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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (10): 1941-1952.doi: 10.3724/SP.J.1006.2021.04227

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2021-10-12 Published:2021-04-01
  • Contact: WU Neng-Biao E-mail:992651097@qq.com;wunb@swu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(30500041)

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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

Table 1

Primers used for this study"

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

Fig. 1

Full length CDS nucleotide sequence and amino acids sequence of PtPAL in Pinellia ternate The coding sequence and the deduced amino acid sequence are shown in uppercase letters in bold. Termination code is denoted by the asterisk; initiation code and termination code are denoted by underlines."

Fig. 2

Multiple alignment of amino acid sequences of PAL in different plants The identical amino acids are shown in white with black background, the conserved amino acids are shown in black with red background. The conserved active site motif (Ala-Ser-Gly) is indicated by filled triangles under the sequences, the other active site residues are indicated by open triangle asterisk, the conserved active site motif is denoted by red box. AtPAL: Arabidopsis thaliana (NP_187645.1); VvPAL: Vitis vinifera (RVW44079.1); LrPAL: Lilium regale (ASV46344.1); GbPAL: Ginkgo biloba (ABU49842.1); EaPAL: Equisetum arvense (AAW80639.1); PaPAL: Plagiochasma appendiculatum (AIU99853.1)."

Fig. 3

Phylogenetic tree analysis of PtPAL and PAL from other plants The number of each interior Branch is the percentage of bootstraps value (1000 replicates). PtPAL is denoted by red dot."

Fig. 4

Deduced protein structure of PtPAL A: monomer; B: homotetramer. MIO: 3,5-dihydro-5-methylene-4H-imidazol-4-one."

Fig. 5

Relative expression levels of PtPAL in different tissues Values with different lowercase letters show significant difference among different tissues at the 0.05 probability level."

Fig. 6

Time gradient induced of the recombinant PtPAL and its purified His-tagged PtPAL M: protein molecular weight standards. A: time gradient induced of reconstructive PtPAL; B: the purified His-tagged PtPAL."

Fig. 7

Functional identification of the recombinant PtPAL The blue, red, and black peak maps are trans-cinnamic acid standard, reaction, and control reaction sample, respectively."

Fig. 8

Optimum pH and temperature of PtPAL for the reaction A: the activity of PtPAL under changing pH levels; B: the activity of PtPAL across a range of temperatures. Bars indicate the standard errors of the means (n = 3)."

Fig. 9

Michaelis-Menten curves for the velocity of PtPAL Bars indicate the standard errors of the means (n = 3)."

Table 2

Comparison of PAL kinetic properties from different plants"

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]

Fig. 10

Effects of metalions on the recombinant PtPAL ** means significant difference at the 0.01 probability level."

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