高山松及其亲本种油松和云南松DHAR基因的功能分化  

Functional Divergence of Dehydroascorbate Reductase Genes in Pinus densata,P.tabulaeformis and P. yunnanensis

作　　者：考洪娜[1,2] 兰婷[1] 王晓茹[1] 曾庆银[1] 

机构地区：[1]中国科学院植物研究所系统与进化植物学国家重点实验室,北京100093 [2]中国科学院研究生院,北京100049

出　　处：《植物学报》2012年第1期1-10,共10页Chinese Bulletin of Botany

基　　金：国家重点基础研究发展计划(No.2009CB119104);国家自然科学基金(No.30830010)

摘　　要：高山松(Pinus densata)是油松(P.tabulaeformis)与云南松(P.yunnanensis)自然杂交产生的同倍性杂种,分布于青藏高原东南缘,占据了油松和云南松两个亲本种都不能正常生长的高海拔地带。为了揭示高山松、油松和云南松脱氢抗坏血酸还原酶(DHAR)基因的组成和功能分化,分别从高山松、油松和云南松中克隆到2类DHAR基因(DHAR1与DHAR2)。组织表达模式分析表明,这6个基因在根、韧皮部、叶和芽中均有表达;通过系统发育分析发现,高山松在物种形成过程中保留了油松的DHAR1拷贝以及云南松的DHAR2拷贝;酶学性质分析则表明,高山松与油松DHAR1蛋白对底物具有相似的催化活性、催化效率、最适pH和热力学稳定性,但其催化活性比云南松DHAR1蛋白高约300倍。高山松DHAR2蛋白对底物的催化活性和热力学稳定性均高于油松DHAR2蛋白。高山松DHAR基因在生化功能上显示出优于或类似亲本DHAR,这种优势功能的选择与杂种独特的生态适应性可能有重要的相关性。Plant dehydroascorbate reductase （DHAR） is a physiologically important reducing enzyme in the ascorbateglutathione recycling reaction. In this study, we cloned 6 DHAR genes from a hybrid pine species complex of Pinus densata, P. yunnanensis and P. tabulaeformis. P. densata originated by natural hybridization of P. yunnanensis and P. tabulaeforrnis. The 6 DHAR genes were divided into 2 types： DHAR1 and DHAR2. Phylogenetic analyses indicated that the 3 DHAR1 and 3 DHAR2 genes from the 3 Pinus species were 2 orthologous groups. DHAR1 and DHAR2 genes originated from an ancestral duplication event that occurred in the most recent common ancestor of the early land plants. P. densata contains a copy of DHARI similar to that of P. tabulaeformis and a copy of DHAR2 similar to that of P. yun- nanensis. RT-PCR revealed that the 6 DHAR were constitutive expression genes in the 3 Pinus species. The recombinant Pinus DHAR proteins were overexpressed in E. coil and purified by Ni-affinity chromatography. P. densata and P. tabulaeformis DHAR1 proteins showed similar enzymatic activities, catalytic efficiency, thermal stabilities and optimal pH pro- files towards substrate DHA but about 300-fold higher enzymatic activities than P. yunnanensis DHAR1 protein. The en- zymatic activity and thermal stability of P. densata DHAR2 protein were higher than those of P. tabulaeformis DHAR2 protein. Joint analyses of sequence structure, phylogenetic relationships, expression patterns, enzymatic properties and protein 3-D structure revealed selective DHAR gene composition in the hybrid genome of P. densata. Such a combination of divergent copies of DHAR gene in P. densata may have adaptive implications for its colonization of novel habitats on the Tibetan Plateau.

关 键 词：适应性 DHAR 功能分化 自然杂种 蛋白质功能 

分 类 号：S791[农业科学—林木遗传育种]