橡胶树HbbZIP74基因的表达分析及蛋白原核表达纯化

Expression analysis and prokaryotic expression purification of HbbZIP74 gene in rubber tree

摘要

为研究巴西橡胶树（Hevea brasiliensis）bZIP 转录因子在天然橡胶生物合成中的作用，本研究在实验室前期工作基础上，从橡胶树中克隆了 1 个 bZIP 类转录因子 HbbZIP74 基因，并对其进行了生物信息学分析和表达分析，成功构建 pET28-HbbZIP74 原核表达载体，在大肠杆菌（Escherichia coli） BL21（DE3）菌株中进行异源表达并对重组蛋白进行纯化。结果表明，HbbZIP74 基因开放读码框（ORF）为 486 bp，编码 161 个氨基酸，含有 bZIP 结构域，属于 bZIP 类转录因子 S 亚家族。HbbZIP74 在胶乳和叶片中表达量较高，茉莉酸、脱落酸、乙烯和水杨酸可诱导胶乳中 HbbZIP74 表达。HbbZIP74-His 重组蛋白异源表达最佳条件为 1 mmol·L⁻¹ IPTG, 37 ℃ 诱导 3 h，但主要存在于包涵体中。通过 Ni-NTA 亲和层析柱纯化获得大小约 22 kDa 的重组蛋白，与预期结果一致。该研究为进一步探究 HbbZIP74 在天然橡胶生物合成中的功能奠定了基础。

Abstract

In order to assess the role of the bZIP transcription factor in the biosynthesis of natural rubber in Hevea brasiliensis, HbbZIP74 gene was successfully cloned based on previous work in our laboratory. Bioinformatics analysis and expression pattern analysis were performed. The pET28-HbbZIP74 expression vector was constructed, and the recombinant protein was expressed in the strain Escherichia coli BL21 (DE3) and then purified. The results showed that the open reading frame (ORF) of the geneHbbZIP74 is 486 bp, encoding 161 amino acids, with a bZIP domain, classifying it as a bZIP transcription factor S subgroup. The expression level of HbbZIP74 is higher in latex and leaves, and its expression can be induced by jasmonic acid, abscisic acid, ethylene, and salicylic acid in latex. The optimal condition for the heterologous expression of the HbbZIP74-His recombinant protein was induced at 1 mmol·L⁻¹ IPTG and 37 ℃ for 3 h. The recombinant protein was mainly accumulated in inclusion bodies. The recombinant protein, approximately 22 kDa in size, was purified using Ni-NTA affinity chromatography, which was consistent with expectations. This study lays the foundation for further exploration of the role of HbbZIP74 in the biosynthesis of natural rubber.

关键词

橡胶树 / bZIP 转录因子 / 基因克隆 / 基因表达 / 蛋白纯化

Key words

Hevea brasiliensis / bZIP transcription factor / gene cloning / gene expression / protein purification

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基金资助

国家自然科学基金面上基金项目(32471926)

国家自然科学基金面上基金项目(31970620)

热带作物生物育种全国重点实验室科研项目(NKLTCBZRJJ6)

中央级公益性科研院所基本科研业务费专项(1630052022-009)