波尔山羊杂交后代及其亲本随机扩增多态DNA研究

利用随机扩增多态DNA技术研究了波尔山羊、唐山奶山羊、青龙本地山羊以及波尔山羊与这两个山羊群体杂交后代共计128个山羊个体的随机扩增多态DNA。结果表明：（1）总群体平均遗传多样性指数（Hsp）为0.6974,群体遗传分化指数为0.9706,山羊群体间平均遗传距离指数（0.1314～0.2052）明显大于群体内的相应值（0.0582～0.1440）,上述结果说明,所研究山羊群体不仅具有较为丰富的遗传多样性,而且其核基因组遗传变异主要存在于群体间。（2）山羊群体间的分子聚类关系与各群体间的亲缘关系基本一致。 Abstract:The random amplified polymorphic DNA（RAPD）of 128 individuals was studied,which were from Boer goat, Tangshan dairy goat,Qinglong native goat and their hybrids crossbred with Boer goat.The average index of genetic polymorphism for whole population（Hsp）and the index of genetic differentiation were 0.6974 and 0.9706,respectively.The average index of genetic distance between populations（0.1314～0.2052）was significantly higher than that within populations（0.0582～0.1440）.All of these indicated that the genetic polymorphism was not only abundant,but also the genetic variation was mainly existed between goat populations.The molecular dendrogram among goat populations was in accord with their genetic relationship.     

我国主要地方绵羊品种随机扩增多态DNA研究

对蒙古羊、湖羊、滩羊、小尾寒羊、乌珠穆沁羊、藏绵羊、阿勒泰羊7个地方绵羊品种和无角陶赛特羊、德国美利奴羊、萨福克羊3个引入品种基因组DNA进行了RAPD分析。结果表明：（1）RAPD可作为一种有效的标记用于绵羊品种之间遗传亲缘关系的分析。（2）在所使用的43种随机引物中,有35种引物扩增出多态谱带,多态频率为66.24%,说明RAPD技术用于研究绵羊核DNA的遗传变异具有较高的检出率和灵敏度。（3）总群体平均遗传多样性指数（HSP）为0.9139,说明绵羊群体具有较为丰富的遗传多样性。（4）我国地方绵羊品种间的分子聚类关系与其所处的地理位置、考古学结果,以及细胞遗传学研究结果基本一致,引入品种间的分子聚类关系也与其育成史基本一致。 Abstract:The genetic polymorphism and relationship of 7 indigenous sheep breeds of China and 3 imported sheep breeds were studied using random amplified polymorphic DNA (RAPD).The results indicated that the RAPD was an effective marker for the analysis of genetic relationship among sheep breeds.Among 43 arbitrary primers,35 were polymorphic.The percentage of polymorphic markers was 66.24%,which indicated that the RAPD had higher efficiency of polymorphism detection and sensitivity in studying the genetic variation among sheep breeds.The average index of genetic polymorphism for whole population (Hsp) was 0.9139,which showed that the genetic polymorphism was abundant between sheep populations.The genetic relationship between different indigenous sheep breeds in China was in accord with their localities,the results from archeology and cytogenetics and the genetic relationship between imported sheep breeds was in accord with their breeding history.     

丁不同群体间形态学差异与随机扩增多态DNA(RAPD)分析

采用聚类分析、主成分分析和判别分析3种数理统计方法,对我国新疆朱家湖丁群体7、3水库丁群体和从捷克引进我国的丁群体的可量性状和框架参数进行分析。聚类分析和主成分分析显示,朱家湖群体与73水库群体较为接近,而捷克群体与前两群体相距较远。判别分析亦可将捷克群体与前两群体分开,准确率达100%。从60个随机引物中筛选出的20个引物对丁朱家湖群体、73水库群体和捷克群体进行了随机扩增多态DNA(RAPD)分析。引物S440在捷克群体扩增出的450bp片段为该群体特有的标记片段。朱家湖群体、73水库群体、捷克群体多态位点比例分别为24%、22.67%和18.42%;群体内遗传相似度分别为0.8967、0.9035和0.9309;遗传多态度(π)分别为0.1539、0.1489和0.1142。表明朱家湖群体保持着较高的遗传变异。三群体间的遗传相似度为0.6868—0.9496,群体遗传分化系数(Fst)为0.048—0.238,分子方差分析发现群体内方差占总方差的83.96%,群体间的方差只占16.04%,由此推断三群体间遗传分化并不大。     

