球孢白僵菌种群野生菌株异核现象的分子验证

球孢白僵菌28S rRNA基因D11域由于一内含子的存在与否,引起这一区域的扩增产物有两种片段类型（约520bp及120bp),将此作为分子标记分析了球孢白僵菌的117个菌株,发现17％菌株的扩增产物为双片段类型,即为异核体,异核体在不同种群中的分布频率因地理生态条件而异,单孢分离分析表明,异核体的分离频率约为35％,RAPD检测结果说明异核体在准性循环中发生了不同程度的遗传交换及遗传重组,球孢白僵菌异核体在继代培养中极不稳定。     

球孢白僵菌混菌培养的遗传学分析

放线酮抗性及34℃耐受性不同的球孢白僵菌两营养亲和单孢株经混合培养,能够形成异核体。在分生孢子形成的单倍化过程中,异核体中的染色体或其片段发生连续丢失,至少经4代准性循环,遗传性状才会趋于相对稳定。遗传标记及RAPD分析表明,异核体中染色体的丢失并非随机的,重组株的遗传性状表现为倾向选择,即子代主要只表现为某一母株的遗传性状,另一亲本型性状被完全抑制或其遗传物质被丢失。混合比例不同、培养介质不同可影响准性生殖过程及倾向选择频率。混菌培养有利于优良性状的保持。     

新疆棉花枯萎病菌异核体及其不同表型分离子核DNA的差异*

为探索丝状真菌异核体的产生机制,从新疆地区患枯萎病的棉杆中分离出棉花枯萎病菌Fusariumoxysporumf.sp.vasinfectum异核体菌株X515,并自其菌落出现的不同角变处分离出三个不同表型的分离子,X515-Ⅰ、X515-Ⅱ和X515-Ⅲ。选用200种随机引物对它们进行核DNA的RAPD分析,只在OPB-16引物对致病力强的分离子X515-Ⅰ核DNA的PCR扩增产物中出现了一条354bp的差异片段,其部分碱基序列与Fusariumoxysporumf.sp.lycopersici的短散布序列(Foxy)末端序列的相似性达88%。本研究结果提示,随着所采用引物数量的增加,可有望找出异核体及其不同分离子之间核DNA的差异,为最终阐明异核体形成的机制提供线索。     

新疆棉花枯萎病菌异核体及其不同表型分离子核DNA的差异

为探索丝状真菌异核体的产生机制,从新疆地区患枯萎病的棉杆中分离出棉花枯萎病菌Fusariumoxysporumf.sp.vasinfectum异核体菌株X515,并自其菌落出现的不同角变处分离出三个不同表型的分离子,X515-Ⅰ、X515-Ⅱ和X515-Ⅲ。选用200种随机引物对它们进行核DNA的RAPD分析,只在OPB-16引物对致病力强的分离子X515-Ⅰ核DNA的PCR扩增产物中出现了一条354bp的差异片段,其部分碱基序列与Fusariumoxysporumf.sp.lycopersici的短散布序列(Foxy)末端序列的相似性达88%。本研究结果提示,随着所采用引物数量的增加,可有望找出异核体及其不同分离子之间核DNA的差异,为最终阐明异核体形成的机制提供线索。     

基于SSR标记的琅琊山球孢白僵菌寄主专化性

球孢白僵菌Beauveria bassiana是一类常见的昆虫病原真菌,其自然侵染的寄主昆虫众多,达15目149科750种。为了解球孢白僵菌自然种群的遗传多样性,探讨种群异质性和寄主来源之间的关系,分析其寄主专化性的强弱,本研究利用SSR分子标记技术,比较了安徽琅琊山国家森林公园的85株球孢白僵菌（寄主种类涉及7目24种）群体遗传多样性差异,通过构建聚类树分析菌株基因型和寄主关联性。结果表明琅琊山球孢白僵菌群体Nei’s基因多样性指数h=0.2906,Shannon信息指数I_s=0.4510,多态位点百分率P为100%。不同寄主目球孢白僵菌遗传多样性水平由高至低为鞘翅目>膜翅目>同翅目>双翅目>鳞翅目>直翅目>半翅目,其中菌株数量较多的鞘翅目、膜翅目和同翅目3个亚种群的遗传多样性较高且水平接近。聚类分析发现8对SSR引物将85株球孢白僵菌分成29个基因型,并在遗传相似系数0.70处分别聚为3个分支。分析寄主类型发现相同基因型的株系可侵染不同目的寄主,而同一类型寄主也可被不同基因型的菌株侵染。球孢白僵菌种群的总体遗传多样性较高,遗传谱系与寄主来源无明显相关性,菌株的寄主专化性弱。     

