甘蔗ScGH3.8基因的克隆、表达及蛋白纯化

黄润; 张木清

doi:10.13417/j.gab.042.001311

甘蔗ScGH3.8基因的克隆、表达及蛋白纯化

黄润^1,2,
张木清^1,2, ,

1. 广西大学,亚热带农业生物资源保护与利用国家重点实验室,广西甘蔗生物学重点实验室;
2. 广西大学农学院

基金项目:

广西科技重大专项基金项目(桂科AA22117003)资助

详细信息

通讯作者:
张木清,zmuqing@163.com

中图分类号: S566.1
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出版历程
- 刊出日期: 2023-12-24

Cloning, Expression and Protein Purification of ScGH3.8 Gene from Sugarcane

HUANG Run^1,2,
ZHANG Mu-qing^1,2, ,

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University;
2. College of Agriculture, Guangxi University

摘要

摘要: Gretchen hagen 3(GH3)基因编码酰基酰胺合成酶，属于植物生长素早期响应基因，通过催化生长素和茉莉酸等激素与氨基酸的偶联反应，使激素失活或激活来维持植物体内激素的动态平衡，从而调节植物生长发育和抵抗逆境胁迫。本研究采用RT-PCR克隆甘蔗(Saccharum officinarum)GH3基因，获得长1 776 bp具有完整开放阅读框的基因序列，命名为ScGH3.8。生物信息学分析表明，ScGH3.8基因编码591个氨基酸，具有GH3高度保守结构域、无跨膜结构和信号肽，亚细胞定位于细胞质，属酸性蛋白。系统进化分析表明，ScGH3.8与南荻(Miscanthus lutarioriparius)、高粱(Sorghum bicolor)和玉米(Zea mays)的GH3进化关系最近，属于同一进化分支。逆境胁迫表达分析表明，ScGH3.8同源基因Sspon.03G0024250-1A、Sspon.03G0024250-2C、Sspon.03G0024250-3D、Sspon.03G0037250-1B受干旱和甘蔗花叶病毒胁迫诱导表达；Sspon.03G0024250-1A受甘蔗镰孢菌(Fusarium sacchari)胁迫诱导表达。通过构建pRSF-ScGH3.8原核表达载体，将其转入大肠杆菌BL21(DE3)菌株并诱导表达，发现重组蛋白最佳表达条件为37℃, 0.5 mmol/L的IPTG诱导2 h。利用亲和层析法对重组蛋白进行纯化获得ScGH3.8蛋白，相对分子质量为64.59 kDa。这些结果为进一步探究ScGH3.8基因在甘蔗发育和抗逆性中的功能提供理论参考和数据支持。
- 甘蔗 /
- 酰基酰胺合成酶基因(GH3) /
- 逆境胁迫 /
- 原核表达
Abstract: The Gretchen hagen 3（GH3） gene encodes acyl acid amido synthetases, which belongs to the early response gene of plant auxin, through catalyzing the coupling reaction of hormones such as indole-3-acetic acid（IAA） and jasmonic acid（JA） with amino acids. The hormone inactivates or activates to maintain the dynamic balance of hormone in plants, thereby regulating plant growth and development and resisting stress. In this study, the sugarcane ScGH3 gene was cloned by RT-PCR to obtain a 1 776 bp gene sequence with a complete open reading frame named ScGH3.8. Bioinformatics analysis showed that the ScGH3.8 gene encoded 591 amino acids, had a highly conserved domain of GH3 without transmembrane structure and signal peptide, localized in the cytoplasm, and belonged to acidic protein. Phylogenetic analysis showed that ScGH3.8 had the closest evolutionary relationship with GH3 of Miscanthus lutarioriparius, Sorghum bicolor, and Zea mays, belonging to the same evolutionary branch. The analysis of stress expression showed that the ScGH3.8 homologous genes of Sspon.03G0024250-1A, Sspon.03G0024250-2C, Sspon.03G0024250-3D, and Sspon.03G0037250-1B were induced by drought and sugarcane mosaic virus stress; Sspon.03G0024250-1A expressed in response to Fusarium sacchari. The prokaryotic expression vector pRSF-ScGH3.8 was constructed, introduced into Escherichia coli BL21（DE3）, and induced to express. The best expression condition of the recombinant protein was induced in 0.5 mmol/L IPTG at 37 ℃ for 2 h. The recombinant protein was purified by affinity chromatography to obtain ScGH3.8 protein with a relative molecular weight of 64.59 kDa. These results provide theoretical reference and data support for further exploring the function of the GH3 gene in sugarcane development and stress resistance.
- Sugarcane /
- Gene encoding acyl acid amido synthetases（GH3） /
- Stress of adversity /
- Prokaryotic expression

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参考文献(36)

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