小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为；第一组4种1变种，小于764碱基对（bp）；第二组2种，765～824bp；第三组2种1变种，大于825bp。所研究的许多种中，特别是第三组，单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI，TaqI；Tru9I，和HinfI4种限制性内切酶，对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱，种内非常一致而种与种之间有差别。反之，在某些种其限制性酶切图谱不仅种间互不相同，在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下，只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义，并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。

PCR-BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISMS IN RIBOSOMAL DNA OF THE GENUS CUNNINGHAMELLA

A pair of oligonucleotide primers ITS1 and ITS4 were used to amplify internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of Cunninghamella species with G+ C of nuclear DNA less than 30%. Twenty-two isolates of ten species and varieties were successfully amplified. The amplified ITS fragments differed greatly in length in different species of the same genus. Three groans were obtained. Numbers of taxa and their DNA length in Group 1, Group 2. and Group 3, respechvely, are: four species and one variety, below 764 base pair (bp); two species,765 to 824 bp; and two species and one veriety, 825 bp and above. Some species studied,especially in Group 3, can be generally identified based on the length of their polymerase chain rearhon (PCR) products. However, in Groups 1 and 2, restriction analysis must be used to further separate species. Restriction endonucleasea Rsa I, Taq I, Tru9 I and Hihf I were used to analyze the restriction fragment polymorphisms (RFLPs) of all amplified Products. Restriction patterns were identical within C elegans, C bertholletiae, and C blakesleeana and distinct among these species.In contrast in some species, the restriction patterns differed greatly not only among species, but high vallation of 1--3 restrichon patterns also existed among strains of the same species. In these cases, different taxa could only be separated using comprehensive data. This study confirms the role Plaaed by PCR-RFLP in dishnguishing species and varieties of Cunninghamella, with the finding of individual differences within species. This is further verified by our later study on sequence analysis.