应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

家庭内红色毛癣菌病患者间菌株差异性分析

目的用PCR技术比较分离自同一家庭红色毛癣菌病患者的菌株差异性,分析家庭内多发的红色毛癣菌病的致病菌株是家内相互感染,还是家外感染。方法以家庭内多发的皮肤癣菌病患者为研究对象,分离致病菌株并以传统方法鉴定菌种。再分别用随机扩增多态性DNA(RAPD)和巢式PCR特异扩增红色毛癣菌的串联重复亚元件(TRSS:TRS-1/TRS-2)产生的指纹图谱分析种内株间有无差异性。结果纳入实验的16株菌分离自8个家庭,用形态学等方法及种特异引物均鉴定为红色毛癣菌。RAPD显示4个家庭内的菌株间有差异性,TRS-1区PCR指纹图谱显示5个家庭内菌株有株间差异,TRS-2区能鉴定出2个家庭内菌株间有差异。综合各方法共区分出6个家庭内的菌株间有带型差异。结论该研究提示家庭内多发红色毛癣菌病从家外途径感染率高于家内感染。TRS-1区PCR指纹图谱对红色毛癣菌的菌株区分度高于RAPD,更适于红色毛癣菌株间分型。结合多种分子分型方法可最大限度发现不同菌株间的差异。     

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。     

斜生褐孔菌多糖诱导人肝癌HepG-2细胞凋亡的研究

研究了斜生褐孔菌多糖对人类肝癌HepG-2细胞凋亡的诱导作用。采用噻唑蓝法(MTT法)观察了斜生褐孔菌多糖对HepG-2细胞生长的影响,用透射电镜观察了细胞形态,用DNA Ladder检测了细胞凋亡,用流式细胞仪检测了细胞凋亡率;同时采用逆转录-聚合酶链反应法(RT-PCR法)研究了不同浓度斜生褐孔菌多糖作用后HepG-2细胞中Bax和Bcl-2基因mRNA转录水平的变化。结果表明斜生褐孔菌多糖能抑制HepG-2细胞增殖,并呈时间剂量依赖关系;电镜观察、DNALadder和流式细胞仪检测均证实了斜生褐孔菌多糖能够诱导HepG-2细胞凋亡;经斜生褐孔菌多糖处理后,HepG-2细胞中Bax基因mRNA转录水平增强,而Bcl-2基因mRNA无明显变化。证明了斜生褐孔菌多糖具有抑制HepG-2细胞生长及诱导HepG-2细胞凋亡的作用,这可能与调节Bcl-2和Bax基因表达水平有关。     

东北地区小麦赤霉病菌DNA随机扩增多态性分析

利用RAPD对我国东北地区引致小麦赤霉病的2种镰刀菌进行种群分析,并与江苏和西北的菌株进行了比较。共筛选出16个引物用于扩增反应,其扩增图谱显示供试菌株在种间和种内均具多态性。多数引物扩增出了种的特征性谱带,可用作种的鉴定。综合所有谱带系统聚类所得树状图明显地将Fusarium graminearum的34个菌株和F.avenaceum的5个菌株各聚为一类,每一类又可区分为不同组,以前者分为3组,后者分为2组差异显著,表明种内存在不同的菌系类型;组的划分与菌株致病力及其寄主品种间没有必然的相关性,但与菌株分布的大区域生态气候类型似有联系,供试的东北地区F.graminearum大多数菌株与西北的菌系有较大的遗传相似性,与江苏的菌系相距甚远。     

东北地区小麦赤霉病菌DNA随机扩增多态性分析*

利用RAPD对我国东北地区引致小麦赤霉病的2种镰刀菌进行种群分析,并与江苏和西北的菌株进行了比较。共筛选出16个引物用于扩增反应,其扩增图谱显示供试菌株在种间和种内均具多态性。多数引物扩增出了种的特征性谱带,可用作种的鉴定。综合所有谱带系统聚类所得树状图明显地将Fusarium graminearum的34个菌株和F.avenaceum的5个菌株各聚为一类,每一类又可区分为不同组,以前者分为3组,后者分为2组差异显著,表明种内存在不同的菌系类型;组的划分与菌株致病力及其寄主品种间没有必然的相关性,但与菌株分布的大区域生态气候类型似有联系,供试的东北地区F.graminearum大多数菌株与西北的菌系有较大的遗传相似性,与江苏的菌系相距甚远。     

