多花黄精查尔酮合酶PcCHS的原核表达、亚细胞定位及表达分析

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

摘要/Abstract

摘要：

【目的】探究查尔酮合酶（chalcone synthase, PcCHS）基因在多花黄精类黄酮合成中的作用，为后续解析PcCHS功能以及多花黄精新品种选育提供可靠的理论依据。【方法】以多花黄精为cDNA模板，克隆多花黄精PcCHS基因的编码序列，对该基因进行生物信息学分析。通过构建PcCHS的原核表达载体，纯化目的重组蛋白，验证该酶的体外表达活性。利用瞬时超表达体系探究该基因过表达后总黄酮的含量变化。利用Gateway技术构建亚细胞定位载体35S::PcCHS-GFP，通过本氏烟草表达系统确定目的蛋白亚细胞定位情况。【结果】PcCHS基因的开放阅读框为1 251 bp，理论分子量为44.63 kD，等电点为5.89，属于亲水蛋白，与石刁柏（Asparagus officinalis）CHS亲缘关系较近。原核表达实验表明，pET28a-PcCHS经IPTG（异丙基-β-D-硫代半乳糖苷）诱导表达可溶性重组蛋白，Western-blot显示大小约为45 kD，与预期大小一致，且纯化的目的蛋白具有一定的酶活性，能催化对香豆酰辅酶A和丙二酰辅酶A转化为柚皮素查尔酮。此外，PcCHS瞬时超表达中，PcCHS组的表达量显著高于空载K组，总黄酮含量也显著高于空载K组，最高可达1.83倍。亚细胞定位结果显示，该基因在细胞膜和细胞核中发挥作用。【结论】PcCHS基因原核表达的酶具有体外酶活性，其亚细胞定位于细胞膜和细胞核，且瞬时超表达能够显著提高多花黄精叶片总黄酮含量。

关键词: 多花黄精, 查尔酮合酶基因（CHS）, 原核表达, 瞬时超表达, 蛋白纯化, 体外酶活, 亚细胞定位

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

潘萍萍, 徐志浩, 张怡雯, 李青, 王忠华. 多花黄精查尔酮合酶PcCHS的原核表达、亚细胞定位及表达分析[J]. 生物技术通报, 2024, 40(5): 280-289.

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.

图/表 8

表1 实验所用引物

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）

图1 PcCHS克隆

Fig. 1 PcCHS cloning

图2 PcCHS进化树（A）、疏水性（B）、磷酸化位点（C）和三级结构（D）

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

图3 PcCHS的亚细胞定位

Fig. 3 Subcellular localization of PcCHS

图4 菌落PCR（A）、原核载体酶切验证（B）和过表达载体酶切验证（C）

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

图5 不同浓度IPTG诱导蛋白、蛋白纯化情况及Western-blot鉴定 A：不同浓度IPTG诱导（1、2为0 mmol/L IPTG诱导的沉淀和上清，3、4为0.5 mmol/L IPTG诱导的沉淀和上清，5、6为1 mmol/L IPTG的沉淀和上清）；B：目的蛋白纯化（1为0.5 mmol/L IPTG诱导的沉淀，2为0.5 mmol/L IPTG诱导的上清，3、4为穿透液，5-7为杂质洗脱液，8-14为50 mmol/L洗脱液洗脱的蛋白）；C：重组蛋白PcCHS表达的Western-blot鉴定

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

图6 体外酶活分析 A：350 nm下的柚皮素查尔酮标品峰图；B：对照组峰图，对香豆酰辅酶A、丙二酰辅酶A和100℃ 10 min纯化的PcCHS酶；C：实验组峰图，对香豆酰辅酶A、丙二酰辅酶A和纯化的PcCHS酶

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

图7 瞬时超表达的DNA验证、PcCHS表达量和总黄酮含量 A：DNA验证；B：PcCHS瞬时超表达的表达量；C：PcCHS瞬时超表达的总黄酮含量；*表示在P<0.05水平差异显著

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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