梨内根-贝壳杉烯合酶基因克隆及表达分析

摘要

中矮1号(Pyrus communis)是中国农业科学院果树研究所选育的梨优良矮化砧木,为研究其矮化机理,应用同源克隆的方法从中矮1号叶片总RNA中克隆赤霉素合成过程中的关键酶——内根-贝壳杉烯合酶(KS)基因的cDNA全长序列,并对该基因在不同梨品种中的表达进行分析.结果表明:基于苹果基因组序列设计的特异引物从中矮1号的总RNA中扩增出了1个编码框全长为2214 bp的cDNA序列,其编码737个氨基酸残基,推断分子量为83.63 kD,等电点为5.37.其氨基酸序列与报道的其他植物KS氨基酸序列的相似性为53.61％～72.63％,其中,与板栗(AEF32083.1)的相似性最高,其氨基酸序列中包含植物KS基因所共有的YDTAWVAWVP和DDXXD保守结构域,因此,将分离到的基因命名为PcKS.早酥、锦香和中矮1号等不同梨品种的PcKS基因的cDNA序列仅有个别碱基差异,但编码的氨基酸序列完全一致,品种间不存在结构差异.RT-PCR表达分析结果表明,PcKS基因在早酥中的表达量低于同时期的锦香和中矮1号,与植株高度相反,初步表明该基因的表达有与梨植株生长势呈负相关的趋势.%Zhongai 1 is an excellent dwarfing inter-stock bred by Research Institute of Pomology, Chinese Academy of Agricultural Sciences. For the study of dwarfing mechanism, ent-kaurene synthase (KS) gene, a critical enzyme in the pathway of gibberellins biosynthesis, was isolated from the young leaves of Zhongai 1 pear by homologous clone way. Its expression intensity in different pears was also analyzed. The results were as followed: by the specific primers designed from the apple genome sequence, a full-length cDNA sequence of 2214 nucleotides was amplified,which encodes a putative protein of 737 amino acids residues; the molecular weight and isoelectric point of this protein were 83.63 kD and 5.37 respectively; the amino acids sequences had a similarity between 53.61% and 72.63% with those of other reported plants and had the highest similarity with Castanea mollissima (AEF32083.1). The protein also contained the conserved domain YDTAWVAWVP and DDXXD of KS in common with other plants, so the isolated gene was named PcKS. The cDNA sequences had only a few nucleotide bases difference in Zhongai 1, Zaosu and Jinxiang, but their encoding amino acids sequences were completely consistent and had no structural differences. Semi-quantitative RT-PCR analysis in different varieties showed that PcKS had lower expression in Zaosu than that in Zhongai 1 and Jinxiang in all the tested growing periods, which was reverse with plant height, showing that the gene expression had negative correlation tendency with the plant vigour.