绿僵菌航天诱变菌株的DNA变异性分析

昆虫病原真菌绿僵菌M2189菌株经航天搭载诱变后,分离得到200多个单孢菌株。以9个随机引物对47个诱变菌株进行PCR扩增,获得了38个位点的共1381条DNA片段。统计分析表明,47个诱变菌株在DNA水平上发生了不同程度的变异,变异系数在0.062~0.406之间,平均为0.176,>0.3的有3个菌株。由聚类分析可将47个诱变菌株划分为14个组群,组间变异率小于0.2,与原始菌株相比变异系数接近的诱变菌株无规律地分散在不同组群中,说明航天诱变导致DNA变异的位点和频率是随机的、分散的,并无特定位点。航天诱变为选育生物防治优良菌株提供了新途径。     

8份广西产绞股蓝种质的RAPD引物筛选及其遗传多样性分析

本研究的目的在于筛选合适的RAPD随机引物,应用RAPD技术对药用植物绞股蓝进行遗传多样性分析,并构建DNA指纹图谱。研究结果表明,我们利用生物信息学方法挑选出的20条引物中有19条引物的扩增条带清晰且多态性好;在清晰稳定出现的354条带中,294条具有多态性;其中有3条引物的扩增条带可清楚区分绞股蓝与混淆品种乌蔹莓,可建立其DNA指纹图谱。按UPGMA法进行聚类分析,计算其遗传相似系数,结果显示,8份绞股蓝供试材料聚为两类,聚类结果与其地理区域远近和生长环境一致。本研究中筛选出的19条引物适用于绞股蓝遗传多样性分析,且获得的DNA指纹图谱可用于鉴别绞股蓝。     

利用核心简单重复序列(SSR)标记分析无籽西瓜品种的DNA指纹及遗传多样性

为构建无籽西瓜品种的DNA指纹,实现无籽西瓜品种的快速准确鉴定,客观评价品种的遗传多样性,本研究利用核心SSR标记对我国54份无籽西瓜(Citrullus lanatus)主栽品种进行了分析。结果显示,23对多态性引物共扩增出63种基因型,基因型数2～5个不等,平均2.74个;平均多态性信息量(PIC)为0.39,变化范围为0.04～0.67,有5个品种具有特征谱带;54个品种遗传相似系数变化范围为0.643 9～1.0,平均0.859 3,参试品种具有较高的遗传相似性;组合23对引物,除无法区分雪峰花皮无籽与郑抗无籽1号、郑抗新1号与广西3号、黒宝无籽与桂冠1号,其余品种均能一一区分开,利用PIC0.4的10对引物构建了5份主要参试品种的DNA标准指纹图谱;采用类平均法进行聚类分析,在相似系数0.82处,可将54个品种分为7大类。本研究建立了参试品种的标准DNA指纹图谱,为我国无籽西瓜品种的真实性鉴定和知识产权保护提供了技术基础。     

利用ISSR揭示不同类型月季遗传多样性

本研究应用正交设计法对ISSR反应体系中的各个主要影响因子进行了优化筛选,确立了适合月季ISSR-PCR反应的最佳体系。结果表明,25μL的ISSR反应体系中各组分的最适浓度分别为：1×PCR缓冲液、1U Taq DNA聚合酶、800pmol／L 引物、0.16mmol／L dNTPs、Mg^2＋ 1.5mmol／L。筛选了33个ISSR引物,共得到了11个多态性比较高的ISSR引物,占所筛引物的33.33％。利用筛选出的11条ISSR引物对3种月季类型的23份月季材料进行遗传多样性分析,共扩增出477条DNA带,其中多态性位点有14个,平均每条引物可以检测到4.5个多态性位点。用NTSYS软件对样品进行了UPGMA聚类分析,聚类结果表明丰花月季基本能聚为一类,切花月季与藤本月季交叉聚在一起。这表明月季种质的遗传差异与其应用分类的相关性不紧密。     

华东竹黄菌不同居群遗传分化的RAPD分析

为了解竹黄地理居群间的遗传分化,本研究采用随机引物扩增多态性DNA分子标记技术对江苏、安徽和浙江3省的8个居群共32个竹黄样本进行了遗传多样性分析。从50个RAPD引物中筛选得到了5个随机引物,对供试材料的DNA进行扩增,共检测出77个位点,其中多态性位点52个,多态性位点比率为67.53%。UPGMA聚类分析结果表明,这8个居群分为三类：安吉居群、临安居群、宜兴居群、广德居群和泾县居群聚为一类;宁国居群和休宁居群聚为一类;淳安居群单独为一类。遗传多样性分析表明8个竹黄居群中,淳安居群的遗传多样性水平最高,安吉居群的遗传多样性水平最低。Nei＇s基因多样性指数和Shannon信息指数均表明竹黄物种水平的遗传多样性高于居群水平。     

