大麦HvRBR基因克隆与序列分析

Molecular Cloning and Sequence Analysis of Novel RBR Gene from Barley

【目的】从栽培大麦(Hordeum vulgare)中分离并克隆对植物细胞周期起负调控作用的RBR(retinoblastoma-related),鉴定大麦RBR(HvRBR)分子特征,明确其与同源基因间的亲缘关系和分类地位,为探索动、植物生长发育过程中细胞增殖和分化相关调控途径的研究提供理论依据。【方法】通过对植物RBR生物信息学分析,根据RBR保守区域序列设计通用引物,采用PCR方法从栽培大麦DNA和苗期总cDNA中分别分段克隆,获得特征序列后在DNAMAN软件下进行序列分析、多重序列比对并构建系统树。【结果】从栽培大麦籽粒皱缩突变材料GSHO1854中获得全长为5547bp的大麦HvRBR序列(GU121481),其cDNA编码区(GU121480)全长3179bp,包含一个编码975个氨基酸的开放阅读框。由其推导的氨基酸序列与已报道的RBR蛋白序列有较高的一致性。在A、B保守区之间有一个间隔区,虽然同源性较低,但是所有氨基酸序列的相似位点都包含一个半胱氨酸残基,这说明该半胱氨酸残基形成的分子内或分子间二硫键可能对整个RBR蛋白的结构和功能产生重要的影响。系统进化分析表明HvRBR与水稻同源性最高(84.3%),与苜蓿、拟南芥等双子叶植物的同源性较低(50%)。【结论】首次从大麦中得到与植物细胞周期起调控、细胞增殖和分化相关的RBR蛋白编码基因HvRBR。对栽培大麦籽粒皱缩突变材料GSHO1854的HvRBR进行了分子克隆和鉴定,并通过系统进化分析将HvRBR归为植物RBR家族C亚族。

[ Objective ] The aim of this study is to isolate and clone RBR （retinoblastoma-related） gene from barley （Hordeum vulgare）, which is a negative regulator during the cell cycle progress, to characterize HvRBR sequence, ensure taxonomic status among the homologous genes, and give more information about cell proliferation and differential regulation pathway in animals and plants. [ Method ] After large numbers of RBR genes were bioinformatically analyzed, specific primers were designed based on the conservative sequences. The RBR gene sequences were obtained from both genomic DNA and cDNA of seedlings. DNAMAN software was used to perform sequence analysis, multiple sequence alignment, and phylogenetic tree construction was also carried out by MEGA. [Result] The 5 547 hp of DNA sequence （GU121481） and 3 179 bp of cDNA sequence （GU121480） of HvRBR were obtained from GSHO1854 （a shrunken endosperm mutational material of cultivated barley）, respectively. The coding region of this gene could encode a protein with 975 amino acid residues. This deduced amino acid had a high identity with the known RBR proteins form plants and animals. Although the lower identity was found in the spacer region between pocket A and B domain, a conservative cysteine residue was found in all the RBR protein family members at similar position. It was suggested that the intra- or inter-molecular disulfide bond probably play an important role in affecting the structure and function of RBR protein. Phylogenetic analysis revealed that the highest degree of identity （84.3%） was between HvRBR and OsRBR2 （Oryza sativa）, and a lower value （50%） between HvRBR and dicot plants such as MsRBR （Medicago sativa） and AtRBR1 （Arabidopsis thaliana）. [Conclusion] It was the first time to obtain the barley HvRBR gene, which regulate cell cycle, proliferation and differentiation. HvRBR is a novel member of subgroup C of plant RBR family by sequence analysis, identification and phylogenetic analysis.