collected at three different altitudes in Tiantai Mountain, Zhejiang Province and its relationships to environmental factors were analyzed by random amplified polymorphic DNA (RAPD) technique. Amplification using 12 random primers of 60 plants and 122 repetitive loci were produced. The percentage of polymorphic loci of three populations ranged from 18.85% to 23.77% with an average of 21.86%, indicating the relatively low genetic diversity of H. miconioides. The average Shannon index of phenotypic diversity (0.1329) and Nei index (0.0925) within populations were relatively low. A distinct genetic differentiation existed among populations of H. miconioides in spite of the relatively small geographical distribution. The average genetic diversity within populations of H. miconioides accounted for 33.58% of the total genetic diversity while the genetic diversity among populations accounted for 66.42% as estimated by the Shannon index of phenotypic diversity, The genetic differentiation among populations of H. miconioides was 0.6546, as estimated by Nei index. The gene flow estimated from G _ST was only 0.2656 and it indicated that gene flow among populations of H. miconioides was relatively low. The mean value of the genetic identity among populations of H. miconioides was 0.7126 and the average of genetic distance of H. miconioides was 0.3412. The genetic identity between populations at the elevation of 990 m and at the elevation of 780 m was the highest. The genetic identity between population at the elevation 500 m and other two populations was relatively low. The correlation analysis showed that the genetic diversity within populations was significantly related with the soil total nitrogen. __________ Translated from Journal of Zhejiang University (science Edition) 2005, 32 (4)[译81EA;: 浙江大学学报(理学版), 2005,32(4)]     

新疆桑属植物栽培居群的遗传多样性研究

应用RAPD分子标记对新疆不同地区栽培的桑属植物2种3个分类群共11个居群进行了遗传多样性研究。结果表明,新疆桑属栽培植物中虽然存在较为丰富的遗传多样性,多态位点比率(PPB)为87．39％,Shannon多样性指数为0．3997,但在栽培居群内的遗传变异水平相对较低;在不同居群间遗传变异水平仔住很人差异,各居群的多态位点比率(PPB)为4．5％至45．95％,Shannon多样性指数为0．0312至0．2339;白桑(Morus alba L．)及其变种鞑靼桑(Morus alba L．var．tatarica)居群内的遗传变异水平远高于黑桑种(Morus nigra)。新疆桑属植物栽培居群内较低的遗传变异水平与其采用扦插等无性繁殖方式有关。分析全部的遗传变异显示,11个栽培居群之间的基因分化系数(Gst)为0．3541,其中桑及其变种9个居群间的基因分化系数为0．4597,黑桑种2个居群问的基因分化系数为0．4728。AMOVA分析表明,在全部遗传变异中,黑桑种和白桑种2个物种之间的遗传变异占59．16％,居群间遗传变异为17．46％。遗传距离和聚类分析也表明,黑桑种和白桑种及其变种鞑靼桑之间存在很大的遗传分化。     

缙云山川鄂连蕊茶在不同群落类型中的RAPD分析

采用随机扩增多态性DNA(RAPD)对常绿阔叶林灌木层广布种川鄂连蕊茶(Camellia rosthorniana)在缙云山3个群落类型(毛竹林、针阔混交林和常绿阔叶林)中的遗传结构和DNA多样性进行了研究。10个引物共扩增出138个产物,其中115个是多态性的,多态位点比率为83．33％。Shannon指数估算的3个种群的遗传多样性为常绿阔叶林0．3345,毛竹林0．3148,针阔混交林0．3085,遗传分化为8．12％;Nei指数计算的3个种群的基因多样性为常绿阔叶林0．2095,毛竹林0．1981,针阔混交林0．1934,遗传分化为7．51％。种群间遗传距离在0．0177～0．0393之间。川鄂连蕊茶在3个群落中的种群大小和生长状况不同,各种群遗传多样性的高低可能是对所处群落生境的适应。     

Random amplified polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered plant Cerastiumfischerianum var. molle than in the widespread conspecific C. fischerianum var. fischerianum (Caryophyllaceae)

Random amplified polymorphic DNA (RAPD) analysis was used to assess the level of genetic diversity in an endangered plant Cerastiumfischerianum var. molle and the widespread conspecific C. fischerianum var. fischerianum. At both the variety and the population level, C. fischerianum var. molle had a lower level of RAPD variation than var. fischerianum. Analysis of molecular variance ( AMOVA ) showed that most of the genetic diversity exists among populations for both varieties. Genetic differentiation among populations probably resulted from the highly selfing habits of both varieties. To conserve C. fischerianum var. molle genetically, preservation of only a part of the population is insufficient.     

亚欧美栗疫病菌群体的遗传多样性

从 12 0个随机引物中筛选出条带清晰、主带明显、重复性好的 9个引物 ,对来自不同地域和寄主的 7个群体的 14 2个栗疫病菌菌株进行 RAPD分析。 9个引物共扩增出条带 12 4条 ,其中多态性条带 111条 ,多态性比率为 89.5 2 %。利用 Popgen3.2软件对供试群体进行遗传多样性分析和 UPGMA聚类。结果表明 ,中国地区 4个群体间的遗传相似性较大 ,与美国、意大利和日本群体间的相似性较小 ;美国和意大利群体间的遗传相似性较大 ,且它们与日本群体间的相似性大于与中国群体间的相似性。病原菌群体的遗传变异率为 0 .2 35 1,其中在地区水平上 ,82 .34%由群体内的变异引起 ,17.6 6 %由群体间的差异引起 ,群体间的基因流动值为 2 .3311;而在寄主水平上 ,则 79.4 2 %由群体内的变异引起 ,2 0 .5 8%由群体间的差异引起 ,群体间的基因流动值为 1.92 97