银杏内生真菌刺盘孢遗传多样性的初步分析

为了研究银杏内生真菌的刺盘孢菌株的遗传分化,选用了分离自江苏5个县市的14个菌株,利用RAPD技术进行遗传多样性分析。从20个随机引物中筛选出5个引物,扩增出62条DNA带,在此基础上分析并建构了遗传相关聚类图。结果表明,不同地区的刺盘孢菌株其遗传差异性不同,14个菌株可聚为两大类,其中启东、邳州的6株聚为一类,南京、南通和泰兴的8株又聚为一类。所有的14个菌株的相似性系数范围在46%~89%。     

杨梅不同品种的ISSR分析

利用ISSR－PCR方法对杨酶（Myrica rubra Sieb.et Zucc．)。植物的7个品种2个无性系进行了基因组多态性分析。选用11个引物扩增出116个DNA片段，其中48个片段呈现多态性，占总扩增片段的41．4％，依据扩增结果进行遗传距离分析，构建了分子树状图，研究结果表明，ISSR分析中产生了一些品种特有的指纹图谱。利用DNA扩增结果进行聚类分析，把供试验酶的7个品种和2个无性系分为3类，并对基本品种及种下品种群的遗传关系进行了探讨。     

基于ISSR指纹的甘薯食用品种的遗传多样性分析

运用ISSR分子指纹技术检测甘薯基因组DNA的多态性,是鉴定甘薯遗传多样性和亲缘关系有效的方法之一。本研究选用6个ISSR标记对17份甘薯品种进行了DNA指纹扩增,共扩增出55条谱带,其中多态性谱带50条,多态性水平为90.91%;UBC825引物扩增出带型最多,有16种;根据Nei’s遗传距离计算获得的聚类树状图表明,在GS为0.625时,参试的17份甘薯品种分为4个组群;遗传相似性分析表明,顶芽菜用品种间遗传相似系数最高0.877,叶柄食用品种和烤薯型品种之内的遗传相似系数分别为0.778和0.740。结果表明甘薯主要食用间具有较好的遗传多样性,但同类型品种间却又有较高的遗传相似性,这类品种需要引进或创制新资源。     

河南粳稻品种SSR指纹图谱的构建及遗传差异分析

为了建立河南粳稻品种的DNA指纹图谱,了解河南粳稻种植品种的遗传多样性,本研究利用均匀分布于水稻(Oryza sativaL.)12条染色体的37对SSR引物,以河南1963～2010年间主要推广种植的37个粳稻(O.sativaL.ssp.japonica)品种为材料,进行简单序列重复(SSR)指纹图谱构建和遗传多样性分析。结果表明,33对引物具有特异多态性片段,共检测到114个等位基因,平均每位点3.53个等位基因;引物多态性频率为0.054～0.739。37个品种的遗传相似系数为0.627～0.992,平均为0.812。17个品种都至少具有1对SSR特征引物,其余20个品种的鉴定需要结合不同的SSR引物。利用19对核心SSR引物构建的指纹图谱能逐一区分出36个粳稻品种。采用非加权类平均法(UPGMA)进行聚类分析,在遗传相似系数0.710处可将37个品种分为2大类,河南选育品种基本都聚到同一大类群,表明河南推广的粳稻品种遗传基础狭窄。     

利用SSR标记分析藜麦品种的遗传多样性

为了解藜麦种质资源的多样性,本研究利用SSR引物对所搜集的41个藜麦种质的多态性及其亲缘关系进行了分析。结果表明,从54对SSR引物中筛选出了16对能明显扩增出稳定的多态性条带的引物,共检测出139个等位基因条带,每一对引物的等位基因个数为3~13,平均为8.7;16对引物的多态信息含量(PIC)变幅为0.208~0.432,平均为0.366。UPGMA聚类分析显示,41份材料的遗传相似系数(GS)在0.374~0.906之间,平均相似系数为0.626。在阀值(GS)约为0.665时,41份材料可分为4大类。其中614929与B.B.Quinoa浙Ⅰ间的遗传相似系数最小,为0.374,表明来源于不同地区的遗传距离较远,遗传基础较广泛。藜麦品种资源间的亲缘关系的揭示为藜麦资源保存和新品种选育提供了理论依据。     

