烟草NtPLR1基因克隆与表达分析

Cloning and expression analysis of the tobacco NtPLR1 gene

维生素B_6(VB_6)在植物体内参与多种生化反应,对植物生长至关重要,吡哆醛还原酶(PLR)是VB_6代谢转换的作用酶,催化吡哆醛(PL)生成吡哆醇(PN),对维持细胞内VB_6的动态平衡发挥重要作用,而PLR在植物中鲜有报道。以拟南芥Arabidopsis thaliana吡哆醛还原酶氨基酸序列At PLR1为模板,在公用数据库通过同源比对获得数条烟草Nicotiana tabacum Nt PLR1基因的片段,结合互补脱氧核糖核酸(c DNA)的末端快速扩增-聚合酶链式反应(RACE-PCR)技术获得了烟草吡哆醛还原酶Nt PLR1基因。该基因全长1 370 bp,编码369个氨基酸残基,预测其编码蛋白的分子量为41 kDa,理论等电点为9.42。氨基酸多序列比对结果表明:Nt PLR1与其他物种的PLR1相似性较高。实时荧光定量PCR(q RT-PCR)分析结果表明:外源吡哆醛PL处理时,Nt PLR1表达先升高后降低,在4 d达到顶峰。相应地,高效液相色谱分析结果表明:烟草叶片中PL含量随时间逐渐降低而吡哆醇PN含量逐渐升高,表明Nt PLR1可像酵母PLR一样,催化PL形成PN。此外,定量分析结果表明:Nt PLR1在烟草根、茎和叶片中均有表达,其中在叶片中表达显著高于其他部位(P<0.05)。在紫外线、氧化和盐害胁迫下,Nt PLR1的表达与对照相比均显著上调(P<0.05),表明Nt PLR1对这3种逆境有响应,可能参与烟草的抗逆过程。将Nt PLR1连入原核表达载体p ET32a,并进行诱导表达,成功表达出目的蛋白。报道的烟草Nt PLR1基因功能为进一步探明植物PLR基因的功能和调控机制以及VB_6的生物合成提供了重要参考。

Vitamin B6（VB6）, essential for plant growth and development and involved in more than 100 biological processes, utilizes pyridoxal reductase（PLR） as the key enzyme in the VB6 salvage pathway, thereby catalyzing pyridoxal（PL） to generate pyridoxine（PN）. Since studies on PLR of plant VB6 are quite limited, PLR genes were cloned and characterized to improve understanding of VB6 biosynthesis in plants. Several Nt PLR1 gene fragments were found in Nicotiana tabacum through a homologous blast with Arabidopsis At PLR1. Full length was obtained using rapid amplification of c DNA ends（RACE）. Real-time quantitative polymerase chain reaction（PCR） and high performance liquid chromatography（HPLC） analysis were conducted; Nt PLR1 expression by ultraviolet, oxidation, exogenous PL, and Na Cl treatments were compared to a control; and prokary otic expression of Nt PLR1 was accomplished. Results of RACE showed that full length c DNA of Nt PLR1 was1 370 bp, which encoded 369 amino acid residues with a protein molecular weight of about 41 k Da and a theoretical isoelectric point of 9.42. Real-time quantitative PCR analysis revealed that an exogenous PL treatment induced Nt PLR1 expression with highest expression at 4 d. The HPLC analysis showed that PL content significantly decreased（P〈0.05）; whereas, PN content significantly increased（P〈0.05） during an exogenous PL treatment. Nt PLR1 was expressed in roots, stems, and leaves with leaves having the highest（P 0.05） expression level. Also, ultraviolet, oxidation, and Na Cl treatments, compared to a control, significantly induced（P〈0.05） Nt PLR1 expression. Furthermore, prokaryotic expression of Nt PLR1 in vector p ET32 a successfully revealed the recombinant protein at the expected size. This study reported the Nt PLR1 gene of N. tabacum for the first time, finding that it catalyzed PL to form PN in tobacco as found in yeast, and it may be induced in response to ultraviolet, oxidation, and Na Cl stress; thus, the Nt PLR1 gene can be an important reference for further plant PLR gene functional characterization and regulation as well as VB6 biosynthesis.