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作物学报 ›› 2021, Vol. 47 ›› Issue (10): 1941-1952.doi: 10.3724/SP.J.1006.2021.04227

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

半夏PtPAL基因的克隆、表达与酶动力学分析

何潇1,2(), 刘兴3, 辛正琦1,2, 谢海艳1,2, 辛余凤1,2, 吴能表1,2,*()   

  1. 1西南大学生命科学学院, 重庆 400715
    2三峡库区生态环境教育部重点实验室, 重庆 400715
    3重庆市大足中学, 重庆 402360
  • 收稿日期:2020-10-14 接受日期:2021-03-19 出版日期:2021-10-12 网络出版日期:2021-04-01
  • 通讯作者: 吴能表
  • 作者简介:E-mail: 992651097@qq.com
  • 基金资助:
    国家自然科学基金项目(30500041)

Molecular cloning, expression, and enzyme kinetic analysis of a phenylalanine ammonia-lyase gene in Pinellia ternate

HE Xiao1,2(), LIU Xing3, XIN Zheng-Qi1,2, XIE Hai-Yan1,2, XIN Yu-Feng1,2, WU Neng-Biao1,2,*()   

  1. 1School of Life Science, Southwest University, Chongqing 400715, China
    2Key Laboratory of Eco-environments in Three Gorges Region, Ministry of Education, Chongqing 400715, China
    3Chongqing Dazu Middle School, Chongqing 402360, China
  • Received:2020-10-14 Accepted:2021-03-19 Published:2021-10-12 Published online:2021-04-01
  • Contact: WU Neng-Biao
  • Supported by:
    National Natural Science Foundation of China(30500041)

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摘要：

本研究以半夏为研究对象, 基于其转录组数据克隆获得1个苯丙氨酸解氨酶基因, 命名为PtPAL, 通过DNAMAN、MEGA等生物信息学软件进行序列结构与系统进化分析表明, 该基因核酸序列长度为2289 bp, 编码762个氨基酸, 终止密码子为TAA, PtPAL与单子叶植物百合PAL相似度达78%, 具有PAL-HAL、PLN02457、phe_am_lyase、Lyase_aromatic及HutH结构域, 属于苯丙氨酸解氨酶家族成员(Lyase_I_like Superfamily)。半夏PtPAL与单子叶植物百合、菠萝和油棕亲缘关系较近, 归于单子叶植物。采用实时荧光定量PCR分析PtPAL在不同组织间的表达情况表明, PtPAL基因在叶中表达量最高, 其次是块茎和根, 花中表达量最低。通过对半夏PtPAL基因进行功能表达分析发现, PtPAL重组蛋白可以高效催化L-Phe生成t-CA, 且催化反应最适pH与温度分别为9.0、70℃; PtPAL的Km、Vmax、Kcat和Kcat/Km分别为0.89 mmol L-1、63.96 nKat mg-1、6.56 s-1和7.37×103 s-1 M-1。进一步探究金属离子对PtPAL酶活性的影响表明, Ba2+显著增强了PtPAL酶活性, Mn2+、Co2+、Cu2+及Zn2+抑制了PtPAL酶活性。本研究为进一步研究PtPAL的功能特点及半夏苯丙胺类生物碱代谢途径奠定基础。

关键词: 半夏, 麻黄碱, 苯丙氨酸解氨酶, 基因克隆, 表达分析, 酶动力学分析

Abstract:

In this study, based on the transcriptome data, a phenylalanine ammonia lyase gene was cloned and named PtPAL using Pinellia ternate as the experimental material. The analysis of sequence structure and systematic evolution revealed that the length of the gene was 2289 bp, encoding 762 amino acids, and the termination codon was TAA. PtPAL was the similarity of 78% compared with monocot Lilium regale PAL, with PAL-HAL, PLN02457, phe_am_lyase, Lyase_aromatic, and HutH domains that belonging to the Lyase_I_like superfamily. Phylogenetic tree analysis showed that PtPAL was closely related to the monocotyledon Lilium regale, Ananas comosusm, and Elaeis guineensis, and thus belonging to Moncotyledons. Real-time fluorescent qPCR indicated that the relative expression level of PtPAL gene was the highest in leaves, followed by tubers and roots, and the lowest in flowers. The functional expression analysis of PtPAL gene revealed that the PtPAL recombinant protein could efficiently catalyze L-Phe to t-CA, and the optimal pH and temperature of the reaction were 9.0℃ and 70℃, respectively. Km, Vmax, Kcat, and Kcat/Km of PtPAL were 0.89 mmol L -1, 63.96 nKat mg-1, 6.56 s-1, and 7.37×103 s-1 M-1. Further exploration of the effect of metal ions on PtPAL enzyme activity showed that Ba2+ could significantly enhance PtPAL enzyme activity, and Mn2+, Co2+, Cu2+, and Zn2+ inhibit the activity of PtPAL enzyme. This study lays the foundation for further research of the functional characteristics of PtPAL and the metabolic pathways of amphetamine alkaloids in Pinellia ternata.

