Department of Biological Chemistry and Food Sciences, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Brewing Society of Japan, 2-6-30, Takinogawa, Kita-ku, Tokyo 114-0023, Japan.
Department of Biological Chemistry and Food Sciences, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
J Biosci Bioeng. 2020 Oct;130(4):367-373. doi: 10.1016/j.jbiosc.2020.06.005. Epub 2020 Jul 7.
Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3-K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11 created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.
通过孢子形成获得单倍体细胞的难度阻碍了清酒酵母的杂交育种。我们之前证明，典型的清酒酵母菌株在减数分裂染色体重组方面存在缺陷，这导致了不良的孢子形成和孢子活力丧失。在这项研究中，我们筛选了实验室酿酒酵母 GRF88 的单拷贝质粒基因组 DNA 文库，以寻找可能弥补来自清酒酵母 Kyokai no.7（K7）的菌株 UTCAH-3 的减数分裂重组缺陷的基因。我们确定了实验室菌株（ScSPO11）的 SPO11 基因，该基因编码一种减数分裂特异性内切酶，可催化减数分裂重组所需的 DNA 双链断裂，是恢复 UTCAH-3 减数分裂重组和孢子活力的基因。K7SPO11 不能恢复 UTCAH-3 和实验室菌株 BY4743 spo11Δ/spo11Δ 的孢子形成效率和孢子活力，表明 K7SPO11 没有功能。对各种清酒酵母（K1 和 K3-K10）的 SPO11 基因进行序列分析表明，除了在所有测试的清酒酵母中存在的另外三个共同突变外，K7 组清酒酵母（K6、K7、K9 和 K10）还存在一个相互的错义突变（C73T）。通过体外诱变产生的 ScSPO11 不能恢复 BY4743 spo11Δ/spo11Δ 的孢子活力。另一方面，除了 C73T 之外还具有另外三个共同突变的 K8SPO11 可以恢复 BY4743 spo11Δ/spo11Δ 的孢子活力。这些结果表明，C73T 可能是 K7SPO11 重组缺陷的致病突变。此外，我们发现引入 ScRIM15 可以恢复孢子形成效率，但不能恢复孢子活力。
