小拟南芥双键还原酶基因OpDBR的克隆及表达分析

Molecular cloning and expression analysis of Op DBR from Olimarabidopsis pumila

从小拟南芥,又名无苞芥(Olimarabidopsis pumila,异种名Arabidopsis pumila)幼苗叶片c DNA文库中获得1条与拟南芥烯醛双键还原酶基因At DBR1(Gen Bank登录号为NP_197202.2)高度相似的EST序列,该序列包含1个1041 bp的最大开放阅读框(ORF),推测编码346个氨基酸(Gen Bank登录号为KU845686)。利用RT-PCR技术从小拟南芥叶片的c DNA中克隆了该基因,命名为Op DBR。比对分析结果表明,该基因属于中链还原/脱氢酶(medium chain reductase/dehydrogenases,MDR)超级家族,具有double bond reductase-like保守蛋白结构域;该蛋白的二级结构包含106个α-螺旋和48个β-转角。系统进化树分析结果表明,Op DBR编码产物与预测的亚麻芥烯醛双键还原酶(Gen Bank登录号为XP_010492631.1)遗传关系最为接近,属于同一进化分支。实时荧光定量RT-PCR(q RT-PCR)结果显示,Op DBR在小拟南芥的不同组织中都有表达,茎中表达量最高,根中最低。其在200 mmol/L Na Cl、4℃、100μmol/L ABA、20%PEG-6000处理下的表达结果分析显示,该基因可能在小拟南芥应对逆境胁迫时起着重要作用。

An expressed sequence tag（EST） which shares high similarity with Arabidopsis thalania alkenal double bond reductase At DBR1（Gen Bank accession number： NP_197202.2） was obtained from the cDNA library of Olimarabidopsis pumila（synonym： Arabidopsis pumila） seedlings leaves. It contained a complete open reading frame（ORF） with 1041 bp, encoding 346 amino acids（Gen Bank accession number： KU845686）. The gene was cloned from O. pumila leaf cDNA using RT-PCR technique and was named as OpDBR. Bioinformatics analysis showed that OpDBR belongs to the medium chain reductase/dehydrogenases（MDR） super family with a double bond reductase-like conserved protein domain. The secondary structural analysis showed that OpDBR protein contains 106 α-helixes and 48 β-turns. Phylogenetic tree analysis revealed that OpDBR showed closer genetic kinship with predicted Camelina sativa alkenal double bond reductase（Gen Bank accession number： XP_010492631.1）, indicating that they belonged to the same evolutionary branch. Quantitative Real-time RT-PCR（q RT-PCR） revealed that OpDBR displayed a wide rage of expression in O. pumila different tissues, with a maximum expression in stem and a minimum expression in root. The expression analysis of OpDBR under 200 mmol/L NaCl, 4 ℃, 100μmol/L ABA and 20% PEG-6000 indicated that OpDBR played an important role in response to stresses in O. pumila.