Prokaryotic Expression, Subcellular Localization and Expression Analysis of PcCHS Gene from Polygonatum cyrtonema Hua

doi:10.13560/j.cnki.biotech.bull.1985.2023-1100

Abstract

Abstract:

【Objective】This work aims to explore the role of chalcone synthase（chalcone synthase，PcCHS）gene in the synthesis of flavonoids in Polygonatum cyrtonema Hua, which may provide a reliable theoretical basis for the subsequent analysis of the function of PcCHS and the breeding of new varieties of P. cyrtonema Hua. 【Method】Using P. cyrtonema Hua as cDNA template, the coding sequence of PcCHS gene was cloned, and the gene was analyzed bioinformatically. The prokaryotic expression vector of PcCHS was constructed and the recombinant protein was purified to verify the expression activity of PcCHS in vitro. Transient overexpression system was used to investigate the changes of total flavonoids content after overexpression of this gene. Gateway technology was used to construct the subcellular localization vector 35S::PcCHS-GFP, and the subcellular localization of target protein was determined by the tobacco expression system. 【Result】The results showed that PcCHS was a hydrophilic protein with an open reading frame of 1 251 bp, a theoretical molecular weight of 44.63 kD and an isoelectric point of 5.89, and was closely related to AoCHS（Asparagus officinalis）. Prokaryotic expression experiment showed that pET28a-PcCHS was induced to express soluble recombinant protein by IPTG（isopropyl-β-d-thiogalactoside）. Western-blot showed that the size of pET28a-PcCHS was about 45 kD, which was consistent with the expected size. The purified protein had certain enzymatic activity and catalyzed the conversion of p-coumaryl-CoA and malonyl-CoA into naringin chalcone. In addition, in the transient overexpression of PcCHS, the expression level of PcCHS group was significantly higher than that of no-load K group, and the total flavonol content was also significantly higher than that of no-load K group, up to 1.83 times. Subcellular localization results showed that the gene plays a role in the cell membrane and nucleus. 【Conclusion】Prokaryotic expression of PcCHS gene has enzyme activity in vitro, and its subcellular location is in cell membrane and nucleus, and instantaneous overexpression significantly increase the total flavonoid content in the leaves of P. cyrtonema Hua.

Key words: Polygonatum cyrtonema Hua, chalcone synthase gene（CHS）, prokaryotic expression, transient overexpression, protein purification, enzyme activity in vitro, subcellular localization

PAN Ping-ping, XU Zhi-hao, ZHANG Yi-wen, LI Qing, WANG Zhong-hua. Prokaryotic Expression, Subcellular Localization and Expression Analysis of PcCHS Gene from Polygonatum cyrtonema Hua[J]. Biotechnology Bulletin, 2024, 40(5): 280-289.

Figures/Tables 8

Table 1 Primers used in the experiment

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Usage
PcCHS-F	ATGGGTTCCATTCCGGAGATG	基因克隆
PcCHS-R	TCACGGACAGCGGAGGAC	基因克隆
28-CHS-F	AATGGGTCGCGGATCCATGGGTTCCATTCCGGAGATGC	重组蛋白制备
28-CHS-R	CCGCAAGCTTGTCGACCGGACAGCGGAGGACGACAGTCTCC	重组蛋白制备
T7	TAATACGACTCACTATAGGG	菌落PCR
T7t	GCTAGTTATTGCTCAGCGG	菌落PCR
D-CHS-F	GGGGACAATGTTGTACAAAAAAGCAGGCTGCATGGGTTCCATTCCGGAGATG	入门载体构建
D-CHS-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCTCACGGACAGCGGAGGAC	入门载体构建
35s-CHS-F	CGGTACCCGGGGATCCATGGGTTCCATTCCGGAGATGC	过表达载体构建
35s-CHS-R	GGGCGAATTGGTCGACCGGACAGCGGAGGACGACAGTCTCC	过表达载体构建
qPCR-CHS	CAAGGTCACAAACAGCGAGC	qPCR
qPCR-CHS	TATGCCGCAATGGATGGGTT	qPCR
UBQ-10-F	GGACCCAGAAGTACGCAATG	qPCR（内参）
UBQ-10-R	AATTACCAGGGATACAGCACC	qPCR（内参）

Fig. 1 PcCHS cloning

Fig. 2 PcCHS evolutionary tree（A）, hydrophobicity（B）, phosphorylation site（C）and tertiary structure（D）

Fig. 3 Subcellular localization of PcCHS

Fig. 4 Colony PCR（A）, prokaryotic vector digestion verification（B）and overexpressed vector digestion verification（C）

Fig. 5 Protein induced by IPTG at different concentrations, protein purification and Western-blot identification A: Induced by different concentrations of IPTG（1 and 2 were 0 IPTG-induced precipitation and supernatant; 3 and 4 were 0.5 mmol/L IPTG-induced precipitation and supernatant; 5 and 6 were 1 mmol/L IPTG precipitating and supernatant）. B: Objective for protein purification（1 was precipitated by 0.5 mmol/L IPTG, 2 was supernatant induced by 0.5 mmol/L IPTG, 3 and 4 were penetrating fluid, 5-7 were impurity eluents, 8-14 were proteins eluted by 50 mmol/L eluent）. C: Expression of recombinant protein PcCHS identified by Western-blot

Fig. 6 Enzyme activity analysis in vitro A: Naringin chalcone standard peak at 350 nm. B: Control group peak, para-coumaryl CoA, malonyl CoA and product enzyme at 100℃ for 10 min. C: Experimental group peak, para-coumaryl CoA, malonyl CoA and product enzyme

Fig. 7 Transient overexpressions of DNA validation, PcCHS expression amount and total flavone content A: DNA verification. B: The amount of transient overexpression of PcCHS. C: Transient overexpressed total flavonoid content of PcCHS. * indicates significant difference at P<0.05 level

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