Key words: Pinellia ternate, ephedrine, phenylalanine ammonia-lyase, gene cloning, expression analysis, enzyme kinetics analysis

表1

本研究所用引物"

引物用途
Primer function		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
基因克隆
Gene cloning		 PtPAL-F
PtPAL-R		 CACCTAAACCTTCTTCCGCAG
GATCTTACTTGTTACTGGTTCCAAT
荧光定量PCR
Fluorescence quantitative PCR		 PtPAL-qF
PtPAL-qR
PtGAPDH-qF
PtGAPDH-qR		 CCAACATCCTGGCTCTGCTCTC
GTCCTGCTTCGGCTTCGTCA
TCGTCTTGAGAAATCGGCGAC
GTCAAAGATGCTCGACCGCT
原核表达载体构建
Construction of prokaryotic expression vector		 PtPAL-Hind III-F
PtPAL-Xho I-R		 CGCAAGCTTTTATGGCGGCCAAGTCGAACGGTCT CGCCTCGAGTTACTGGTTCCAATAACCCCTT

图1

半夏PtPAL 基因全长CDS核酸及氨基酸序列 编码区核酸序列和翻译的氨基酸序列用大写字母表示。终止密码子用*表示, 起始密码子和终止密码子用横线表示。"

图2

不同植物PAL氨基酸序列多重比对 一致氨基酸残基用白色字体黑色背景表示, 保守氨基酸残基用黑色字体红色背景表示。保守的Ala-Ser-Gly催化三联体用表示实心三角, 其他保守活性位点用空心三角表示, 保守活性基序用红色方框表示。AtPAL: 拟南芥(NP_187645.1); VvPAL: 葡萄(RVW44079.1); LrPAL: 百合(ASV46344.1); GbPAL: 银杏(ABU49842.1); EaPAL: 问荆(AAW80639.1); PaPAL: 背苔(AIU99853.1)。"

图3

半夏PtPAL和其他植物PAL系统进化树 各节点处数值表示Bootstrap值百分比(重复1000次)。半夏PtPAL用红色点表示。"

图4

PtPAL蛋白结构预测 A: 单体; B: 同源四聚体。MIO: 3,5-二氢-5-亚甲基-4H-咪唑-4-酮。"

图5

不同组织中半夏PtPAL基因表达分析 不同小写字母表示不同组织间在0.05水平差异显著。"

图6

PtPAL重组蛋白时间梯度诱导表达和融合His标签PtPAL重组蛋白纯化 M为蛋白marker。A: PtPAL重组蛋白时间梯度诱导表达; B: 融合His标签PtPAL重组蛋白纯化。"

图7

PtPAL重组蛋白功能鉴定 蓝色、红色和黑色峰图分别表示反式肉桂酸标品、反应样品、反应对照样品。"

图8

PtPAL催化反应最适pH和温度 A: 不同pH下PtPAL催化活性测定; B: 不同温度下PtPAL催化活性测定。误差线表示生物学重复的标准误差(n = 3)。"

图9

PtPAL米曼氏动力学曲线 误差线表示生物学重复的标准误差(n = 3)。"

表2

不同植物PAL酶动力学参数比较"

酶
Enzyme		 温度
Temperature
(℃)		 米氏常数
Km
(mmol L-1)		 最大酶促反应速率
Vmax
(nkat mg-1)		 催化常数
Kcat
(s-1)		 催化效率
Kcat/Km
(s-1 M-1)		 参考文献
References
PtPAL 70 0.89 63.96 6.56 7.37×103 本研究 This study
37 0.80 37.67 3.75 4.69×103
EsPAL1 37 0.144 7.45×10-3 0.588 3.87×103 Taketo et al. (2008) [20]
EsPAL2 37 0.152 5.77×10-3 0.457 3.18×103
BoPAL1 50 1.01 — 10.11 1.0 ×104 Hsieh et al. (2011) [21]
BoPAL2 50 0.33 — 16.04 4.86×104
CdPAL 55 0.101 — 3.36 3.32×103 Hu et al. (2011) [22]
RgPAL 50 1.3 — 25×103 19.20 Zhu et al. (2013) [23]
RcPAL 45 7.90 — 52.31 6.62 ×103 Ma et al. (2013) [24]

图10

金属离子对PtPAL重组蛋白活性的影响 **表示在0.01水平差异显著。"

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pmid: 8534851
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