茉莉花<i>JsMYB</i>305转录因子的原核表达及蛋白纯化

万超; 张月; 胡莉; 伍炳华; 袁媛

doi:10.19303/j.issn.1008-0384.2022.002.005

茉莉花JsMYB305转录因子的原核表达及蛋白纯化

doi: 10.19303/j.issn.1008-0384.2022.002.005

福建农林大学园艺学院，福建　福州　350002

基金项目: 国家自然科学基金项目（31902050）；福建农林大学科技创新专项基金项目（CXZX2020025A）

详细信息

作者简介:
万超（1995−），男，硕士研究生，研究方向：花卉香气合成和代谢调控（E-mail：wanc1356@163.com）

通讯作者:
袁媛（1988−），女，博士，研究方向：观赏植物花香和花色相关次生代谢（E-mail：yuanyuan@fafu.edu.cn）

中图分类号: S 685.16
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出版历程
- 收稿日期: 2021-10-21
- 修回日期: 2022-01-11
- 网络出版日期: 2022-02-07
- 刊出日期: 2022-02-25

Prokaryotic Expression and Purification of JsMYB305 Transcription Factor in Jasminum sambac

College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的获得茉莉花香气调控转录因子JsMYB305的重组蛋白，为深入研究JsMYB305调控茉莉萜类香气代谢的分子机理及筛选其他互作蛋白提供基础。方法通过酶切连接的方式，将JsMYB305的编码序列构建到原核表达载体pGEX-4T-1，转化BL21（DE3）表达菌株，通过IPTG诱导蛋白表达、GST亲和树脂分离纯化，最后Western Blot鉴定重组蛋白。结果重组蛋白的诱导条件为0.2 mmol·L⁻¹ IPTG，最适宜纯化的温度和时间为28 ℃诱导4 h，经20 mmol·L⁻¹ GSH洗脱的蛋白纯度较好，Western Blot结果表明重组蛋白的大小正确。结论成功获得了JsMYB305重组蛋白，为后期利用GST-pull down技术筛选互作蛋白及EMSA研究JsMYB305对特定启动子位点的结合提供基础。
- 茉莉花 /
- MYB转录因子 /
- 原核表达 /
- 蛋白纯化
Abstract: Objective The recombinant protein of fragrance-regulating transcription factor JsMYB305 was obtained in preparation for studies on the molecular mechanism of terpenoid-regulation and interacting proteins of the gene in Jasminum sambac. Methods Coding sequence of JsMYB305 was constructed into prokaryotic expression vector pGEX-4T-1 by enzyme digestion and ligation, then, transformed into the BL21 (DE3) expressing strain. The protein expression was induced by IPTG, and the recombinant protein separated and purified using GST affinity resin followed by western blot for positive identification. Result After induction by 0.2 mmol·L⁻¹ IPTG, the recombinant protein was purified at the optimized temperature of 28 ℃ for 4 h and eluted with 20 mmol·L⁻¹ GSH for purification. The western blot confirmed the protein weight to be as expected. Conclusion The recombinant protein of JsMYB305 was successfully obtained to pave the way for further studies on the search for the interacting proteins by GST-pull down technique and the binding of the gene to specific promoter sites by EMSA.
- Jasminum sambac /
- MYB transcription factor /
- prokaryotic expression /
- protein purification

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图 1 茉莉JsMYB305基因编码序列的扩增、酶切回收及菌液PCR鉴定

注：M为DL2000 Marker ；A为茉莉JsMYB305基因编码序列的扩增，其中2为JsMYB305基因；B为JsMYB305基因扩增产物酶切回收后的产物，其中5为目的基因，1、3、4为其他基因条带；C为重组载体pGEX-4T-1-JsMYB305的菌液PCR鉴定，1～5为阳性条带。

Figure 1. Amplification of JsMYB305 coding sequence, recovery of PCR product after enzyme digestion, and PCR identification of bacteria

Note: M: DL 2000 marker; A: amplification of JsMYB305 encoding sequence, 2: JsMYB305; B: amplification product of JsMYB305 after enzyme digestion and recovery; 5: JsMYB305; 1, 3, and 4: bands of other genes; C: identification of recombinant vector pGEX-4T-1-JsMYB305; 1-5: positive bands.

