小麦烯醇化酶基因ENO2的可变翻译分析和原核表达

doi:10.13560/j.cnki.biotech.bull.1985.2023-1223

摘要/Abstract

摘要：

【目的】鉴定重要农作物小麦（Triticum aestivum L.）上参与糖酵解的烯醇化酶家族成员ENO2编码基因TaENO2，分析TaENO2的可变翻译特征及产物蛋白的烯醇化酶活性。【方法】采用生物信息学方法鉴定小麦基因组中的TaENO2基因；选择1个TaENO2为代表，以原生质体为受体通过外源表达方法分析该基因的可变翻译产物，通过原核表达和亲和层析过程纯化该基因的重组蛋白产物并测定其烯醇化酶活性。【结果】小麦的六倍体基因组中含有3个部分同源的TaENO2基因，3个基因的产物蛋白含有保守的烯醇化酶活性中心，编码烯醇化酶蛋白序列第94位甲硫氨酸残基（M94）的ATG密码子（ATG^M94）是潜在的内部可变翻译起始位点。在原生质体中，外源导入的TaENO2表达出定位于细胞质和细胞核的全长产物烯醇化酶和定位于细胞核的N端截短产物转录抑制因子TaMBP-1两种蛋白，而外源导入的突变ATG^M94后的TaENO2基因只表达全长产物烯醇化酶。获得了纯度较高的可溶性重组蛋白GST-TaENO2，该重组蛋白在体外能够催化由2-磷酸-甘油酸（2-PGA）向磷酸烯醇丙酮酸（PEP）转变的烯醇化酶反应。【结论】小麦基因组含有3个烯醇化酶ENO2编码基因TaENO2。在外源表达的情况下，TaENO2表现可变翻译特征，可分别从第一起始密码子和内部ATG^M94密码子处起始表达出全长形式的烯醇化酶蛋白和N端截短形式的TaMBP-1蛋白。原核表达的重组蛋白GST-TaENO2具有体外烯醇化酶活性。

关键词: 小麦, 烯醇化酶, ENO2, 可变翻译, 原核表达

Abstract:

【Objective】The objective of this study is to identify the genes（TaENO2s）encoding the enolase family member ENO2 in the important crop plant of wheat（Triticum aestivum L.）and to determine the alternative translation feature and protein enolase activity of TaENO2s.【Method】Wheat TaENO2s were identified by bioinformatics. One of the identified TaENO2s was selected as a representative for protoplast-based exogenous expression and characterization of alternative translation products. This selected TaENO2 was also subjected to prokaryotic expression for purification of recombinant protein. The enolase activity of the purified recombinant protein was determined with in vitro assays. 【Result】The hexaploid wheat genome had three homeologous TaENO2s which sequences encoded a conserved enolase catalytic center and had a putative alternative translation start site at the internal ATG codon encoding the residue M94（ATG^M94）of the enolase sequence. When exogenously expressed in protoplasts, the representative TaENO2 encoded two proteins, one full-length form of cytoplasmic and nuclear enolase protein and one N-terminal truncated form of nuclear transcriptional repressor TaMBP-1, while the variant of the same gene containing mutated ATG^M94only encoded the full-length form of enolase protein. Soluble recombinant GST-TaENO2 protein expressed in Escherichia coli was successfully purified and verified to have an in vitro enzymatic activity to catalyze the conversion from 2-phosphoglycerate（2-PGA）to phosphoenolpyruvate（PEP）. 【Conclusion】The wheat genome has three TaENO2 genes encoding the enolase protein ENO2. Under the condition of exogenous expression, TaENO2 encodes two protein products via mRNA alternative translation, one enolase protein translated from the first start codon and one TaMBP-1 protein translated from the internal start codon ATG^M94. The prokaryotically expressed recombinant protein GST-TaENO2 possesses in vitro enolase activity.

Key words: wheat, enolase, ENO2, alternative translation, prokaryotic expression

张娜, 刘梦楠, 屈展帆, 崔祎平, 倪嘉瑶, 王华忠. 小麦烯醇化酶基因ENO2的可变翻译分析和原核表达[J]. 生物技术通报, 2024, 40(5): 112-119.

ZHANG Na, LIU Meng-nan, QU Zhan-fan, CUI Yi-ping, NI Jia-yao, WANG Hua-zhong. Alternative Translation of Wheat Enolase-encoding Gene ENO2 and Its Prokaryotic Expression[J]. Biotechnology Bulletin, 2024, 40(5): 112-119.

图/表 5

表1 实验所用引物

Table 1 Primers used in this study

引物名称 Prime name	引物序列 Primer sequence（5'-3'）
ENO-m-F	AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC
ENO-m-R	GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC
Δ93-m-F	AAGTCCGGAGCTAGCTATGGTTCAGCAGCTTGATGGA
Δ93-m-R	GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC
M94A-F1	AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC
M94A-R1	AACAGCAAAGTTGTCGAGCTCAGTTTGAG
M94A-F2	GCTCGACAACTTTGCTGTTCAGCAGCTTGATGGAACCAAG
M94A-R2	GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC
ENO-GST-F	GATCTGGTTCCGCGTGGATCCATGGCGGCGACGATCCAGTC
ENO-GST-R	GTCACGATGCGGCCGCTCGAGGTATGGCTCCACCGGTGCAC

