荔波连蕊茶GA20氧化酶基因的克隆及表达分析

Cloning and Expression Analysis of Gibberelin 20 Oxidase Gene from Camellia lipoensis Chang et Xu

根据植物GA20ox基因编码区的保守序列设计引物,以山茶属荔波连蕊茶幼嫩茎段为材料,提取总RNA,进行RT-PCR。采用RACE技术扩增获得1 567 bp的GA20氧化酶基因全长cDNA序列,命名为ClGA20ox2(GenBank登录号KF823787)。序列分析表明,ClGA20ox2开放阅读框(ORF)为1 146 bp,编码382个氨基酸,5'非编码区115 bp,3'非编码区303 bp。预测的蛋白质分子量为43.56 kD,等电点为7.02,所推导的蛋白氨基酸序列与夹竹桃和杨树GA20ox蛋白的同源性分别为73%和72%。ClGA20ox2与其它植物GA20ox蛋白比较,构建系统进化树,结果显示山茶GA20ox蛋白与夹竹桃和杨树的GA20ox蛋白的亲缘关系最为密切。实时定量PCR结果显示,该基因在荔波连蕊茶的根、茎、叶和种子中均有表达,其表达模式却不同:ClGA20ox2基因在二年生茎段中的表达丰度最高,在顶端分生组织中表达丰度最低,在嫩叶和根中表达丰度较高,成熟叶片和种子表达丰度较低。

In this study,gibberellin 20 oxidase gene was cloned from the stems of Camellia lipoensis Chang et Xu using RT-PCR and RACE methods. The full length of the GA20 oxidase gene,named ClGA20ox2,was1 567 bp（ GenBank accession No. KF823787）,which contained a 1 146 bp open reading frame（ ORF）encoding 382 amino acid residues,contained a 5＇-UTR with 115 bp and 3＇-UTR with 303 bp. The putative protein molecular weight was 43. 56 kD and its theoretical isoelectric point was 7. 02. The deduced amino acid sequence of the ClGA20ox2 protein shares 73% and 72% identities with those of Nerium indicum and Populus trichocarpa,respectively. The phylogenetic tree constructed on the basis of amino acid sequences suggested that the relationship of GA oxidase from C. lipoensis was most intimate with those from N. indicum and P. trichocarpa.The results from the analysis on tissue specific expression showed that the endogenous ClGA20ox2 gene expression levels in the root,stem,leaf and seed of C. lipoensis were different. The ClGA20ox2 transcripts were the most abundant in two-year old stem,followed by young leaf and root,and then mature leaf and seed,the lowest transcripts were found in apical shoot meristems.