首页|暗紫贝母甲羟戊酸激酶基因的重组表达与定点突变研究

暗紫贝母甲羟戊酸激酶基因的重组表达与定点突变研究

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目的 研究暗紫贝母Fritillaria unibracteata甲羟戊酸激酶(FUMK)基因的重组表达与定点突变.方法 基于暗紫贝母转录组中FUMK基因的ORF序列,全化学合成FUMK-pET28a重组质粒,在大肠杆菌BL21中表达其重组蛋白,纯化后检测其酶活性.利用定点突变验证关键氨基酸.结果 FUMK基因ORF全长为1158 bp,编码一条长度为385个氨基酸的多肽.FUMK与卷叶贝母的甲羟戊酸激酶(MK)同源性最高,序列相似性可达77.14％.FUMK与百合科贝母属植物的MK聚类形成单系群,具有最近的亲缘关系.在大肠杆菌BL21中成功表达出FUMK重组蛋白,纯化后的重组蛋白能够催化腺嘌呤核苷三磷酸(ATP)与甲羟戊酸反应,形成甲羟戊酸磷酸.FUMK对ATP的酶促反应最大速度(Vmax)与米氏常数(Km)值分别为1.72 μmol·min-1·mg-1、15.84 μmol·L-1;对甲羟戊酸的Vmax与Km值分别为0.71 μmol·min-1·mg-1、28.58 µmol·L-1.His198Ala和Ser137Ala两种突变蛋白的酶促活性分别显著减少49.08％、23.91％,表明其是FUMK行使催化功能的关键氨基酸残基.结论 成功鉴定了FUMK基因,并验证了His198与Ser137两个关键氨基酸残基,为研究其基因在暗紫贝母甾体类生物碱生物合成途径中的生物学作用奠定了理论基础.
Study on recombinant expression and site-specific mutation of mevalerate kinase gene in Fritillaria unibracteata
OBJECTIVE To study the recombinant expression and site-directed mutagenesis of the mevalonate kinase(MK)(FUMK)gene of Fritillaria unibracteata(FU).METHODS Based on the ORF sequence of the FUMK gene in the transcriptome of FU,a recombinant plasmid FUMK-pET28a was chemically synthesized,and its recombinant protein was expressed in E.coli BL21.The enzymatic activity of the purified FUMK protein was then evaluated.Finally,site-directed mutagenesis was used to validate key amino acids.RESULTS The full-length of the ORF of the FUMK gene is 1158 bp,encoding a polypeptide with a length of 385 amino acids.FUMK had the highest homology with the Fritillaria cirrhosa MK with sequence identity of 77.14％.Phylogenetic results showed that FUMK was closely clustered with those of Fritillaria species,indicating that these species had the close relation ship.The recombinant FUMK protein was successfully expressed in E coli BL21,and the purified recombinant protein could catalyze ATP and mevalonic acid(MVA)to form mevalonic acid phosphoric acid,and the maximum Vmax and Km values of FUMK for ATP and MVA were 1.72 μmol·min-1·mg-1,15.84 µmol·L-1 and 0.71 pmol·min-1·mg-1,28.58 μmol·L-1,respectively.The His198Ala and Ser137Ala mutants decreased by 49.08％and 23.91％,respectively,indicating that both were key residues involved in the catalytic process of FUMK.CONCLUSION In this paper,the FUMK gene was successfully identified.His198 and Ser137 were verified as key residues.These results provides a theoretical basis for studying the biological role of the FUMK gene in the biosynthetic pathway of steroidal alkaloids in FU.

Fritillaria unibracteata P.K.Hsiao & K.C.HsiaMevalonate kinaseBioinformaticsRecombinat expressionSite-directed mutationSequence alignmentCatalytic reactionReaction rate

黄德娅、邹萌、黄斌翰、周嘉裕、廖海

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西南交通大学生命科学与工程学院,四川成都 610000

暗紫贝母 甲羟戊酸激酶 生物信息学分析 重组表达 定点突变 序列比对 催化反应 反应速率

四川省科技项目四川省科技项目四川省中医药管理局面上项目成都市科技局项目中央高校医工结合项目

2018SZ00612021ZHFP01702021MS1162022-YF05-01357-SN2682022ZTPY053

2024

10.13375/j.cnki.wcjps.2024.01.010

华西药学杂志
四川大学,四川省药学会

华西药学杂志

CSTPCD北大核心
影响因子：0.624
ISSN：1006-0103
年,卷(期)：2024.39(1)
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