青稞酸性磷酸酶基因HvnACP2克隆和亚细胞定位研究

Cloning and Subcellular Localization of Acid Phosphatase-encoding Gene HvnACP2 in Hulless Barley

为了探索青稞酸性磷酸酶基因 HvnACP2的基因和蛋白结构特点,为青稞磷吸收利用机制研究提供基础。以青稞‘肚里黄’叶片为材料,根据植物基因组数据库Gramene中的大麦 HvACP2基因和启动子区域序列设计引物,通过PCR获得青稞 HvnACP2基因和启动子区域序列。采用生物信息学软件对 HvnACP2基因启动子区域元件以及蛋白理化性质、跨膜结构、磷酸化位点、信号肽、二级、三级结构进行分析。结果表明: HvnACP2有2个外显子和1个内含子,启动子区域有与分生组织、光响应、厌氧、茉莉酸和脱落酸相关的顺式作用元件。HvnACP2蛋白由454个氨基酸组成,分子质量为51 285.15 u,总原子数为7 058,亲水系数为-0.451,理论等电点为6.17,不稳定指数34.54,脂溶性指数为67.05。具有12个磷酸化位点和信号肽,不具有跨膜结构。 HvnACP2中α螺旋、延长链、β转角、无规则卷曲所占比例分别为18.72%、53.96%、23.57%、3.74%。 HvnACP2和其他物种的同源蛋白都有紫色酸性磷酸酶N末端结构域和金属酸性磷酸酶结构域,将 HvnACP2与其他植物的氨基酸序列进行对比并构建进化树,发现青稞 HvnACP2与小麦TaACP2亲缘关系最近。亚细胞定位结果显示 HvnACP2定位在细胞膜上。

The gene and protein structure characteristics of HvnACP2 were explored to provide a basis for the study of phosphorus uptake and utilization mechanism in hulless barley.In this study,the leaves of the hulless barley‘Dulihuang’were used as materials,and primers were designed according to barley HvnACP2(HORVU.MOREX.r2.4HG0340110.1)gene and promoter region sequences in Gramene(http://www.gramene.org/),and the HvnACP2 gene and promoter region sequences were obtained by PCR.The gene structure,promoter region elements,physicochemical properties,transmembrane structure,phosphorylation sites,signal peptide,secondary and tertiary structure,homologous protein amino acid sequence alignment and phylogenetic tree were constructed by bioinformatics software,the subcellular localization of HvnACP2 was also studied.The results showed that HvnACP2 contains 2 exons and 1 intron and the promoter region contained a large number of cing-acting elements related to meristem,light,anaerobic,methyl jasmonate and abscisic acid.HvnACP2 protein had 454 amino acids,molecular mass was 51285.15 u,total number of atoms was 7058,GRAVY was-0.451,theoretical pI was 6.17,instability index was 34.54,aliphatic index was 67.05.HvnACP2 protein contained 12 phosphorylation sites,1 signal peptide and no transmembrane structure.The proportions ofα-helix,extended strand,β-turn and random coil of HvnACP2 were 18.72%,53.96%,23.57%and 3.74%,respectively.Both HvnACP2 and other homologous proteins of other species contained purple acid phosphatase N-terminal and metallophosphatase domain.By comparing HvnACP2 amino acid sequences with those of other plants and constructing phylogenetic tree,it is found that hulless barley HvnACP2 is closely related to TaACP2.HvnACP2 is localized in the cell membrane.