随机扩增多态性DNA技术对结核分支杆菌菌株鉴别及其耐药相关性分析

用引物G1和L1扩增的16S-23SrDNA间隔区作为模板DNA,再用引物AP50进行随机扩增多态性DNA（Random Amplified PolymorphicDNA,RAPD)分棉线AP50扩增出的各菌株DNA片段,用1.2%的琼脂糖凝胶电泳EB染色,可获得1或2个大小不同的DNA片段（390-1031bp)。各株间差异明显,结果表明,不同菌株有不同的DNA多态性,药物敏感株与耐药株之间多态性差异明显,耐药株中耐药谱相似或相同的菌株间其多态性也相似,RAPD有助于简便,快速,有效地了解菌株间的克隆相关性及耐药性传播。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD方法扩增出的高变异产物含有重复序列.用含重复序列的个体特异性RAPD产物作探针,与无关个体鸡基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变异性较高的DNA指纹图谱.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

丁(鱼岁)不同群体间形态学差异与随机扩增多态DNA(RAPD)分析

采用聚类分析、主成分分析和判别分析3种数理统计方法,对我国新疆朱家湖丁(鱼岁)群体、73水库丁(鱼岁)群体和从捷克引进我国的丁(鱼岁)群体的可量性状和框架参数进行分析.聚类分析和主成分分析显示,朱家湖群体与73水库群体较为接近,而捷克群体与前两群体相距较远.判别分析亦可将捷克群体与前两群体分开,准确率达100%.从60个随机引物中筛选出的20个引物对丁(鱼岁)朱家湖群体、73水库群体和捷克群体进行了随机扩增多态DNA(RAPD)分析.引物S440在捷克群体扩增出的450bp片段为该群体特有的标记片段.朱家湖群体、73水库群体、捷克群体多态位点比例分别为24%、22.67%和18.42%;群体内遗传相似度分别为0.8967、0.9035和0.9309;遗传多态度(π)分别为0.1539、0.1489和0.1142.表明朱家湖群体保持着较高的遗传变异.三群体间的遗传相似度为0.6868-0.9496,群体遗传分化系数(Fst)为0.048-0.238,分子方差分析发现群体内方差占总方差的83.96%,群体间的方差只占16.04%,由此推断三群体间遗传分化并不大.     

布氏田鼠封闭群遗传结构的随机扩增多态DNA分析

目的使用随机扩增多态DNA标记建立标准化的布氏田鼠封闭群遗传质量控制分子标记库。方法使用高盐沉淀法从鼠尾中提取布氏田鼠基因组DNA。采用40条PRAD引物对布氏田鼠封闭群进行PCR扩增,琼脂糖电泳分离条带,参考标准分子量标记计算条带大小,并使用多态位点数、单态位点数以及多态位点比率评价种群的遗传多样性。结果筛选出8个能获得清晰稳定扩增条带的RAPD标记。这8个RAPD标记检测到的多态位点数存在明显差异。8个引物得到的遗传多态位点的数据之和能揭示种群的遗传结构。结论本实验建立了检测布氏田鼠封闭群遗传结构的RAPD标记。     

幽门螺杆菌临床分离株的随机扩增多态性DNA指纹图分析

目的:建立武汉及周边地区幽门螺杆菌(Helicobacter pylori,Hp)感染病人胃内分离的Hp的DNA指纹图谱,并进行数理统计分析,探讨Hp基因型与疾病的相互关系,为临床诊断、防治及致病机制提供理论与实践基础.方法:采取随机扩增多态性DNA指纹法(Random amplified polymorphic DNA,RAPD)对48例病人Hp基因组DNA进行PCR反应,其随机引物为:1290 5'-GTGGATGCGA-3'.反应产物经2%琼脂糖凝胶电泳,成像存盘.用统计分析软件(Statistic analysis software,SAS)对Hp DNA指纹图的相似性以及与疾病的相关性进行分析.结果:每个菌株都有其独特的DNA指纹图,显示其基因的多态性;计算机聚类分析显示:Hp DNA指纹图可分为两大类,其与宿主疾病之间有一定程度的相关性(P＜0.05).结论:(1)RAPD对 Hp DNA扩增结果是稳定、可靠的,是一种较好的分型方法.(2)幽门螺杆菌感染所致疾病可能与其基因型相关.     

