苹果矮化砧木GA20氧化酶基因的克隆及其表达分析

Cloning and Expression Analysis of GA20ox1 Gene from Apple Dwarfing Rootstocks

为了研究GA20氧化酶基因在苹果矮化砧木中的分子特征和表达特征,利用RT-PCR方法从苹果矮化砧木2号和36号c DNA中克隆了GA20氧化酶基因(GA20ox1),并对该基因及其编码氨基酸序列以及在不同时期砧木及嫁接品种中的表达分别进行了分析。结果表明,GA20ox1基因c DNA编码序列长1 179 bp,推导编码393个氨基酸(包括终止密码子),预测蛋白相对分子质量44.3 k Da,理论等电点5.89,编码的蛋白质包含GA20ox1基因家族所具有的特征保守结构域,系统进化分析表明该蛋白序列与苹果SH40的同源性达到96.9%。实时荧光定量PCR分析显示:36号砧木在6月份GA20ox1基因表达强度显著低于八棱海棠对照,而7-8月份的均显著高于对照,其上嫁接的品种7月份表达强度显著低于对照。在7-8月份2号砧木及嫁接品种GA20ox1基因的表达强度均显著低于对照。

In order to study the molecular characterization and expression patterns of GA20 oxidase gene in apple dwarfing rootstocks,RT-PCR method was used to clone GA20 oxidase gene（ GA20ox1） from c DNA of the apple dwarfing rootstocks No. 2 and No. 36,and the genes and the amino acid sequences,as well as gene expression on rootstocks and the grafted varieties at various times,was analysed,respectively. Results showed that c DNA of GA20ox1 gene coded a 1 179 bp sequence,and derived a protein with 393 amino acids including termination codon,which the predicted protein molecular weight was 44. 3 k Da and theoretical isoelectric point was 5. 89. The protein contained conserved domain characteristics of GA20ox1 gene family,and phylogenetic analysis showed that the protein sequence homology with SH40 was at 96. 9%. Real-time fluorescent quantitative PCR analysis revealed that the GA20ox1 expression of No. 36 rootstock was significantly lower than the control（ Malus robusta Rehd.） in June and higher than that in July to August,the expression of the varieties grafted on it was significantly lower than control in July. From July to August,GA20ox1 gene expression levels of No. 2 rootstock and the grafted varieties were significantly lower than control.