荔波连蕊茶GA2ox1基因的克隆及表达分析

Cloning and Expression Analysis of GA2ox1 Gene from Camellia lipoensis

[目的]GA2氧化酶是赤霉素生物合成代谢过程中关键酶之一,GA2ox家族基因常被用于植物矮化基因工程育种。[方法]根据植物GA2氧化酶基因编码区的保守序列设计引物,以山茶属荔波连蕊茶嫩枝为材料,提取总RNA,进行RT-PCR。采用RACE技术扩增获得1 371 bp的GA2ox基因全长c DNA序列,命名为Cl GA2ox1(Gen Bank登录号KJ502290)。[结果]序列分析表明,Cl GA2ox1放阅读框(ORF)为1 002 bp,编码333个氨基酸,5’非编码区59 bp,3’非编码区310 bp。预测的蛋白质分子量为37.31 k D,等电点为5.92,具有GA2ox超基因家族的保守结构域和特有的氨基酸残基。Cl GA2ox1蛋白与Gen Bank中收录的其它植物GA2ox蛋白氨基酸的相似性达到80%。构建系统进化树,结果显示山茶GA2氧化酶与烟草GA2ox蛋白的亲缘关系最为密切。实时定量PCR结果显示,该基因在荔波边蕊茶不同器官及发育不同时期的均有表达,表达量有所不同:Cl GA2ox1基因在2年生茎段中的表达量最高,在嫩枝和根中也有较高的表达,而在新抽生的嫩叶中最低。[结论]试验结果为进一步明确Cl GA2ox1基因的功能特征及揭示其参与调控植物生长的分子机制奠定基础。

Gibberellin 2 oxidase is one of the key enzymes in GA biosynthesis and metabolism,and GA2 ox genes are often used to dwarf plant engineering breeding. In this study,the gibberellin 2 oxidase gene was cloned from the stems of Camellia lipoensis using RT-PCR and RACE methods. The full length of the GA2 ox gene, named Cl GA2ox1,is 1 371 bp（ Gen Bank accession No. KJ502290）,which contains a 1 002 bp open reading frame encoding 333 amino acid residues,contains a 5＇-UTR with 59 bp and 3＇-UTR 310 bp. The putative protein molecular weight is 37. 31 k D and its theoretical isoelectric point is 5. 92. The sequence includes GA2 ox superfamily domain and the family signature amino acid residues. Homologous alignment shows that it shares 80% of amino acid identities with GA2 ox from other plants in Gen Bank. The phylogenetic tree constructed on the basis of amino acid sequences of GA2 ox suggests that the relationship between C. lipoensis and Nicotiana tabacum is the most intimate.The results showed that the Cl GA2ox1 gene expression level in root,young stem,two-year-old stem,young leaf,mature leaf and seed of C. lipoensis were different. The Cl GA2ox1 transcripts were the most abundant in two-yearold stem,followed by new stem and root,and the lowest transcripts were found in the young leaf. This study mayprovide references for further research on the functional characteristics of Cl GA2ox1 and its molecular mechanism involved in the regulation of plant growth.