新疆棉花枯萎病菌异核体及其不同表型分离子核DNA的差异

为探索丝状真菌异核体的产生机制,从新疆地区患枯萎病的棉杆中分离出棉花枯萎病菌Fusarium oxysporum f.sp．vasinfectum异核体菌株X515,并自其菌落出现的不同角变处分离出三个不同表型的分离子,X515-I、X515-Ⅱ和X515-Ⅲ。选用200种随机引物对它们进行核．DNA的RAPD分析,只在OPB-16引物对致病力强的分离子X515-I核DNA的PCR扩增产物中出现了一条354bp的差异片段,其部分碱基序列与Fusarium oxysporum f．sp．lycopersici的短散布序列(Foxy)末端序列的相似性达88％。本研究结果提示,随着所采用引物数量的增加,可有望找出异核体及其不同分离子之间核DNA的差异,为最终阐明异核体形成的机制提供线索。     

双孢蘑菇遗传多样性分析

应用AFLP指纹技术对双孢蘑菇的20个野生菌株和5个栽培品种的遗传多样性进行了研究。AFLP指纹揭示出20个野生异核体菌株所固的基因型。5个商业品种表现出比较一致的AFLP指纹,但也显示出它们之间的一些差别。由单孢分离获得的同核体菌株携带着部分异核体菌株的AFLP指纹;由同一子实体分离得到的大部分单孢菌株是异核体菌株,它们具有与其亲本一致的AFLP指纹。UPGMA分析揭示出2个与地理分布（美洲、欧洲）和相对应的组。研究结果表明：（1）在野生菌株之间存在着明显的遗传差异;（2）大多数单孢分离的菌株具有与母本一致的遗传物质;（3）野生菌株间的遗传变异大于栽培品种间的变异;（4）在双孢蘑菇的遗传多样性分析中,AFLP技术是非常有效的工具。     

粟长蠕孢菌的异核现象及异核体核型比影响因素的研究

本试验通过23株带有遗传标记的粟长蠕孢菌突变菌株,获得生理性状及生长势不同于亲本的异核体。利用营养缺陷型标记菌株研究的结果表明,粟长蠕孢菌异核体的形成及核型成分的变化受选择压力的影响。原生质体检测结果表明,在异核菌丝体中,异核细胞占46.7%,同核细胞占53.3%。分生孢子检测结果表明,只有0.06%的分生孢子保持异核状态。     

利用RAPD-PCR检测三种白僵菌及球孢白僵菌种内变异

采用PCR-RAPD技术研究了球孢白僵菌2株野生型多孢株及其各1株单孢分离株的 RAPD扩增片段的多态性,比较了彼此间的相似率。结果显示,相似率在63.6～99.3％之间变化很大,表明各分离子间的基因组DNA已发生变化。球孢白僵菌与另一种白僵菌布氏白僵菌之间的相似率在64.9％～80.9％之间,与粘孢白僵菌之间的相似率在50.0～61.9％之间。     

