Preparation and Function Verification of Recombinant Protein of Chalcone Isomerase Gene from Rhododendron Delavayi
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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。 Abstract:Objective Recombinant protein of chalcone isomerase (CHI) gene in Rhododendron delavayi was prepared and its activity was verified. This will provide theoretical basis for analyzing the function of CHI, and also lays a foundation for improving flower color and increasing medicinal ingredientst. Method Primers were designed based on the sequence of RdCHI1, and the prokaryotic expression vector constructed. Conditions to induce the expression of soluble recombinant protein were optimized. Activity of the prepared protein was verified by an in vitro enzymatic assay. Result The successfully constructed RdCHI1 prokaryotic expression vector was expressed in the supernatant under the optimized induction that applied 0.35 mmol·L−1 IPTG at 15 ℃ for 36 h. The nickel column-purified recombinant protein significantly hastened the conversion of naringin chalcone to naringin in an in vitro assay. Conclusion As a type I CHI, RdCHI1 significantly accelerated the convertion from naringin chalcone to naringin and increase the accumulation od flavonoids. -
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.
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 
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