铁皮石斛2个F家族ABC转运蛋白基因的克隆和表达研究  

Molecular cloning and expression analysis of two genes encoding F family ATP-binding cassette transporters in Dendrobium officinale

作　　者：李依民[1] 雷根平[2] 颜永刚[1] 彭亮[1] 张娜[1] 刘亮亮[1] 黑小斌 李欢[1] 张岗[1,3] 郭顺星[3] 

机构地区：[1]陕西中医药大学药学院陕西省中药基础与新药研究重点实验室,陕西西安712046 [2]陕西中医药大学附属医院第一临床医学院,陕西咸阳712000 [3]中国医学科学院北京协和医学院药用植物研究所,北京100193

出　　处：《中草药》2017年第15期3153-3159,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(31101608);陕西省自然科学基金项目(2017JM8030);陕西省青年科技新星项目(2012KJXX-44)

摘　　要：目的克隆铁皮石斛Dendrobium officinale ABC(ATP-binding cassette)转运蛋白F家族基因并进行生物信息学分析。方法利用RACE从铁皮石斛叶片cDNA中分离ABC基因,并进行编码蛋白相对分子质量、等电点、结构域、信号肽、跨膜域及亚细胞定位等生物信息学分析;采用DNASTAR和MEGA6进行氨基酸多序列比对和分子进化分析;借助实时荧光定量PCR技术检测基因组织表达模式。结果从铁皮石斛中分离到2个F家族ABC转运蛋白基因DoABCF1和DoABCF2(Gen Bank注册号KU160474和KU160475),全长为2 104 bp和2 193 bp,各编码1条由600和659个氨基酸组成的肽链,相对分子质量67 030和74 140,等电点6.20和5.71;DoABCF1和DoABCF2蛋白均包含2个保守的ABC结构域(分别为74~314、385~600和65~323、392~607)和多个基元;2个蛋白不含信号肽或跨膜域,预测均定位在叶绿体。2个基因与植物F家族ABC转运蛋白基因相似性高达80%以上,与玉米和水稻等单子叶植物F家族ABC转运蛋白基因亲缘关系较近。DoABCF1和DoABCF2基因转录本在石斛3个器官中差异表达且均在叶中高度表达,茎和根中相对表达量差异不显著;以茎为校正样本,前者在根中相对表达量为茎中的1.74倍,后者在叶中表达量为茎中的3.44倍。结论成功获得DoABCF1和DoABCF2基因全长cDNA,二者在铁皮石斛叶中的高表达特征暗示其可能在铁皮石斛生长发育过程中起一定作用。Objective To clone and characterize the F family ATP-binding cassette（ABC） transporter genes in Dendrobium officinale. Methods RACE was used to isolate ABC transporter genes from the leaf cDNA of D. officinale. Characteristics including molecular weight, theoretical p I（isoelectric point）, conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed by serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed by DNASTAR and MEGA6. Tissue specific expression patterns were determined by real-time quantitative PCR（q PCR） analyses. Results Two full length genes DoABCF1 and DoABCF2（Gen Bank accessions KU160474 and KU160475）, 2 104 and 2 193 bp in length, respectively, were obtained. DoABCF1 was deduced to a 600 aa（amino acid） protein with a molecular weight of 67 030 and p I of 6.20, while DoABCF2 encoded a 659 aa protein with a molecular weight of 74 140 and p I of 5.71. The two deduced DoABCF1 and DoABCF2 proteins both had two conserved ABC domains（74—314 and 385—600 for the former, 65—323 and 392—607 for the latter） and several functional motifs. The proteins did not contain any signal peptide or transmembrane domain, and were predicted to locate in the chloroplast. The two genes were highly identical to the plant F family ABC transporter genes with more than 80% similarity, and were mostly close to monocots ABC F family members from maize and rice. DoABCF1 and DoABCF2 showed different expression among the three organs and both had relatively highest expression levels in the leaves, whereas no significant difference could be observed in the roots and stems. Taken the stem as the calibrator sample, DoABCF1 transcripts were 1.74 fold over that in the stems and DoABCF1 transcripts were 3.44 fold, respectively. Conclusion Two F family DoABCF1 and DoABCF2 genes with full length cDNAs are successfully cloned in this study. The highest expression levels of the two

关 键 词：铁皮石斛 ATP-BINDING CASSETTE 转运蛋白 克隆 表达模式 

分 类 号：R282.12[医药卫生—中药学]