藏药川西獐牙菜中SmMK基因的克隆及生物信息分析
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摘要
目的了解川西獐牙菜Swertia mussotii Franch甲羟戊酸途径中的关键酶甲羟戊酸激酶基因(MK)的功能,并进一步推进川西獐牙菜中的甲羟戊酸途径的研究。方法根据川西獐牙菜的转录组信息,获得了川西獐牙菜MK(SmMK)基因的全长c DNA序列,并且设计了特异性引物,利用RT-PCR对该基因进行了克隆;利用生物信息学方法,对Sm MK基因的序列进行分析;构建原核表达载体MBP-SmMK,转入大肠杆菌Rosetta(DE3)中,然后对Sm MK基因进行了原核表达,并对其进行了纯化。结果 Sm MK基因cDNA全长为1 164 bp,共编码387个氨基酸。SmMK与其他植物中的MK具有较高相似性。对其信号肽、跨膜区域、蛋白定位及二级结构和三维构象进行了预测分析。SDS-PAGE检测表明所纯化蛋白与预期蛋白的大小相一致,为40970。结论为进一步研究川西獐牙菜中MK的功能奠定了基础,并为进一步研究川西獐牙菜中的甲羟戊酸途径,提高川西獐牙菜中类异戊二烯化合物的产量提供了依据。
Objective In order to identify the function of the mevalonate kinase(MK) which is a key enzyme of the mevalonate pathway(MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta(DE3) for expression. Results The results showed that SmMK cDNA complete sequences had a length of1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.
Objective In order to identify the function of the mevalonate kinase(MK) which is a key enzyme of the mevalonate pathway(MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta(DE3) for expression. Results The results showed that SmMK cDNA complete sequences had a length of1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.
引文
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[11]王鹏杰,陈丹,曹红利,等.茶树甲羟戊酸激酶基因Cs MVK克隆及表达特性分析[J].南方农业学报,2017,48(10):219-223.
[12]赵乐,马利刚,俎梦航,等.独行菜LaMK基因克隆、生物学信息分析及原核表达[J].中国药学杂志,2017,52(11):924-930.
[13]张晓东,李彩霞,赵静,等.滇龙胆甲羟戊酸激酶基因的克隆与表达分析[J].贵州农业科学,2015,43(12):196-202.
[14]乌云塔娜,王淋,叶生晶.杜仲甲羟戊酸激酶(Eu MK)基因鉴定及生物信息学分析[J].经济林研究,2014,32(1):6-12.
[15]Xiang B,Li X,Wang Y,et al.Cloning and characterization of two iridoid synthase homologs from Swertia mussotii[J].Molecules,2017,22(8):1387-1391.
[16]陈桂琛,卢学峰,孙菁,等.藏药川西獐牙菜的引种栽培研究[J].安徽农业科学,2005,33(2):272-273.
[17]赵纪峰,刘翔,王昌华,等.珍稀濒危药用植物川西獐牙菜的资源调查[J].世界科学技术-中医药现代化,2014,16(4):845-850.
[18]Simkin A J,Guirimand G,Papon N,et al.Peroxisomal localisation of the final steps of the mevalonic acid pathway in plant[J].Planta,2011,234(5):903-914.
[2]宇妥·元丹贡布,马世林.四部医典[M].上海:上海科学技术出版社,1987.
[3]孟宪华,陈德道,张樱山,等.川西獐牙菜的化学成分、药理作用和临床应用研究进展[J].现代药物与临床,2012,27(2):176-179.
[4]井灵,张玉娟,陈志.川西獐牙菜的化学成分及药理研究进展[J].青海农林科技,2014(4):55-57.
[5]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.
[6]王秋军,张犇,王剑文.药用植物类异戊二烯代谢途径及其活性产物合成调控研究进展[J].植物学研究,2012,1(2):23-29.
[7]Liao P,Hemmerlin A,Bach T J,et al.The potential of the mevalonate pathway for enhanced isoprenoid production[J].Biotechnol Adv,2016,34(5):697-713.
[8]郭溆,罗红梅,陈士林.三七甲羟戊酸激酶Pn MVK1基因的克隆和生物信息学分析[J].药学学报,2012,47(8):1092-1097.
[9]Riou C,Tourte Y,Lacroute F,et al.Isolation and characterization of a cDNA encoding Arabidopsis thaliana mevalonate kinase by genetic complementation in yeast[J].Gene,1994,148(2):293-297.
[10]王宝莲.小麦甲羟戊酸激酶基因克隆及功能分析[D].曲阜:曲阜师范大学,2011.
[11]王鹏杰,陈丹,曹红利,等.茶树甲羟戊酸激酶基因Cs MVK克隆及表达特性分析[J].南方农业学报,2017,48(10):219-223.
[12]赵乐,马利刚,俎梦航,等.独行菜LaMK基因克隆、生物学信息分析及原核表达[J].中国药学杂志,2017,52(11):924-930.
[13]张晓东,李彩霞,赵静,等.滇龙胆甲羟戊酸激酶基因的克隆与表达分析[J].贵州农业科学,2015,43(12):196-202.
[14]乌云塔娜,王淋,叶生晶.杜仲甲羟戊酸激酶(Eu MK)基因鉴定及生物信息学分析[J].经济林研究,2014,32(1):6-12.
[15]Xiang B,Li X,Wang Y,et al.Cloning and characterization of two iridoid synthase homologs from Swertia mussotii[J].Molecules,2017,22(8):1387-1391.
[16]陈桂琛,卢学峰,孙菁,等.藏药川西獐牙菜的引种栽培研究[J].安徽农业科学,2005,33(2):272-273.
[17]赵纪峰,刘翔,王昌华,等.珍稀濒危药用植物川西獐牙菜的资源调查[J].世界科学技术-中医药现代化,2014,16(4):845-850.
[18]Simkin A J,Guirimand G,Papon N,et al.Peroxisomal localisation of the final steps of the mevalonic acid pathway in plant[J].Planta,2011,234(5):903-914.