| Utilizing sugar cane molasses as the main substrate in the alcohol fermentation industry in South China proves a meaningful way for advantages of low cost of production, being free of the grain consumption and solving of pollution problem caused by cane sugar manufacturing.The alcohol fermentation with sugar cane molasses as substrate tends to be the focus of the study all the way, of which the obtaining of high-performance industrial strain is the key factor. Identification of strains and breeding for strains with ideal traits both tend to be meaningful work. So this study proceeded in two directions.First seven S. cerevisiae industrial strains with diverse single cell morphology and alcohol-producing ability in the fermentation with sugar cane molasses as substrate were selected for the analysis of the genomic polymorphism with RAPD method and further studying the relation between genomic polymorphism and related qualities. On the other hand, based on the genome information, then this study conducted gene modification of S. cerevisiae for the breeding of high-performance strains utilizing sugar cane molasses for alcohol fermentation. To meet the requirement of the study, a high throughout PCR template-making method was established, which was capable of obtaining a strain’s genome DNA as PCR template in less than three minutes. ITS amplification and sequencing of S. cerevisiae strains was conducted, demonstrating the specifity of the method in amplifying the target gene, and then the amplification of fragments with diverse size located in genome and the RAPD amplification proceeded, when compared with CTAB genome DNA method, revealing unique qualities, including short time expenditure and facilitation of operation.Based on high throughout PCR template-making method in this study,22primers were designed for screening, of which8primers were selected for RAPD analysis of7S. cerevisiae industrial strains. RAPD analysis of series strains showed a similarity coefficient ranging from0.47to0.96. High-yield strain1015-04-01and high-yield strain1002-03-03showed the highest similarity while high-yield strain1015-04-01and low-yield strain1415showed the lowest. Utilizing UPGMA method, high-yield strain1016-02-04, low-yield strain1313and low-yield strain1415were clustered into a large group, further with1313and1415clustered into a small one. Meanwhile the other four high-yield strains were clustered into another large group. The nuclear genome diversity among the series of strains, revealed by RAPD analysis, showed the similar trend with the diversity of single cell morphology and alcohol-producing trait.Genetic modification was conducted with high-yield strain1015-10-01and low-yield strain as receptor and gene donor respectively, and the mutant034-19was obtained based on high throughout PCR template-making method. Mutation was introduced to site11and site333, leading to corresponding substitution of valine with glycine in site310and substitution of isoleucine in site203respectively. Compared with the contrast, the mutant showed higher growth rate and gas-producing rate while the highest alcohol concentration remained the same or less. In YPD medium, the growing rate of034-19accelerated, among that of the contrast and1415, and the cell concentration reached the highest value6.36×108/mL when it was cultured for16hours. The relative respiration intensity of034-19got enhanced greatly. In the alcohol fermentation with molasses as the substrate, the alcohol-producing rate accelerated with the highest alcohol concentration value of14.52%, somewhat higher than that of the contrast. The corbon utilizing ability and sugar utilizing ability in fermentation of molasses nearly remained the same. |
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