羟甲基戊二酰辅酶A合酶(3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS)是甲羟戊酸途径(MVA)中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进...羟甲基戊二酰辅酶A合酶(3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS)是甲羟戊酸途径(MVA)中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进行生物信息学分析,通过荧光定量PCR的方法分析其组织表达特性。IrHMGS基因cDNA全长1 382bp,编码460个氨基酸,序列分析结果表明该基因编码的氨基酸序列含有羟甲基戊二酰辅酶A合成酶N末端、C末端等保守结构域。荧光定量PCR表明,IrHMGS在冬凌草组培苗叶和根中的表达量显著高于在组培苗花、茎和愈伤组织中的表达量。本研究为后续深入研究IrHMGS在冬凌草二萜类物质合成途径中的功能奠定了基础。展开更多
3-Hydroxy-3-methylglutaryl-CoA synthase(HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target frag...3-Hydroxy-3-methylglutaryl-CoA synthase(HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the p H7 WG2 D and p K7 GWIWG2 D vectors to respectively overexpress and silence, Tw HMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to70% in the expression level compared to cells with empty vector controls. During Tw HMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7 v2 was significantly upregulated to the control. In the RNAi group, the expression of Tw HMGR,TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of Tw HMGS determined the production of triptolide in T.wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-Co A was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.展开更多
Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase（HMGS） and 3-hydroxy-3-methylglutaryl coenzyme-A reductase（HMGR） genes from Panax notoginseng of four-year old during the flowering peri...Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase（HMGS） and 3-hydroxy-3-methylglutaryl coenzyme-A reductase（HMGR） genes from Panax notoginseng of four-year old during the flowering period, the key genes involved in the mevalonic acid pathway for saponin biosynthesis. Methods The cDNA sequences of PnHMGS1 and PnHMGR2 were obtained by reverse transcription PCR（RT-PCR） and rapid amplification of cDNA ends（RACE） methods and were analyzed in their secondary structures, subcellular localizations, domains, and the three-dimensional structures of putative proteins by the bioinformatics tools. Fusion genes were constructed by the prokaryotic expression system. Results The two genes were cloned, named as PnHMGS1 and PnHMGR2, respectively, and were both predicted to be located in the chloroplast. PnHMGS1（1410 bp） encoded a predictive unstable protein with 469 amino acids and covered hydroxymethylglutaryl-Co A synthase domain. PnHMGR2（1690 bp） also encoded an unstable protein with 589 amino acids and possessed a hydroxymethylglutaryl-coenzyme A reductase domain and two transmembrane regions. Both of the genes were expressed most in flowers followed by roots, stems, and least in leaves. Conclusion PnHMGS1 and PnHMGR2 are firstly cloned from P.notoginseng as the new member of the HMGR family,and they show the same expression profile as P.ginseng and P.quinquefolius.展开更多
肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药...肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药性仍是肺癌死亡率高的原因,其预后不尽如意[2]。环状RNA(CircRNAs)是具有保守、稳定性的一类非编码单链RNA分子[3]。大量的研究表明,CircRNAs在高迁移率族蛋白(high mobility group protein,HMG)家族成员中调节不同的信号通路,在肺癌等多种疾病中发挥关键作用。CircRNAs异常高或低表达可激活或抑制HMG蛋白分子,调节不同生物学过程[4]。随着下一代基因测序技术(next-generation sequencing,NGS)的探索,CircRNAs成为RNA领域肿瘤抑制靶点的研究新热点,为生物医药领域开拓了全新思路,然而其在肺癌中的病理生理机制尚未系统阐明。本文通过总结HMG蛋白家族成员相关CircRNAs在肺癌中的分子作用机制的研究进展,有助于为以CircRNAs为基础的作用靶点及新型抗癌药物的研发提供指导思路,提出潜在有效的治疗策略。展开更多