东北地区小麦赤霉病菌DNA随机扩增多态性分析

利用RAPD对我国东北地区引致小麦赤霉病的2种镰刀菌进行种群分析。并与江苏和西北的菌株进行了比较,共筛选出16个引物用于扩增反应,其扩增图谱显示供试菌株在种间和种内均具多态性,多数引物扩增出了种的特征性谱带,可用作种的鉴定,综合所有谱带系统聚类所得树状图明显地将Fusarium graminearum的34个菌株和F.avenaceum的5个菌株各聚为一类,每一类又可区分为不同组,以前者分为3组,后者分为2组差异显著,表明种内存在不同的菌系类型。组的划分与菌株致病力及其寄主品种间没有必然的相关性,但与菌株分布的大区域生态气候类型似有联系,供试的东北地区F.graminearum大多数菌株与西北的菌系有较大的遗传相似性,与江苏的菌系相距甚远。     

深层培养的斜生褐孔菌酚类化合物对铅处理小鼠耐力的增强作用

本文研究了不同条件下深层培养的斜生褐孔菌Phaeoporus obliquus与野生桦褐孔菌在酚类化合物组成及其含量上的差异以及对铅处理小鼠常压缺氧和游泳耐力影响。野生桦褐孔菌主要含有水溶性黑色素、硬毛素衍生物,两者各占总酚类化合物的12.1%和42.2%。而深层培养的桦褐孔菌则主要积累黄酮类化合物,其中正常生理条件下积累量达到总酚的35.5%,加入H2O2后积累量达到总酚的92.5%,同时加入H2O2和熊果苷后则为总酚的76.9%。正常培养基中水溶性黑色素的积累量达到总酚的20.2%,而加入H2O2或同时加入H2O2和熊果苷后水溶性黑色素则很少合成。口服野生斜生褐孔菌或不同条件下深层培养的斜生褐孔菌酚类化合物都能使铅处理小鼠忍耐缺氧和游泳的时间延长12–15min。同时,口服不同来源的斜生褐孔菌酚类化合物可提升铅处理小鼠外周淋巴细胞的活力和总超氧化物歧化酶活性,并降低丙二醛的积累。因此,斜生褐孔菌酚类化合物对铅处理小鼠耐缺氧和游泳的提升作用在于抑制了脂质过氧化反应,增强了过氧化物歧化酶的表达,从而保护了铅处理小鼠的细胞和器官免于氧化胁迫的损伤。     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method

The genetic relationship between twenty-six strains of Agaricus bisporus were analysed by the RAPD (random amplified polymorphic DNA) method. DNA amplification was performed with the use of twelve arbitrary 10-mer primers. Four primers, which gave polymorphic band patterns were chosen for RAPD analysis. In total, they gave 24 distinguishable bands, of which nine were polymorphic. The conducted research showed that there is a great genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a limited genetic pool used in the cultivation of those strains.     

Assessment of genetic diversity among 16 promising cultivars of ginger using cytological and molecular markers

Ginger (Zingiber officinale Roscoe) is an economically important plant, valued all over the world. The existing variation among 16 promising cultivars as observed through differential rhizome yield (181.9 to 477.3 g) was proved to have a genetic basis using different genetic markers such as karyotype, 4C nuclear DNA content and random amplified polymorphic DNA (RAPD). The karyotypic analysis revealed a differential distribution of A, B, C, D and E type of chromosomes among different cultivars as represented by different karyotype formulas. A significant variation of 4C DNA content was recorded in ginger at an intraspecific level with values ranging from 17.1 to 24.3 pg. RAPD analysis revealed a differential polymorphism of DNA showing a number of polymorphic bands ranging from 26 to 70 among 16 cultivars. The RAPD primers OPC02, OPA02, OPD20 and OPN06 showing strong resolving power were able to distinguish all 16 cultivars. The extent of genetic diversity among these cultivars was computed through parameters of gene diversity, sum of allele numbers per locus and Shannon's information indices. Cluster analysis, Nei's genetic similarity and genetic distances, distribution of cultivars into special distance classes and principal coordinate analysis and the analysis of molecular variance suggested a conspicuous genetic diversity among different cultivars studied. The genetic variation thus detected among promising cultivars of ginger has significance for ginger improvement programs.     

