本土酿酒酵母低温耐受性的主效QTL定位克隆及其功能研究

Cold fermentation has been a hot spot in modern oenology for its capability in increasing the flavor diversity of wine, which contributes to wine quality improvement. The success of cold fermentation largely depends on the fermentation properties of wine yeasts, especially on cold tolerance. Cold tolerance is a complex trait dominating by quantitative trait loci（QTL）. To illuminate the genetic mechanism of complex trait is one of the front problems in biology research fields. With an aim to interpreting the genotype-phenotype relationship in cold tolerance, in this project, cold sensitive (S-type) and tolerant (T-type) strains will be isolated from the germplasms of native Saccharomyces strains deposited in the applicant’s laboratory. Heterozygous diploid strains (F1), which are derived from the hybridization of the two parent strains, are fully sporulated and these haplospores（F2）are back-crossed with S-type parent strains for cold stress selection. RBS（recurrent selection and backcross）strategy is adopted to purify the genetic background of the population. By sequencing the pooled strains in several selected generations, QTLs will be identified based on allele frequency estimation, which is facilitated with computational simulations. The function of each candidate gene in QTL is validated by gene knockout, over-expression and homologous gene replacement, as well as gene expression. The project is of great importance for a deeper understanding of the mechanism of native wild yeast’s tolerance to cold stress, fully excavating genetic resources in native yeast strains, and promoting the breeding of wine yeasts with excellent cold fermenting properties and high aroma quality.

葡萄酒低温发酵是提高葡萄酒品质、丰富葡萄酒香气的重要手段，已成为现代葡萄酒工艺革新的研究热点。低温发酵能否顺利进行，主要取决于酿酒酵母的低温耐受性。为阐明基因型与低温耐受表型间的对应关系，本项目以前期研究获得的优良本土野生酿酒酵母为材料，筛选低温耐受型和敏感型菌株，分离单倍体并进行杂交、生孢，获得子代单倍体；继而采用重复选择回交策略，纯化选择群体的遗传背景；同时施加梯度低温胁迫，得到用于混池测序的定向选择群体；结合混池测序方法，辅以计算机模拟确定等位基因频率阈值，定位克隆低温耐受主效QTL；通过目标基因的敲除、同源基因置换、超表达及基因表达检测等方法，研究主效QTL功能。本项目的开展，对于深刻解析本土酿酒酵母的低温耐受机制，挖掘本土酿酒酵母中的优质基因资源，促进优良葡萄酒酵母菌种改良和选育，具有重要意义。

低温发酵是现代葡萄酒酿造工艺革新的研究热点。低温发酵能否顺利进行，主要取决于酿酒酵母的低温耐受性。为了深入研究本土优质低温耐受酿酒酵母的遗传结构与特征，阐释酿酒酵母低温耐受机制，本项目以我国5个主要葡萄酒产区的68株本土酿酒酵母为研究对象，利用全基因组测序技术与分离群体分组分析（BSA）相结合的QTL定位方法，对酿酒酵母低温耐受性的主效QTL进行定位，通过相互半合子分析（RHA）、序列多态性分析以及等位基因替换等方法对主效基因进行鉴定，最终发现并证实Nα-末端乙酰基转移酶NatA的辅助亚基NAT1基因与功能未知的YOR365C基因是控制本土酿酒酵母低温耐受性的主效基因。序列多态性分析发现，两个亲本的NAT1编码区存在一个突变位点（T1187C），在其它28个已知全基因组序列的酿酒酵母菌株中均不存在。经过定点突变和突变型等位基因的替换试验验证，该位点的核苷酸多态性是控制酿酒酵母低温耐受性的一个数量性状核苷酸（QTN），该QTN引起相应的氨基酸变化（F396S）能够提高菌株耐受低温的能力。将原低温敏感二倍体菌株NX9412的NAT1基因替换为等位基因NAT1ZX11(6)后，能显著增强其低温耐受能力。研究结果为进一步解析酿酒酵母低温耐受机制奠定了重要的理论基础和提供了新的调控靶点，同时也为低温耐受酵母遗传改良和应用提供了理论支持，具有重要的理论和实践意义。