鲍曼不动杆菌随机扩增多态性DNA法基因分型的研究

目的 利用随机扩增多态性DNA(RAPD)技术对鲍曼不动杆菌进行基因分型建立DNA指纹图谱,调查该菌在各临床科室的流行情况,并将其与药敏谱进行比较.方法 随机收集中南大学湘雅二医院2007年9月到2008年9月分离出的86株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行药物敏感试验,建立药敏谱,同时利用随机扩增多态性DNA法(RAPD)技术进行基因分型,建立DNA指纹图谱,对二者进行比较,然后结合药敏谱和指纹图谱分析各临床科室鲍曼不动杆菌的感染情况.结果 药物敏感试验将86株鲍曼不动杆菌分为47型,RAPD技术将其分为14型,其中A、F、D、B和L型为5种优势型,其菌株数分别为22、15、11、9和7株.本院ICU,老年病科,神经内科,呼吸内科,神经外科鲍曼不动杆菌检出率较高.结论 RAPD分型方法优于药敏分型,其在流行病学研究上更能证实菌株的相关性,在早期发现和预防感染暴发流行中起重要作用.     

白僵菌不同菌株DNA扩增图谱与其来源的相关性分析

采用RAPD技术对28个不同来源的球孢白僵菌菌株的DNA指纹图谱进行了测定。菌株间显示了DNA图谱的多态性,但系统聚类分析结果表明,其多态性与菌株的原寄主和原采集地之间未表现出相关性。     

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.     

鼠李糖乳杆菌遗传转化方法的优化

采用原生质体电转化和PEB电转化方法向鼠李糖乳杆菌LA-04-01中导入外源基因。在培养过程中使用甘氨酸和青霉素辅助溶菌酶处理细胞制备原生质体,得到300个/μg的转化子。使用PEB缓冲液处理细胞,使转化率稳定达到6×105个/μg,建立了针对鼠李糖乳杆菌简便高效的转化方法,为进一步的基因操作提供了便利。     

番茄随机扩增DNA多态性体系的条件优化

利用改进的SDS法提取代号为03748的栽培番茄叶片基因组DNA。对影响番茄随机扩增DNA多态性（RAPD）扩增结果的因素进行了分析,确定了模板、Mg^2＋、dNTPs、引物和Tap DNA聚合酶的适宜浓度及反应的最佳循环次数。实验结果表明,在以下条件下,番茄的RAPD扩增效果较好：20μL反应体系中使用20-40ng的模板、1．5-2．0mmol／L的Mg^2＋、0．15＋0．20μmol／L的dNTPs、0．15-2．0μmol／L的引物、1．0U的Taq DNA聚合酶;94℃预变性5min,然后经94℃变性1min、360℃ 1min、720℃ 1．5min,进行35个循环,最后在72℃时再延伸10min。     

Determination of paternity in dragonflies by Random Amplified Polymorphic DNA fingerprinting

We used Random Amplified Polymorphic DNA (RAPD) fingerprinting to address issues of paternity in two odonate species. Amplification artifacts of RAPD markers were controlled by assessing paternity patterns relative to the banding patterns generated by quantitative mixtures of DNA from putative parents ('synthetic offspring'). In the aeshnid dragonfly Anax parthenope , for which the mating histories of both males and females were unknown, we found strong evidence for complete paternity success for the contact guarding male. In the highly polygamous libellulid dragonfly Orthetrum coerulescens , we detected and quantified mixed paternity in sequentially produced offspring clutches and demonstrated that fertilization success is correlated with the duration of copulation. Our results suggest that RAPD fingerprinting is suitable to address issues of paternity in systems which are genetically uncharacterized and produce large offspring clutches.     

