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马缨杜鹃查尔酮异构酶(RdCHI1)重组蛋白的制备及功能验证

王聿晗 孙世宇 鞠志刚 孙威 徐小蓉

王聿晗,孙世宇,鞠志刚,等. 马缨杜鹃查尔酮异构酶(RdCHI1)重组蛋白的制备及功能验证 [J]. 福建农业学报,2023,38(5):574−582 doi: 10.19303/j.issn.1008-0384.2023.05.009
引用本文: 王聿晗,孙世宇,鞠志刚,等. 马缨杜鹃查尔酮异构酶(RdCHI1)重组蛋白的制备及功能验证 [J]. 福建农业学报,2023,38(5):574−582 doi: 10.19303/j.issn.1008-0384.2023.05.009
WANG Y H, SUN S Y, JU Z G, et al. Preparation and Function Verification of Recombinant Protein of Chalcone Isomerase Gene from Rhododendron Delavayi [J]. Fujian Journal of Agricultural Sciences,2023,38(5):574−582 doi: 10.19303/j.issn.1008-0384.2023.05.009
Citation: WANG Y H, SUN S Y, JU Z G, et al. Preparation and Function Verification of Recombinant Protein of Chalcone Isomerase Gene from Rhododendron Delavayi [J]. Fujian Journal of Agricultural Sciences,2023,38(5):574−582 doi: 10.19303/j.issn.1008-0384.2023.05.009

马缨杜鹃查尔酮异构酶(RdCHI1)重组蛋白的制备及功能验证

doi: 10.19303/j.issn.1008-0384.2023.05.009
基金项目: 国家自然科学基金项目(31760076);贵州省教育厅特色领域项目(黔教合KY字〔2021〕059);贵州省自然科学基金项目(ZK〔2023〕270);贵州省科技支撑计划项目(黔科合支撑〔2020〕4Y028号);贵州省高等学校高山杜鹃病虫害绿色防控重点实验室项目(黔教技〔2022〕044号)
详细信息
    作者简介:

    王聿晗(2002−),女,主要从事植物细胞与分子生物学研究(E-mail:wyhdyx422@163.com

    通讯作者:

    孙威(1985−),女,教授,博士,主要从事植物次生代谢与调控研究(E-mail:sunwei889@163.com

    徐小蓉(1977−),女,教授,博士,主要从事植物生理生态学、修复生态学研究(E-mail:512742911@qq.com

  • 中图分类号: Q558

Preparation and Function Verification of Recombinant Protein of Chalcone Isomerase Gene from Rhododendron Delavayi

  • 摘要:   目的  制备马缨杜鹃(Rhododendron delavayi)查尔酮异构酶(Chalcone isomerase,CHI)基因表达的重组蛋白并验证其活性,为解析查尔酮异构酶功能提供理论依据,为改良植物花色、增加药用成分奠定基础。  方法  根据所获得的马缨杜鹃查尔酮异构酶RdCHI1基因的序列信息设计引物,构建其原核表达载体,优化RdCHI1可溶性重组蛋白最佳诱导表达条件,制备可溶性重组蛋白并检测其活性。  结果  成功构建RdCHI1原核表达载体,RdCHI1重组蛋白可在上清中表达,最佳诱导条件为:15 ℃、36 h,IPTG浓度0.35 mmol·L−1。经镍柱纯化得到质量较好的RdCHI1重组蛋白,通过体外酶活反应确定,与对照组相比,RdCHI1可以更快地催化柚皮素查尔酮(Naringenin chalcone)反应生成柚皮素(Naringenin)。  结论  RdCHI1为I型CHI,可极大提高柚皮素查尔酮生成柚皮素的速率,增加黄酮类物质积累量。
  • 图  1  酶切位点的引入

    M为Marker;A中,1为RdCHI1大量PCR;B中,1为RdCHI1胶回收;C中,1~2为pMD18-T-RdCHI1菌液PCR。D中,1为pMD18-T-RdCHI1质粒,2为pMD18-T-RdCHI1酶切。

    Figure  1.  Introduction of cleavage sites

    M: Marker; A,1: PCR product of RdCHI1. B, 1: Gel recovery of RdCHI1. C, 1 and 2: pMD18-T-RdCHI1 bacterial solution PCR. D, 1: pMD18-T-RdCHI1 plasmid; 2: Products of pMD18-T-RdCHI1 digestion.

    图  2  RdCHI1原核表达载体的构建及验证

    M为Marker;A中,1为pMD18-T-RdCHI1大量酶切;B中,1为pET32a大量酶切;C中,1~2为pET32a-RdCHI1菌液PCR;D中,1为pET32a-RdCHI1质粒,2为pET32a-RdCHI1酶切产物。

    Figure  2.  Construction and verification of RdCHI1 prokaryotic expression vector

    M: Marker; A, 1: Mass enzyme digestion of pMD18-T-RdCHI1. B, 1: Mass enzyme digestion of pET32a. C, 1 and 2: pET32a-RdCHI1 liquid PCR. D, 1: pET32a-RdCHI1 plasmid; 2: Products of pET32a-RdCHI1 digestion.

