Hikage Takashi, Saitoh Yasushi, Tanaka-Saito Claire, Hagami Hisakazu, Satou Fumi, Shimotai Yoshitaka, Nakano Yuki, Takahashi Miho, Takahata Yoshihito, Tsutsumi Ken-Ichi
Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate, 020-8550, Japan.
Mol Genet Genomics. 2007 Jul;278(1):95-104. doi: 10.1007/s00438-007-0230-4. Epub 2007 Apr 12.
Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to alpha/beta hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants.
此前，我们鉴定出两种密切相关的蛋白，分别称为W14和W15，它们在龙胆科植物三花龙胆的越冬芽中富集。后一种蛋白W15的表达与耐寒性有关，因为在一个冷敏感突变体中不存在该蛋白。在此，我们对这两种蛋白及其编码基因进行了表征。W14和W15蛋白的氨基酸序列仅在三个氨基酸位置上存在差异，并且它们都与α/β水解酶折叠超家族具有同源性。一致地，在细菌中表达的GST融合W14和W15蛋白对乙酸1-萘酯表现出水解酶活性。对包括花药培养衍生的纯合二倍体在内的七个不同龙胆菌株/品种中的这两个基因进行结构分析，发现W14和W15基因是等位基因。发现了三种基因型；两个菌株携带两个等位基因（W14/W15），一个菌株两个等位基因均携带W15基因（W15/W15），其他菌株是W14的纯合子（W14/W14）。有趣的是，这两种蛋白的表达表现出等位基因特异性。在一个W14/W15菌株中，W-15等位基因的表达几乎受到抑制。此外，在不同品种中观察到等位基因的器官特异性表达。结合龙胆植物的抗寒性对这些观察结果进行了讨论。