Amelioration of regulatory T cells by Lactobacillus delbrueckii and Lactobacillus rhamnosus in pristane-induced lupus mice model

Regulatory T cells (Tregs) play an indispensable role in the control of immune responses and induction of peripheral tolerance. Dysregulation of Tregs is involved in the pathogenesis of systemic lupus erythematosus (SLE). Tolerogenic probiotics have shown beneficial effects in the control of autoimmune diseases. We evaluated the prophylactic and therapeutic effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on Tregs and their related molecules in pristane-induced lupus mice model. Fifty-four female BALB/c mice (3–5 weeks) were randomly divided into nine groups. Lupus was induced in all groups using pristane. Prophylactic groups were treated from Day 0 (at the time of pristane injection) and treatment groups were treated 2 months later with L. rhamnosus, L. delbrueckii, mix of both probiotics, and prednisolone. One group was considered as SLE-induced control group without any treatment. Presence of antinuclear antibodies (ANA), antidouble-stranded DNA (anti-dsDNA), antiribonucleoprotein (anti-RNP), proteinuria, and serum level of creatinine, urea, the expression of forkhead box P3 (Foxp3), interleukin 6 (IL-6), IL-10, transforming growth factor β, and the number of Tregs were determined. SLE induction by pristane led to the formation of lipogranuloma, presence of ANA, anti-dsDNA, and anti-RNP. Probiotics consumption decreased the level of lipogranuloma, ANA, and anti-dsDNA. In addition, in probiotics receiving groups, Tregs and the expression level of Foxp3 increased, while IL-6 decreased. The effect of probiotics in the prophylactic group was more prominent. The results may indicate the effectiveness of L. delbrueckii and L. rhamnosus in the enhancement of Tregs and the decrease of inflammatory cytokines and disease severity in SLE-induced mice.     

用RAPD法研究火炬松愈伤组织发生的遗传特性(英文)

体外培养对于植物的快速繁殖是非常有效的。和其它一些松果类硬木植物一样,火炬松的体外培养成功率却一直很低。本工作研究了不同的基本培养基和低温条件对于火炬松Ｊ－５６, Ｓ－１００３, ａｎｄ Ｅ－４４０等三个品系的成熟合子胚形成愈伤组织、分化出芽、成苗的影响。在不同的基本培养基条件下芽分化的程度差异很大。合子胚经过９－１２周培养,开始分化,形成具有器官发生的愈伤组织（Ｆｉｇ．２ａ）。分化后３周,开始诱导出芽（Ｆｉｇ．２ｂ）,芽的生长快慢不同（Ｆｉｇ．２ｃ,ｄ）。同一个愈伤组织上会生出几个芽来（Ｆｉｇ．２ｅ）。在添加有ＩＢＡ和ＢＡ的ＴＥ培养基上芽生长最快（Ｆｉｇ．１）。低温条件持续 １５天,能增加芽的数量和分化的程度（Ｔａｂｌｅ１）。上述培养基中增加ＧＡ３时表明,ＧＡ３对于根的诱导有决定性的作用。将９８株再生苗转移到特殊的混合土壤上;成活了７５株苗（Ｆｉｇ．２ｆ）。以这三种火炬松的再生苗尖为材料制备ＤＮＡ。用２０个引物进行ＲＡＰＤ分析,结果表明：这三种火炬松苗的扩增产物是相同的（Ｆｉｇ．２ｇ,ｈ＆ｉ）。这说明：用愈伤组织克隆植株的过程中没有引起植物遗传变异。     

大鼠RAPD标记的观察

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）技术,分析ＳＤ和Ｗｉｓｔａｒ二种大鼠的基因多态性,探讨用ＲＡＰＤ标记鉴别二种大鼠及其血标本实验中的认证,结果表明,二种大鼠表现出了各自不同的多态性ＲＡＰＤ标记,作为大鼠的分子标记,可在基因水平区别二种大鼠,故认为是一种大鼠研究的分子依据。     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.