曲霉菌的RAPD分析及其在酿造工业中的应用

RAPD analysis of Aspergilli and its application in brewing industry

以米曲霉沪酿3.042（AS3.951）、黄曲霉GIM3.18、酱油曲霉AS3.495为参照,利用RAPD分子标记技术对16株曲霉菌进行系统发育分析.通过改进提取方法,获得了质量较好的模板DNA,凝胶电泳结果和分光光度法检测结果表明其适合用于进一步的RAPD-PCR试验.从9个待选引物中筛选到3个扩增产物谱带多、特征好、覆盖面广的引物：Primer1、Primer2、Primer5,重复实验证明其RAPD-PCR扩增图谱具有较好的稳定性,扩增产物谱带一般8～14条,各试验菌株主带4～9条,次带丰富.由此构建的系统进化树较好地吻合了传统的形态分类学,证实了RAPD分子标记技术在此类微生物系统发育分析中应用的可行性,也为酿造工业中检出产黄曲霉毒素的污染菌株提供了理论基础.

Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology, using AspergiUus oryzae AS3.951, Aspergillus flavus GIM3.18 and Aspergillus sojae AS3. 495 as controls. First, genome DNA of the sixteen test strains were prepared by improved extraction method, and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band （approximately 20kb） in agarose gel electrophoresis, which conformed to the fact that these strains all belong to Aspergillus. OD/260 OD280 of the prepared DNA ranged from 1.80 to 1.90,illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then, three appropriate primers （Primerl, Primer2, Primer5） for RAPD-PCR were screened from nine random primers, and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8- 14 bands in their RADP-PCR patterns, where the number of the main bands was 4- 9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns, where the percentage of polymorphic bands reached to 40.9 % （74 bands）. The similarity coefficient between the strains was calculated based on their RAPD- PCR patterns, ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primerl, Primer2 and PrimerS. It basically corresponded to traditional morphological taxonomy, demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides, the aflatoxin-producing strains （GIM3.17, CICC2219, CICC2357, CICC2390, CICC2402,CICC2404） could be easily discriminated by RAPD molecular marker, whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently, RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.