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内根-贝壳杉烯合成酶基因(KS)、内根-贝壳杉烯酸氧化酶基因(KOA)是赤霉素合成途径的关键基因。通过touchdown PCR从短枝富士茎尖组织中克隆得到KS和KOA基因的开放阅读框(ORF),分别命名为MdKS(GenBank登录号KF437681)和MdKOA1(GenBank登录号为KF437682)。MdKS和MdKOA1基因的ORF分别为2211 bp、1509 bp。氨基酸同源性分析表明,MdKS与其他物种的氨基酸序列具有45.2%~98.8%的同源性;而MdKOA1与其他物种的氨基酸序列同源性为42.8%~99.4%。亚细胞定位显示,MdKS蛋白定位于细胞核、细胞质和细胞质膜;而MdKOA1蛋白定位于细胞质和细胞质膜。荧光定量表明,MdKS与MdKOA1基因在SH40、SH28嫁接品种短枝富士不同组织中的表达模式基本一致。MdKS和MdKOA1在半矮化类型SH28嫁接短枝富士的茎尖、幼果与枝条中的表达量高于矮化类型SH40的嫁接品种。
Neither the root-and-kaurene synthase gene (KS) nor the germane-kaurene oxidase gene (KOA) is the key gene in the gibberellin biosynthesis pathway. The open reading frames (ORFs) of KS and KOA genes were cloned by touchdown PCR from the shoot tips of Fujiminori, and named as MdKS (GenBank accession number KF437681) and MdKOA1 (GenBank accession number KF437682), respectively. The ORFs of MdKS and MdKOA1 genes were 2211 bp and 1509 bp, respectively. Amino acid homology analysis showed that the amino acid sequence of MdKS shared 45.2% -98.8% homology with other species. The amino acid sequence identity of MdKOA1 with other species was 42.8% -99.4%. Subcellular localization showed that MdKS protein was located in the nucleus, cytoplasm and cytoplasmic membrane, whereas MdKOA1 protein localized in cytoplasm and plasma membrane. Fluorescence quantitative analysis showed that the expression patterns of MdKS and MdKOA1 in SH40 and SH28 grafts were similar. The expression level of MdKS and MdKOA1 in the shoot tips, young fruits and shoots of the semi-dwarf SH28 grafted short branch Fuji was higher than that of the dwarfing type SH40.