云南六种实蝇的RAPD快速鉴定

采用RAPD技术构建了果实蝇属（Bactrocera）的南瓜实蝇(B. tau)、黑漆实蝇(B.scutellaris)、具条实蝇(B. scutellata)、瓜实蝇(B. cucurbitae)、桔小实蝇(B. dorsalis)和番石榴实蝇(B. correcta)6种实蝇的指纹图谱.从131个引物中筛选出5个重复性好、多态性高的引物,共扩增出302条谱带,其中111条谱带具有遗传多态性.引物OPC-01、OPI-17、OPL-07、OPL-08以及OPL-16可以用于6种实蝇的分类鉴定.     

Induced mutagenesis in Jatropha curcas L. using gamma rays and detection of DNA polymorphism through RAPD marker

The aim of this study is to examine the effect of different doses (control, 5, 10, 15, 20 and 25 Kr) of gamma irradiation on seed germination, flowering, fruit and seed traits of Jatropha curcas and to identify DNA polymorphism among the mutants through a Randomly Amplified Polymorphic DNA (RAPD) marker analysis. The improved agronomic traits such as flowering, fruits and seeds were recorded in 5 Kr dose and seed germination percentage in 10 Kr dose treated plants, while corresponding parameters were reduced significantly (P>0.05) in 25 Kr dose gamma rays treated plants when compared to that of control. All the twenty-three random primers used except six primers, namely OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 were showed polymorphic bands. The primers: OPAW16, OPAK07, OPAK15, OPS01, OPAK20 and OPAL09 produced only one band each across the six mutants, while the primers: OPU13, OPAB 15, OPF01 and OPAB11 were produced with maximum number of bands (8). The number of amplicons varied from 1 to 8 with an average of 3.9 bands, of which 2.3 were polymorphic. The percentage of polymorphism per primer ranged from 0 to 100 with an average of 55.16%. The Jaccard's coefficients of dissimilarity varied from 0.324 to 0.397, indicative of the level of genetic variation among the mutants studied. The maximum dissimilarity value (0.397) was observed in 5 Kr mutant while the minimum value (0.250) was observed in 20 Kr mutant when compared to that of control. In a dendrogram constructed based on genetic similarity coefficients, the mutants were grouped into three main clusters; (a) control, 10, 15 and 20 Kr dose mutants clustered together, (b) 25 Kr dose grouped alone, (c) 5 Kr dose also grouped alone. The mutants showing the differences in morphological traits showed DNA polymorphism in PCR profile amplified by RAPD marker. It is concluded that DNA polymorphism detected by RAPD analysis offered a useful molecular marker for the identification of mutants in gamma radiation treated plants.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Chromosome polymorphism in the yeast Saccharomyces

The variability of chromosomal band patterns was determined by pulse electrophoresis. The natural strains differed by the quantity and electrophoretic mobility of chromosomal DNA bands. The strains of independent genetic stocks originated from the XII race of Saccharomyces cerevisiae showed less significant difference in band patterns than the strains of different species of the Saccharomyces genus. The progeny of among strains with different karyotypes hybrid showed non-regular segregation of parental bands, the occurrence of new bands and the bands with altered mobility. Reverse crosses of hybrid progeny with strains of Peterhoff genetic stocks of S. cerevisiae led to decrease in chromosomal polymorphism. Homozygotization for ski5 allele and selection for increasing the copy number of killer plasmids was accompanied with repeated splash of polymorphism in 1-2 generations of intratetrad and intrafamily crossed hybrid progeny. Subsequent stabilization of electrophoretic karyotype took place, excluding the mendelian dimorphism of chromosome III, with was a stable trait of the last 6 generations of that progeny.