文摘羟甲基戊二酰辅酶A合酶(3-Hydroxy-3-methylglutaryl-coenzyme A synthase,HMGS)是甲羟戊酸途径(MVA)中的第一个催化酶。根据冬凌草转录组数据库中HMGS基因序列设计特异引物,采用RT-PCR技术克隆冬凌草IrHMGS基因cDNA全长,并对其序列进行生物信息学分析,通过荧光定量PCR的方法分析其组织表达特性。IrHMGS基因cDNA全长1 382bp,编码460个氨基酸,序列分析结果表明该基因编码的氨基酸序列含有羟甲基戊二酰辅酶A合成酶N末端、C末端等保守结构域。荧光定量PCR表明,IrHMGS在冬凌草组培苗叶和根中的表达量显著高于在组培苗花、茎和愈伤组织中的表达量。本研究为后续深入研究IrHMGS在冬凌草二萜类物质合成途径中的功能奠定了基础。
基金supported by the National Natural Science Foundation of China(No.81773830)Beijing Natural Science Foundation Program+3 种基金Scientific Research Key Program of Beijing Municipal Commission of Education(No.KZ201710025022)the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five–year Plan(No.CIT&TCD20170324)National Program for Special Support of Eminent ProfessionalsKey project at central government level:The ability establishment of sustainable use for valuable Chinese medicine resources(No.2060302)
文摘3-Hydroxy-3-methylglutaryl-CoA synthase(HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the p H7 WG2 D and p K7 GWIWG2 D vectors to respectively overexpress and silence, Tw HMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to70% in the expression level compared to cells with empty vector controls. During Tw HMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7 v2 was significantly upregulated to the control. In the RNAi group, the expression of Tw HMGR,TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of Tw HMGS determined the production of triptolide in T.wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-Co A was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.
基金National Nature Science Foundation of China(30900113)
文摘Objective To clone and analyze 3-hydroxy-3-methylglutaryl coenzyme-A synthase（HMGS） and 3-hydroxy-3-methylglutaryl coenzyme-A reductase（HMGR） genes from Panax notoginseng of four-year old during the flowering period, the key genes involved in the mevalonic acid pathway for saponin biosynthesis. Methods The cDNA sequences of PnHMGS1 and PnHMGR2 were obtained by reverse transcription PCR（RT-PCR） and rapid amplification of cDNA ends（RACE） methods and were analyzed in their secondary structures, subcellular localizations, domains, and the three-dimensional structures of putative proteins by the bioinformatics tools. Fusion genes were constructed by the prokaryotic expression system. Results The two genes were cloned, named as PnHMGS1 and PnHMGR2, respectively, and were both predicted to be located in the chloroplast. PnHMGS1（1410 bp） encoded a predictive unstable protein with 469 amino acids and covered hydroxymethylglutaryl-Co A synthase domain. PnHMGR2（1690 bp） also encoded an unstable protein with 589 amino acids and possessed a hydroxymethylglutaryl-coenzyme A reductase domain and two transmembrane regions. Both of the genes were expressed most in flowers followed by roots, stems, and least in leaves. Conclusion PnHMGS1 and PnHMGR2 are firstly cloned from P.notoginseng as the new member of the HMGR family,and they show the same expression profile as P.ginseng and P.quinquefolius.
文摘肺癌是全球癌症死亡的主要原因之一,具有极高的发病率及死亡率,严重威胁了人类的生命健康安全[1]。目前肺癌的治疗方法主要有手术、放化疗、靶向及免疫治疗,随着精准医疗的兴盛,一定程度改善了肺癌患者的生活质量,但癌症复发和药物耐药性仍是肺癌死亡率高的原因,其预后不尽如意[2]。环状RNA(CircRNAs)是具有保守、稳定性的一类非编码单链RNA分子[3]。大量的研究表明,CircRNAs在高迁移率族蛋白(high mobility group protein,HMG)家族成员中调节不同的信号通路,在肺癌等多种疾病中发挥关键作用。CircRNAs异常高或低表达可激活或抑制HMG蛋白分子,调节不同生物学过程[4]。随着下一代基因测序技术(next-generation sequencing,NGS)的探索,CircRNAs成为RNA领域肿瘤抑制靶点的研究新热点,为生物医药领域开拓了全新思路,然而其在肺癌中的病理生理机制尚未系统阐明。本文通过总结HMG蛋白家族成员相关CircRNAs在肺癌中的分子作用机制的研究进展,有助于为以CircRNAs为基础的作用靶点及新型抗癌药物的研发提供指导思路,提出潜在有效的治疗策略。