Cadmium-resistant oyster mushrooms from North China for mycoremediation

Macrofungi have been advocated to be a promising remediation material for heavy metal pollution. This study aimed to screen for cadmium(Cd)-tolerant oyster mushroom strains which may be used as biomaterials for Cd remediation in North China. To this end, 43 oyster mushroom strains were collected across North China and subject to Cd inhibition test. Phylogenetic affiliation of the tolerant strains was identified using molecular fingerprinting, and representative Cd-tolerant strains were characterized in terms of their Cd sorption capacity. Two strains, JINONG21 and SUYIN6 affiliated with Pleurotus ostreatus, were found to be highly tolerant to Cd, with a minimal inhibitory concentration of 4 mg L~(-1) Cd and a maximal tolerant concentration of 80 mg L~(-1) Cd under the test conditions. Meanwhile, JINONG21 and SUYIN6 were able to absorb up to 676 mg kg~(-1) and 970 mg kg~(-1) Cd in their mycelia in the culture medium with 20 mg L~(-1) Cd, respectively. In conclusion, the P. ostreatus strains identified in this study presented an outstanding capacity of Cd tolerance and sorption and could be promising biomaterials for Cd remediation in North China.     

出口平菇菌棒品种筛选试验

对7个平菇菌株的菌盖颜色、菌盖直径、菌柄长度、子实体着生方式和生物学效率进行了观测和分析，结果表明，鲁植1号菌株综合农艺性状和商品性状最好，基本符合韩国市场对平菇的商品要求：菌盖蓝黑色，平均直径3cm，平均柄长8cm，子实体丛生，产量最高，生物学效率达到100％。     

高产漆酶平菇的筛选及其在降解黄曲霉毒素B1中的应用

通过选择培养基平板培养法和液体发酵培养法筛选得到2株高产漆酶的平菇菌株P1和P2,并对平菇菌株产漆酶的培养基进行筛选,得到产漆酶的最适培养基为最低盐MSM培养基。菌株P1不仅产漆酶能力最高,而且降解黄曲霉毒素的能力也最好。在MSM培养基中培养10d时,产漆酶量高达769.44U/L,在800μl的反应体系中,790μl粗酶液可以将1000ng黄曲霉毒素B₁降解到222.62ng,降解率为77.74%,并且平菇粗酶液降解黄曲霉毒素B₁的能力与其中漆酶的含量呈一定的正相关性。     

SCAR标记技术鉴别榆黄蘑品种

为了建立一套基于DNA分子标记技术快速鉴定榆黄蘑菌株的有效方法,本研究通过对生产上常用的16个榆黄蘑菌株进行SRAP多态性分析,从榆黄蘑2号菌株中扩增获得了一个片段长为537bp的特异片段,将其克隆、测序并设计引物,成功转化为稳定的SCAR标记。试验结果表明,利用该特异SCAR标记,能在1d时间内准确鉴定出榆黄蘑2号菌株。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定榆黄蘑菌株的新方法。     

濒危黑颈长尾雉圈养种群的RAPD遗传多样性分析

运用RAPD技术对黑颈长尾雉圈养种群的遗传多样性进行了分析。从50条随机引物中筛选出14条引物,对24个个体的基因组DNA进行了PCR扩增,从检测出的119个位点中有98个多态位点,占总位点的82．35％,标记的分子量大小范围是0．2～3kb。24个个体问的遗传距离幅度0．1597～0．4874,平均是0．2810;用软件NTsys2．10e构建了24个个体相互关系的分支图,24个个体可分为3个类群。实验表明：黑颈长尾雉圈养种群的遗传多样性水平较高,圈养种群内遗传差异性较大。     

平菇栽培废料等有机肥对土壤活性有机质和土壤酶活性的影响

通过大田试验研究了不施有机肥(CK)、施用平菇栽培废料(T1)、施用干腐熟牛粪(T2)和烘干鸡粪(T3)在种植黄瓜01～50.d内土壤中活性有机质和4种土壤酶活性的变化。结果表明:施入不同有机肥对土壤总有机质含量的影响为烘干鸡粪平菇栽培废料干腐熟牛粪对照;对活性有机质含量的影响为平菇栽培废料烘干鸡粪干腐熟牛粪对照;施用平菇栽培废料的土壤中脲酶、转化酶和脱氢酶活性最高,施用干腐熟牛粪的土壤中过氧化氢酶活性最高。相关性分析显示,脲酶、转化酶和脱氢酶活性与土壤活性有机质显著相关。用平菇栽培废料做有机肥能有效提高土壤活性有机质含量和土壤酶活性。     

Acylhomoserine lactone production by bacteria associated with cultivated mushrooms

