毛果杨赤霉素氧化酶基因家族鉴定与功能分析

Identification and functional analysis of gibberellin oxidase gene family in Populus trichocarpa

【目的】赤霉素氧化酶(GAox)是不同赤霉素之间相互转化的关键酶,属于2-酮戊二酸依赖性双加氧酶基因家族(2ODD),选择毛果杨GAox基因家族为研究对象,探究木本植物GAox基因家族成员的进化关系和功能分化机制。【方法】在Phytozome和PLAZA数据库中搜索到水稻、拟南芥和毛果杨2ODD基因,通过系统进化分析确认毛果杨2ODD基因中参与赤霉素代谢的GAox基因,然后整合基因组定位、基因结构、基因表达模式和酶生化活性等数据对GAox基因家族进行研究。【结果】从毛果杨基因组中鉴定得到124个2ODD成员,通过系统进化分析确认其中包含27个GAox基因,它们可能参与到赤霉素合成。毛果杨27个GAox基因分成4个亚家族,分别是C20-GA2ox、GA20ox、C19-GA2ox和GA3ox。基因组定位的结果发现27个毛果杨GAox基因分散定位在19条染色体中的14条染色体上,其中有11对GAoxs基因是由基因组的大片段重复产生的。酶学活性测定的结果表明,PtGA2ox3和PtGA2ox5催化GA1生成GA8,GA9生成GA51,而PtGA20ox6、PtGA20ox7和PtGA20ox8催化GA15生成GA24,GA24生成GA9,GA53生成GA20,显示该基因家族成员在底物特异性方面发生了变化。其中PtGA2ox3对GA9的活性是PtGA2ox5的104倍,但是PtGA2ox3和PtGA2ox5具有相似的三维结构。通过活性中心的序列比对发现,PtGA2ox3存在多个非极性氨基酸,PtGA2ox5存在多个带羟基的氨基酸,推测PtGA2ox3活性中心位置附近的非极性基团是引起其催化活性高的原因。【结论】毛果杨GAox基因家族的基本特征、进化关系和部分基因的功能得到了初步解析,为加深对毛果杨赤霉素代谢途径的了解提供新的视角。

【Objective】Gibberellin oxidase(GAox)is a key enzyme for mutual conversion between different gibberellin.GAox genes are members of the 2-oxoglutarate–dependent dioxygenases(ODDs)family.The Populus trichocarpa GAox gene family was selected to explore the phylogenetic relationship and functional differentiation mechanism of members of the GAox gene family of woody plants.【Method】The ODD genes of Arabidopsis thaliana,Oryza sativa and Populus trichocarpa were download in the Phytozome and PLAZA database.GAox genes involved in gibberellin synthesis were identified from the Populus trichocarpa 2ODD gene family by phylogenetic analysis.Then the function of GAox gene family was investigated by integrating genome localization,gene structure,gene expression pattern and enzyme biochemical activity analysis.【Result】A total of 27 GAox genes involved in gibberellin synthesis were identified from 124 members of the Populus richocarpa 2ODD gene family by phylogenetic analysis.The 27 GAox genes from P.trichocarpa belonged to 4 subfamilies,C20-GA2ox,GA20ox,C19-GA2ox and GA3ox.The chromosomal localization analysis showed that 27 GAox genes were assigned to 14 of 19 chromosomes,among which 11 gene pairs were located on the duplicated genomic blocks,indicating they were created by the whole genome duplication events.The enzyme activity assays showed that PtGA2ox3 and PtGA2ox5 can catalyze GA1 to GA8,and GA9 to GA51.PtGA20ox6,PtGA20ox7 and PtGA20ox8 can catalyze GA15 to GA24,GA24 to GA9 and GA53 to GA20.The results showed that the substrates specificity of the gene family members changed.Although PtGA2ox3 and PtGA2ox5 had similar threedimensional structures,the activity of PtGA2ox3 against GA9 is 104 times compared with PtGA2ox5.Through sequence alignment of the active center,it’s found that PtGA2ox3 has multiple non-polar amino acids and PtGA2ox5 has multiple hydroxyl-containing amino acids.It is speculated that the non-polar group near the active center of PtGA2ox3 might be responsible for its high catalytic activity.【Conclusion】This study preliminarily analyzed the characters,evolutionary relationships and function of some genes of the Populus GAox gene family,which could shed light on the further understanding of the gibberellin synthesis in Populus.