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图 2 JsMYB305重组蛋白的诱导表达及可溶性检测

注：M：Protein ladder。A：JsMYB305重组蛋白的诱导表达，1：未诱导的菌体蛋白，2：0.2 mmol·L⁻¹ IPTG诱导后的菌体蛋白。B：37 ℃诱导重组蛋白表达，1：未诱导的菌体蛋白；2、3、4分别为37 ℃ 0.2 mmol·L⁻¹ IPTG诱导的菌体蛋白、上清、沉淀。C：28 ℃诱导重组蛋白表达，5：未诱导的菌体蛋白，6、7、8分别为28 ℃ 0.2 mmol·L⁻¹ IPTG诱导的菌体蛋白、上清、沉淀。

Figure 2. Inducible expression and soluble detection of JsMYB305 recombinant protein

Note: M: protein ladder; A: induced expression of recombinant protein of JsMYB305; 1: uninduced bacterial protein; 2: bacterial protein induced by 0.2 mmol·L⁻¹ IPTG; B: recombinant protein expression induced at 37 ℃; 1: uninduced bacterial protein; 2, 3 and 4: bacterial protein, supernatant, and precipitate, respectively, induced by 0.2 mmol·L⁻¹ IPTG at 37 ℃; C: recombinant protein expression induced at 28 ℃; 5: uninduced bacterial protein; 6, 7, and 8: bacterial protein, supernatant, and precipitate, respectively, induced by 0.2 mmol·L⁻¹ IPTG at 28 ℃.

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图 3 重组蛋白的纯化及Western Blot 检测

注：M：protein ladder 。A：重组蛋白的纯化，1：未诱导的菌体蛋白；2：0.2 mmol·L⁻¹ IPTG诱导后的菌体蛋白；3：超声破碎后的菌体蛋白上清；4：流出液；5：洗涤液；6：10 mmol·L⁻¹ GSH洗脱液；7：20 mmol·L⁻¹ GSH洗脱液；8：30 mmol·L⁻¹ GSH洗脱液；箭头标注的是纯化的重组蛋白。B：纯化蛋白的Western Blot 检测，箭头标注的是JsMYB305重组蛋白与GST单克隆抗体杂交后的条带。

Figure 3. Purification and western blot identification of recombinant protein

Note: M: 15-180 kDa protein ladder; A: purification of recombinant protein; 1: uninduced bacterial protein; 2: bacterial protein induced by 0.2 mmol·L⁻¹ IPTG; 3: supernatant of bacterial protein after ultrasonic crushing; 4: effluent; 5: washing liquid; 6: eluent of 10 mmol·L⁻¹ GSH; 7: eluent of 20 mmol·L⁻¹ GSH; 8: eluent of 30 mmol·L⁻¹ GSH; arrow points at purified recombinant protein; B: detection of purified protein by western blot; arrow points at band after hybridization of JsMYB305 recombinant protein with GST monoclonal antibody.

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表 1 构建原核表达载体时扩增JsMYB305编码序列的引物序列

Table 1. Primer sequences for amplifying JsMYB305 coding sequence in prokaryotic expression vector construction

引物名称 Primer name	引物序列 Primer sequence（5′-3′ ）
JsMYB305-F	gagagaGGA TCCATGGACAAGAAA ATATGCAAT AGCTCTC
JsMYB305-R	gagagaGTCGACTTAATCCCCATTAAGTAA CTGGATG
注：上游引物中划线部分为Bam H I酶切位点，下游引物中划线部分为Sal I酶切位点。 Note: The underlined sequence in the forward primer is the Bam H I restriction site, in the reverse primer is the Sal I restriction site.

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