一种松材线虫醛脱氢酶的基因克隆及其生化性质

doi:10.13560/j.cnki.biotech.bull.1985.2021-1450

摘要/Abstract

摘要：

松材线虫（Bursaphelenchus xylophilus）是松萎蔫病的病原,可引起数十种松属树种的毁灭性破坏。通过RT-PCR 成功克隆了松材线虫体内一种醛脱氢酶基因aldh,aldh的长度为1 353 bp,编码451 个氨基酸残基,前期研究表明,杀线剂甲吡唑处理能使aldh表达水平显著上调。序列比对表明,与大多数其他醛脱氢酶中依靠半胱氨酸（Cys）残基发挥催化功能不同,aldh 编码的蛋白质保守的位置是亮氨酸（Leu）残基,但其C-端紧邻Leu残基的是组氨酸残基（His）,而不是常见的Leu残基。构建了表达载体pET-15b-aldh并转化大肠杆菌BL21（DE3）,IPTG诱导aldh在工程菌中实现了高表达,重组醛脱氢酶通过Ni-NTA亲和层析纯化后进行表征。以甲醛为底物的醛脱氢酶Km为27.87 mmol/L,最适pH值为7.5,最适温度为25℃,Fe³⁺和Ni²⁺可提高酶活性,Ca²⁺、Mn²⁺、Na⁺和K⁺可降低酶活性。重组醛脱氢酶对5种醛类化合物显示出不同的催化活性,其中香草醛是其最佳底物。该研究为深入了解醛脱氢酶在松材线虫致病过程中的作用打下了基础,也为新型工业用醛脱氢酶的研究提供了参考。

关键词: 松材线虫, 醛脱氢酶, 纯化, 性质

Abstract:

Pine wood nematode（PWN）,Bursaphelenchus xylophilus,is a causal pathogen of pine wilt disease（PWD）that is devastating to dozens of pine species. An gene aldh of aldehyde dehydrogenase from PWNs was successfully cloned through RT-PCR,its length was 1 353 bp and encoded a protein containing 451 amino acids residues. Prior studies demonstrated a nematicide fomepizole resulted in the up-regulation of aldh expression. Sequences alignment showed that the conserved catalytic position was Leu residue,which was different to Cys residue in most other aldehyde dehydrogenases,and its adjacent residue at C-terminal was His rather than the common nonpolar amino acid residue such as Leu. The aldh-encoding aldehyde dehydrogenase was over expressed in E. coli BL21（DE3）transformed with pET-15b-aldh via IPTG induction,and the recombinant aldehyde dehydrogenase was purified by Ni-NTA affinity chromatography. The Km of the aldehyde dehydrogenase with formaldehye as substrate was 27.87 mmol/L,the optimal pH value and temperature was 7.5 and 25℃,respectively. The enzyme activities were enhanced by Fe³⁺and Ni²⁺,while inhibited by Ca²⁺,Mn²⁺,Na⁺ and K⁺. The recombinant aldehyde dehydrogenase showed different catalytic activities towards 5 aldehydes,and vanillin was found to be its optimal substrate in the 5 tested aldehydes. This study lays a foundation for investigating the roles of aldehyde dehydrogenase in PWD and offers a clue to explore aldehyde dehydrogenases utilized in industry.

Key words: Bursaphelenchus xylophilus, aldehyde dehydrogenase, purification, properties

李文硕, 王林松, 杜桂彩, 郭群群, 张廷婷, 杨宏, 李荣贵. 一种松材线虫醛脱氢酶的基因克隆及其生化性质[J]. 生物技术通报, 2022, 38(9): 207-214.

LI Wen-shuo, WANG Lin-song, DU Gui-cai, GUO Qun-qun, ZHANG Ting-ting, YANG Hong LI Rong-gui. Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization[J]. Biotechnology Bulletin, 2022, 38(9): 207-214.

图/表 10

表1 本试验所用引物

Table 1 PCR primers used in this study

引物名称 Name of primer	序列Sequence（5'-3'）
F1	aaaCATATGggacagctccagagtttggt
R1	aaaGGATCCtcactcgatggagatccgctttc

图1 松材线虫aldh基因序列和推测的氨基酸序列

Fig.1 Sequences and predicted amino acid sequences of gene aldh from PWN（pine wilt disease）

图2 醛脱氢酶三维结构（A）和二级结构预测（B）

Fig.2 Prediction of the 3-dimensional structure（A）and 2 -dimensional structure（B）of aldehyde dehydrogenase

图3 重组醛脱氢酶的表达与纯化 M：Marker;1：大肠杆菌E.coli BL21（DE3）空菌全蛋白;2：工程菌全蛋白;3：纯化的醛脱氢酶

Fig. 3 Expression and purification of the recombinant alde-hyde dehydrogenase M：Protein marker;1：total proteins of E. coli BL21（DE3）;2：total proteins of E. coli BL21（DE3）harboring pET-15b-aldh;3：the purified recombinant aldehyde dehydrogenase

图4 通过Lineweaver-Burk计算醛脱氢酶的Km值图中误差线表示标准偏差。小写字母表示不同测试结果间差异达到（P<0.05）显著水平,下同

Fig. 4 Km of the recombinant aldehyde dehydrogenase calculated by Lineweaver-Burk plot The error line in the figure refers to the standard deviation. The lowercase letters indicate that the difference among different test reached a significant level（P <0.05）. The same meaning is suitable for the rest of this paper

图5 不同pH值对醛脱氢酶反应速度的影响

Fig. 5 Effects of pH values on aldehyde dehydrogenase reaction rates

图6 不同温度对醛脱氢酶活性的影响

Fig. 6 Effects of temperatures on aldehyde dehydrogenase activity

图7 不同金属离子对醛脱氢酶反应速度的影响

Fig. 7 Effects of metal ions on aldehyde dehydrogenase reaction rates

图8 醛脱氢酶对不同醛类的催化活性 Vanillin：香草醛;Formaldehyde：甲醛;Acetaldehyde：乙醛;Glutaraldehyde：戊二醛;4-（dimethylamino）-benzaldehyde（PDAB）：4-（二甲氨基）-苯甲醛

Fig. 8 Catalyzed activities of aldehyde dehydrogenase on different aldehydes

图9 多序列对比不同来源的醛脱氢酶的一级结构

Fig.9 Multiple sequences alignments of primary structures of aldehyde dehydrogenases from different sources Bursaphelenchus xylophilus：GenBank NO.CAD5152421.1;Bursaphelenchus okinawaensis：GenBank NO.CAD5218566.1;Trypanosoma grayi：GenBank NO.XP_009312368.1;Caenorhabditis elegans：GenBank NO. NP_741553.1;Mus musculus：GenBank NO.NP_001318043.1;Strongyloides ratti：GenBank NO. XP_024502805.1;Pseudomonas sp. HR199：GenBank NO. CAA06926.1;Schistocerca gregaria：GenBank NO.QVD39485.1;Meloidogyne graminicola：GenBank NO. KAF7633361.1;Ancylostoma caninum：GenBank NO.RCN51637.1;Micrococcus luteus NCTC 2665：GenBank NO. ACS29670.1

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