    图  3  BL21-pET32a-RdCHI1重组蛋白诱导表达

    M:蛋白分子量Marker;1:BL21(加IPTG诱导);2:pET32a空载体(加IPTG诱导);3:pET32a-RdCHI1(未加IPTG诱导);4:pET32a-RdCHI1全菌(加IPTG诱导)。

    Figure  3.  Induced expression of recombinant BL21-pET32a-RdCHI1

    M: Protein molecular weight marker; 1: BL21 (with IPTG induction); 2: pET32a empty vector (with IPTG induction); 3: pET32a-RdCHI1 (without IPTG induction); 4: pET32a-RdCHI1 whole bacteria (with IPTG induction).

    图  4  免疫印迹验证

    M:蛋白分子量Marker;1:BL21(加IPTG诱导);2:pET32a空载体(加IPTG诱导);3:pET32a-RdCHI1(未加IPTG诱导);4:pET32a-RdCHI1全菌(加IPTG诱导)。

    Figure  4.  Function verification of recombinant protein by western blot

    M: Protein molecular weight marker; 1: BL21 (with IPTG induction); 2: pET32a empty vector (with IPTG induction); 3: pET32a-RdCHI1 (without IPTG induction); 4: pET32a-RdCHI1 whole bacteria (with IPTG induction).

    图  5  15 ℃ 下不同培养时间可溶性重组蛋白的诱导表达

    M:蛋白分子量Marker;1:BL21(加IPTG诱导);2:pET32a空载体(加IPTG诱导);3:pET32a-RdCHI1(未加IPTG诱导); 4~8:pET32a-RdCHI1(24、36、48、60、72 h加IPTG诱导)。

    Figure  5.  Induced expression of soluble recombinant protein at 15 ℃ under varied culture time

    M: Protein molecular weight marker; 1: BL21 (with IPTG induction); 2: pET32a empty vector (with IPTG induction); 3: pET32a-RdCHI1 (without IPTG induction); 4~8: pET32a-RdCHI1 (24, 36, 48, 60, 72 h with IPTG induction).

    图  6  15 ℃ 下不同IPTG浓度可溶性重组蛋白的诱导表达

    M:蛋白分子量Marker;1:pET32a空载体(加IPTG诱导);2:pET32a-RdCHI1菌(未加IPTG诱导);3~8:pET32a-RdCHI1菌(IPTG浓度为0.05、0.15、0.25、0.35、0.50、0.65 mmol·L−1)。

    Figure  6.  Induced expression of soluble recombinant protein at 15 ℃ under varied IPTG concentrations

    M: Protein molecular weight marker; 1: pET32a empty vector (with IPTG induction); 2. pET32a-RdCHI1 (without IPTG induction); 3-8: pET32a-RdCHI1 (IPTG concentrations of 0.05, 0.15, 0.25, 0.35, 0.50, and 0.65 mmol·L−1).

    图  7  RdCHI1重组蛋白的梯度洗脱

    M:蛋白分子量Marker;A中,1:pET32a-RdCHI1上清蛋白,2:上Ni柱后溶液,3:10 mmol·L−1咪唑洗脱,4:20 mmol·L−1 咪唑洗脱,5~8:50 mmol·L−1咪唑洗脱。B中,1:50 mmol·L−1咪唑洗脱,2~6:100 mmol·L−1咪唑洗脱,7~8:200 mmol·L−1咪唑洗脱。C中,1~3:200 mmol·L−1咪唑洗脱,4~8:500 mmol·L−1咪唑浓度洗脱。

    Figure  7.  Gradient elution of RdCHI1 recombinant protein

    M: Protein molecular weight marker; A. 1: pET32a-RdCHI1 supernatant protein, 2: solution after Ni column, 3: 10 mmol·L−1 imidazole elution, 4: 20 mmol·L−1 imidazole elution, 5–8: 50 mmol·L−1 imidazole elution; B. 1:50 mmol·L−1 imidazole elution, 2–6: 10 mmol·L−1 imidazole elution, 7 and 8: 20 mmol·L−1 imidazole elution; C. 1–3: 200 mmol·L−1 imidazole elution, 4–8: 500 mmol·L−1 imidazole elution.

    图  8  目的蛋白的纯化与浓缩

    M:蛋白分子量Marker;1:pET32a空载体菌(加IPTG诱导); 2:pET32a-RdCHI1菌(未加IPTG诱导);3:pET32a-RdCHI1菌(加IPTG诱导);4:RdCHI1纯化蛋白。

    Figure  8.  Purification and concentration of target protein

    M: Protein molecular weight marker; 1: pET32a empty carrier bacteria (with IPTG induction); 2: pET32a-RdCHI1 (without IPTG induction); 3: pET32a-RdCHI1 (with IPTG induction); 4: RdCHI1 purified protein.

    图  9  RdCHI1体外酶活检测

    Figure  9.  In vitro enzymatic assay on recombinant RdCHI1

    表  1  马缨杜鹃原核表达载体构建所用引物

    Table  1.   Primers used in constructing prokaryotic expression vector of R. delavayi

    引物名称 Primer name引物序列(5′-3′)
    Primer sequence(5′-3′)
    RdCHI1(F)CGGAATTCATGTCTTCACCACTGGCG
    RdCHI1(R)CCCAAGCTTTTATGTCTCCTTGAATAA
    下载: 导出CSV
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  • 收稿日期:  2022-09-14
  • 录用日期:  2022-09-14
  • 修回日期:  2023-03-10
  • 网络出版日期:  2023-05-24
  • 刊出日期:  2023-05-28

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