Resoure Diversity And Molecular Systematics Studies On Yeasts Which Produce GABA

Yeast is an important microbial resource in China, which has been used for 2,000 years. But there are lots of controversy and confusion in the classification and identification of yeast, which will be detrimental to yeasts'application and research.y-Amino Butyric Acid (GABA), is a natural active ingredient in some organisms. GABA has variety of physiological functions, such as lowering blood pressure, functioning as an anti-anxiety substance, tranquilizing the brain, promoting long-term memory and so on. GABA's research and development have become new hot spots. By screening the high-yield and safe strains of yeast and other microorganisms, food ingredients rich in GABA have been produced by fermenting, which have many benefits, such as low cost, high levels and safety. In this study, the research object is the yeast which can produce GABA, and morphology and molecular biology methods were used in the identification of yeast, to provide alternative methods for strains differentiation and genetic analysis of yeast. Results are as follow:1.256 strains of yeast were isolated from different fruit surfaces and places, from which screening 17 strains that can produce GABA, and another 2 yeast strains were purchased in the study. Based on the morphological characteristic, the 19 tested strains were identified as six different species primarily through traditional identification methods. Y-5, Y-8, Y-9, Y-10, Y-11, Y-16, Y-17 and Y-19 were Saccharomyces cerevisiae. Y-6 and Y-13 were Rhodotorula glutinis. Y-14 and Y-18 were Rhodosporidium toruloides. Y-2, Y-4 and Y-7 were Kluyveromyces lactis. Y-3 and Y-15 were Saccharomyces uvarum. And Y-12 was Saccharomyces telluris.2. Ten primers selected from 27 ISSR primers were used for ISSR amplification. A total of 108 bands were produced with 10 primers, among which 90.7% were polymorphic. According the dendrogram which based on ISSR fingerprints, result of cluster analysis showed that 19 strains were divided into five groups at 0.69 similarity level. Y-1, Y-3, Y-5, Y-10, Y-19, Y-8, Y-16, Y-17, Y-9 and Y-11 were in a group. Y-2, Y-4 and Y-7 were in a group. Y-6, Y-13, Y-18 and Y-14 were in a group. Y-15 and Y-12 represented two groups, respectively.3. Fourteen pairs of SRAP primers screened from 88 SRAP primers were used for SRAP amplification of 19 tested strains. A total of 279 bands were produced with 14 primers, among which 92.5% were polymorphic, each pair of SRAP primers amplified 20 bands. According the dendrogram which based on SRAP fingerprints, result of cluster analysis showed that 19 strains were divided into four groups at 71% similarity level. Y-6, Y-18, Y-14, Y-7, Y-2 and Y-4 were in a group. Y-3, Y-11, Y-16, Y-5, Y-8, Y-13, Y-17, Y-1, Y-19 Y-10 and Y-9 were in a group. Y-15 and Y-12 represented two groups, respectively.4. Nineteen tested strains'5.8 S rDNA-ITS region were amplified by PCR and sequenced. Through blastn analysis, the 19 yeasts were identified:Y-1, Y-3, Y-5, Y-8, Y-9, Y-10, Y-11, Y-16, Y-17 and Y-19 were Saccharomyces cerevisiae. Y-2, Y-4 and Y-7 were Kluyveromyces lactis. Y-6, Y-13, Y-14 and Y-18 were Rhodotorula mucilaginosa. Y-15 was Metschnikowia. Y-12 couldn't be confirmed.5. Finally, basis on the morphological identification, ISSR, SRAP and 5.8 S rDNA-ITS sequenced results,19 tested yeasts were identified exactly. Y-1, Y-3, Y-5, Y-8, Y-9, Y-10, Y-11, Y-16, Y-17 and Y-19 were Saccharomyces cerevisiae. Y-2, Y-4 and Y-7 were Kluyveromyces lactis. Y-6, Y-13, Y-14 and Y-18 were Rhodotorula mucilaginosa. Y-15 was Saccharomyces uvarum. Y-12 was Saccharomyces telluris.The results of this study showed that ISSR, SRAP and ITS analysis could distinguish different yeast in a certain extent, but each technology has its advantages and disadvantages, when used individually and can not classify and identify strains completely. To obtain reliable results, various technologies should be combined with each other for mutual authentication.