球孢白僵菌Pr1蛋白酶基因cDNA克隆与序列分析

从球孢白僵菌菌株Bb202中克隆了昆虫蛋白酶Pr1的基因。使用特异性引物,分别以基因组DNA和mRNA为模板进行PCR反应,得到了球孢白僵菌的Pr1蛋白酶基因cDNA和结构基因序列。测序结果表明球孢白僵菌Pr1蛋白酶结构基因全长大小为1606bp,含有3个内含子,分别为69、61和68bp。其ORF全长1140bp,预测编码379个氨基酸残基。成熟蛋白理论分子量为38.8kD,理论等电点为8.06。为进一步研究球孢白僵菌Pr1基因,并构建高毒力工程菌株提供了理论基础。     

香菇自然群体中个体间的空间分布及其遗传联系*

应用体细胞不亲和性反应、交配型因子分析和基因组ＤＮＡ的ＲＡＰＤ分析,研究了一个分布于方圆约１ｋｍ的６根倒木上的１８个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（８０．４％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达 ６２．５％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在 １１个特异的Ａ因子和７个特异的Ｂ因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。ＲＡＰＤ分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于ＤＮＡ相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.     

内蒙古沙棘共生菌Frankia的遗传多样性

利用RFLP分子标记方法,在自然条件下对内蒙古东、西部8个地点采集的24个沙棘根瘤样品进行沙棘共生菌Frankia的遗传多样性分析。结果表明,供试样品nifD-nifK基因间隔区(IGS)扩增片段的大小约1 100bp;不同样品的酶切图谱有明显差异;有些样品产生复合RFLP型,揭示在自然状态下不同基因型的Frankia菌株可共同侵染同一沙棘寄主。聚类分析显示,来源于相同地点和不同地点的根瘤样品内的Frankia菌株间均有遗传多样性;没有发现Frankia菌株遗传多样性的分布与采样地点有相关性。     

苏云金芽胞杆菌YBT1520杀虫晶体蛋白基因的属性

通过Ｓｏｕｔｈｅｒｎ杂交发现高毒力苏云金芽胞杆菌（Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ）ＴＢＴ－１５２０菌株含有两个杀虫晶体蛋白基因片段,其５’＝末端所在ＨｉｎｄⅢ片段分别为６．８ｋｂ和４．６ｋｂ,它们对应的基因分别命名为ｃｒｙ２１８和ｃｒｙ４．６。经ＰＣＲ鉴定,该菌含有ｃｒｙ１Ａａ、ｃｒｙ１Ａｂ和ｃｒｙ１Ａｃ基因,以及ｃｒｙ２基因,其中ｃｒｙ２１８属于ｃｒｙ１Ａｃ。分析了ｃｒｙ１Ａｃ基因     

Analysis of two theta-replicating plasmids of Streptococcus thermophilus

We report the characterization of two new theta-replicating plasmids of Streptococcus thermophilus (pSMQ-312b and pSMQ-316) as well as the further analysis of pSMQ-308. The nucleotide sequences of pSMQ-312b and pSMQ-316 were determined and both contained 6710 bp. In fact, the two sequences were identical, despite that the plasmids were isolated from two different S. thermophilus strains as demonstrated by pulsed-field gel electrophoresis. Comparative analyses indicated that the two plasmids were highly related to the previously characterized S. thermophilus plasmid pSMQ-308 (8144 bp). Plasmid stability tests showed that pSMQ-312b/316 was more stable in LM17 medium while pSMQ-308 was the most stable in milk. The presence of the plasmids did not modify the acidification profile of the S. thermophilus strains during growth in milk and under time-temperature conditions mimicking an industrial process. These theta-replicating plasmids are unique genetic material for the construction of stable cloning vectors for industrially relevant strains of S. thermophilus.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

Genetic recombination in auxotrophic strains ofPhanerochaete chrysosporium

Four auxotrophic strains of the ligninolytio basidiomycetePhanerochaete chrysosporium were obtained by UV mutagenesis. The heterokaryotic mycelium formed by complementation of different auxotrophic isolates was able to fruit and produce basidiospores. From the hasidiospore progeny of the heterokaryons prototrophic strains and strains with a recombined set of parental nutritional requirements were isolated. Genetic recombination hence takes place in fruit bodies produced by the heterokaryotic mycelium.