图1 小麦和拟南芥烯醇化酶蛋白的多序列比对 TaENO2-5A、TaENO2-5B和TaENO2-5D为3个部分同源的小麦烯醇化酶ENO2蛋白，AtENO1、AtENO2和AtENO3为拟南芥烯醇化酶家族的3个成员。红色实线框示活性中心的负责结合镁离子的丝氨酸残基（S），红色虚线框示ENO2序列中的第二个甲硫氨酸残基（M），编码该残基的ATG密码子是可变翻译内部起始密码子

Fig. 1 Protein sequence alignment of wheat and Arabidopsis enolases TaENO2-5A, TaENO2-5B, and TaENO2-5D are wheat enolases of homeologous ENO2 proteins, and AtENO1, AtENO2, and AtENO3 are Arabidopsis enolase proteins. Solid red box indicates the serine residue（S）responsible for binding of Mg2+ in the catalytic center. Dashed red box indicates the second methionine residue（M）in the ENO2 sequences. The ATG codon encoding this residue is an internal start codon of alternative translation

图2 TaENO2的可变翻译产物分析 A：3种（I、II和III）融合mCherry的TaENO2编码序列结构模式图；B：TaENO2可变翻译产物的亚细胞定位。将图A中所示的不同mCherry融合基因转化到原生质体中进行表达，对转化后24 h的原生质体进行细胞核的Hoechst 33342染色，随后在共聚焦显微镜下观察原生质体中红色荧光的亚细胞分布。图中比例尺为10 μm；C：Western杂交鉴定在原生质体中表达的TaENO2基因的可变翻译产物

Fig. 2 Alternative translation products of TaENO2 A: Schematic representation of the three forms（I, II, and III）of mCherry-fused TaENO2 coding sequences. B: Subcellular localization of the alternative translation products of TaENO2. Different mCherry fusion genes shown in（A）were respectively transformed into protoplasts for expression. The subcellular distribution of red fluorescence and the Hoechst 33342-stained nuclei in protoplasts were observed at 24 h after protoplast transformation. Scale bar indicates 10 μm. C: Western blot analysis on the alternative translation products of TaENO2 expressed in protoplasts

图3 GST-TaENO2重组蛋白的原核表达与纯化 A：GST-TaENO2重组蛋白的原核表达。将携带有GST-TaENO2基因的大肠杆菌BL21（DE3）菌株培养至对数生长期，然后在培养物中加入IPTG（+）诱导重组蛋白的表达至图中所示时间点，同时设置不加IPTG（-）对照培养物。煮沸裂解大肠杆菌培养物释放总蛋白，取等量的总蛋白进行SDS-PAGE分离。箭头示诱导表达的GST-TaENO2重组蛋白条带；B：原核表达重组蛋白的可溶性分析。对经IPTG诱导的大肠杆菌培养物进行超声破碎和可溶、不溶性成分的离心分离，对总蛋白、不溶性蛋白和可溶性蛋白进行电泳分离，箭头示GST-TaENO2重组蛋白条带；C：GST-TaENO2重组蛋白的纯化。M：蛋白分子量标准；1：未经IPTG诱导的大肠杆菌总蛋白；2：经IPTG诱导4 h的大肠杆菌总蛋白；3-6：亲和层析纯化过程中收集的流穿液（3）和连续单管洗脱液（4-6），箭头示重组蛋白条带；D：纯化重组蛋白的Western杂交鉴定

Fig. 3 Prokaryotic expression and purification of recombinant GST-TaENO2 protein A: Prokaryotic expression of recombinant GST-TaENO2 protein. The E. coli strain BL21（DE3）carrying the fusion gene GST-TaENO2 was grown to the log growth phase. The heterologous protein expression was then induced with IPTG for the indicated time periods. Cells were lysed by boiling and an equal aliquot of protein was resolved by SDS-PAGE. Arrows indicate the bands of recombinant protein. B: Solubility analysis on the prokaryotically expressed recombinant GST-TaENO2 protein. E. coli culture induced by IPTG was lysed by sonication. The soluble and insoluble protein fractions were separated by centrifugation and resolved by SDS-PAGE. Arrows indicate the bands of recombinant protein GST-TaENO2. C: Purification of recombinant GST-TaENO2 protein. M: Protein marker; 1: Total extracted protein from the E. coli culture which was not subjected to IPTG induction of gene expression; 2: Total extracted protein from the E.coli culture which had been subjected to IPTG induction of gene expression for 4 h; 3-6: Collected flow-through（3）and successive elution fractions（4-6）in the process of protein purification by affinity chromatography. D: Western blot analysis of the purified recombinant GST-TaENO2 protein. Arrows indicate the bands of recombinant protein

图4 重组蛋白GST-TaENO2的体外烯醇化酶活性分析烯醇化酶催化产物PEP在230 nm波长处有最大吸收峰。图中所示为体外烯醇化酶活性分析的反应液在230 nm 处的吸光值（OD230）随时间的变化趋势。红色虚线框示OD230的近似线性增长阶段，该阶段的数据用于计算酶活力

Fig. 4 In vitro assays on the enolase activity of the recombinant protein GST-TaENO2 The catalytic product of enolase, PEP, has maximum optical absorption at a wavelength of 230 nm. The figure shows the time-dependent changes in the optical density of the in vitro enolase assay solutions at 230 nm（OD230）. Red dash box indicates the near linear increase stage of OD230. The data of this stage were used for calculation of enolase activity

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