The main bacterial pathogens of cultivated mushroom as well as mushroom-associated bacteria, which were isolated from Agaricus bisporus, Pleurotus ostreatus and Pleurotus eryngii mushroom niches, were evaluated for the production of N-acyl-L-homoserine lactones (AHLs) by using four bioreporters. Furthermore, identification of AHLs by LC-ESI-FTICR MS was performed on culture filtrates of selected pathogens and mushroom-associated bacteria strains, which resulted in inducing at least one of the four bioreporters. Strains of Burkolderia gladioli pv. agariciola, Pseudomonas agarici and Pseudomonas gingeri, but not those of Pseudomonas tolaasii and Pseudomonas reactans, produced an array of AHLs depending on the strain. This is the first report of AHL production by mushroom bacterial pathogens. Forty-four of 236 bacterial isolates obtained from different niches of cultivated mushrooms, in part identified by the Biolog identification system, were demonstrated to produce AHLs. Among them, seven mushroom-associated bacterial species were for the first time demonstrated to produce the above signal molecules. In the culture filtrates of a certain number of isolates/strains the AHL-hydrolyzed forms were also present. The minimal signal inducing concentration (MSIC) of selected pure AHLs was also determined for the four bioreporters used in this study.     

Constructing DNA fingerprinting of Hemarthria cultivars using EST-SSR and SCoT markers

DNA fingerprinting of known cultivars may provide much-needed data to assist with the identification of such cultivars. From 86 pairs of expressed sequence tag SSRs (EST-SSR) and 45 start-codon targeted polymorphism (SCoT) primers, eight pairs of EST-SSR primers and seven SCoT primers were chosen to construct the DNA fingerprinting of six Hemarthria cultivars in this study. Using genomic DNA from six cultivars, a total of eight pairs of EST-SSR primers were able to amplify 193 bands, producing an average of 24.1 bands per primer pair. The percentage of polymorphic bands (PPB) was 83.4 %, and the polymorphism information content (PIC) ranged from 0.480 to 0.695, with an average of 0.602. A total of seven SCoT primers amplified 105 bands with an average of 15 bands per sample. The PPB was 84.8 %, and the PIC ranged from 0.471 to 0.758, with an average of 0.612. The unweighted pair-group method with arithmetic mean dendrogram from EST-SSR and SCoT markers grouped the six Hemarthria cultivars into two major groups of the same. These clusters are in accordance with their known species and origin. Our results indicate a rich genetic diversity in these six Hemarthria cultivars. The six cultivars we assayed could be easily identified using these eight pairs of EST-SSR and seven pairs of SCoT primers.     

不同致病型水稻细条病菌的遗传多样性分析

采用引物BOX和ERIC对来自4省的35个细条病菌菌株进行Rep-PCR扩增,BOX引物扩增出14条指纹带,10种谱型,以致病型为单位计算遗传多样性值为0.63～1.00;ERIC引物扩增出19条指纹带,17种谱型,遗传多样性值为0.86～1;引物ERIC比BOX在遗传多样性方面有更好的分辨率;树状聚类图反映的遗传分簇差异,与菌株的致病型及其地理的来源之间没有相关性。     

Speciation of thermotolerant Campylobacter isolates involved in foodborne disease by means of DNA restriction analysis and molecular probes

The molecular identification of several strains of Campylobacter jejuni and Campylobacter coli involved in foodborne disease was carried out by investigating the restriction profiles of their chromosomal DNA and by DNA/DNA hybridization. Cleavage with EcoRV allowed the visualization of a 3 kb DNA fragment characteristic of C. jejuni, whereas restriction with ClaI allowed the identification of a 9.3 kb DNA fragment, also characteristic of C. jejuni, and a DNA duplet of 9.5-10 kb, specific to C. coli.Restriction analysis with enzyme BglII allowed the visualization of DNA fragments of 3.5, 4, and 6.7 kb, characteristic of C. jejuni. C. jejuni subsp. doylei strains investigated shared a higher genetic homology among themselves-as determined by DNA/DNA hybridization-than with C. jejuni subsp. jejuni. A DNA probe, initially designed by Korolik et al. (Korolik, V.; Coloe, P. J.; Krishnapillai, V. J. Gen. Microbiol. 1988, 134, 521-529), including a DNA fragment encoding an antigenic membrane protein of 31.5 kDa in C. jejuni, when used as probe, allowed the specific identification of all strains of C. jejuni through the detection of strong hybridization signals in two BglII DNA fragments of 2.3 and 2.5 kb, which were not observed in C. coli. Cleavage of chromosomal DNA with BglII-either alone or coupled with probing assays with specific probes-proved to be a valuable tool for the speciation of Campylobacter isolates involved in foodborne disease.