雷公藤乙酰CoA酰基转移酶基因全长cDNA克隆及表达分析(1)
[摘要] 研究根据雷公藤悬浮细胞转录组数据库设计引物，对雷公藤悬浮细胞乙酰辅酶A酰基转移酶(acetyl-CoA C-acetyltransferase)基因TwAACT进行全长cDNA(GeneBank：KP297934)克隆以及生物信息学分析和基因表达分析。克隆得到TwAACT cDNA全长为1 704 bp，编码405个氨基酸，等电点为6.10，相对分子质量为41.20 kDa，在线预测表明TwAACT具有2个催化活性位点。雷公藤悬浮细胞经茉莉酸甲酯(MeJA)外源诱导后，通过荧光定量PCR表明，TwAACT相对表达量明显增加，在24 h达到最高。研究首次克隆得到雷公藤AACT基因，为进一步阐述雷公藤萜类生物合成途径奠定基础。
[关键词] 雷公藤;乙酰 CoA 酰基转移酶;克隆;生物信息学分析;基因表达分析
[收稿日期] 2014-12-24
[基金项目] 国家自然科学基金项目(81422053，81325023，81373906);全国优秀博士学位论文作者专项(201188)
, 百拇医药
[通信作者] 陈忻，教授，研究方向为中药对帕金森病神经保护作用的基础研究，Tel：(010)83911633， E-mail：chenxin4283@126.com;高伟，副教授，研究方向为分子生药学，Tel：(010)83911633， E-mail： weigao@ccmu.edu.cn
[作者简介] 赵瑜君，硕士研究生，E-mail： diana1989119@sina.com
Cloning and expression analysis of a acetyl-CoA C-acetyltransferase
gene (TwAACT) from Tripterygium wilfordii
ZHAO Yu-jun， ZHANG Meng， LIU Yu-jia， SU Ping， TONG Yu-ru， HU Tian-yuan，CHEN Xin， GAO Wei， HUANG Lu-qi
, 百拇医药
(1. School of Traditional Chinese Medicine， Capital Medical University， Beijing 100069， China;
2.State Key Laboratory of Dao-di Herbs， National Resource Center for Chinese MateriaMedica， China Academy of
Chinese Medical Sciences， Beijing 100700， China)
[Abstract] In this study， based on the transcriptome data， we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells， and then analyzed the bioinformation of the sequence， detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10， a calculated molecular weight was about 41.20 kDa， and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA， the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly， that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.
, http://www.100md.com
[Key words] Tripterygium wilfordii; AACT; clone; bioinformatics analysis; expression analysis
doi：10.4268/cjcmm20150513
中药雷公藤Tripterygium wilfordii Hook. f.系卫矛科Celastraceae植物雷公藤的根，其味辛、苦，性凉，具有活血化瘀、清热解毒、消肿散结、杀虫止血等功效[1]。在治疗免疫性疾病、抗肿瘤等方面得到广泛应用。雷公藤甲素(triptolide)是中药雷公藤公认的主要活性成分之一，自1972年Kupchan[2]等首次分离并发现雷公藤甲素具有明显抗白血病作用以来，研究发现雷公藤甲素具有明显的抗炎、免疫抑制、抗肿瘤等重要生物活性[3]，最近其抗肿瘤研究取得了突破性进展[4-6]，成为国内外研究人员的关注热点，并进入临床评价研究阶段。, 百拇医药(赵瑜君 张萌 刘雨佳 苏平 童宇茹 胡添源 陈忻 高伟 黄璐琦)
[关键词] 雷公藤;乙酰 CoA 酰基转移酶;克隆;生物信息学分析;基因表达分析
[收稿日期] 2014-12-24
[基金项目] 国家自然科学基金项目(81422053，81325023，81373906);全国优秀博士学位论文作者专项(201188)
, 百拇医药
[通信作者] 陈忻，教授，研究方向为中药对帕金森病神经保护作用的基础研究，Tel：(010)83911633， E-mail：chenxin4283@126.com;高伟，副教授，研究方向为分子生药学，Tel：(010)83911633， E-mail： weigao@ccmu.edu.cn
[作者简介] 赵瑜君，硕士研究生，E-mail： diana1989119@sina.com
Cloning and expression analysis of a acetyl-CoA C-acetyltransferase
gene (TwAACT) from Tripterygium wilfordii
ZHAO Yu-jun， ZHANG Meng， LIU Yu-jia， SU Ping， TONG Yu-ru， HU Tian-yuan，CHEN Xin， GAO Wei， HUANG Lu-qi
, 百拇医药
(1. School of Traditional Chinese Medicine， Capital Medical University， Beijing 100069， China;
2.State Key Laboratory of Dao-di Herbs， National Resource Center for Chinese MateriaMedica， China Academy of
Chinese Medical Sciences， Beijing 100700， China)
[Abstract] In this study， based on the transcriptome data， we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells， and then analyzed the bioinformation of the sequence， detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10， a calculated molecular weight was about 41.20 kDa， and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA， the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly， that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.
, http://www.100md.com
[Key words] Tripterygium wilfordii; AACT; clone; bioinformatics analysis; expression analysis
doi：10.4268/cjcmm20150513
中药雷公藤Tripterygium wilfordii Hook. f.系卫矛科Celastraceae植物雷公藤的根，其味辛、苦，性凉，具有活血化瘀、清热解毒、消肿散结、杀虫止血等功效[1]。在治疗免疫性疾病、抗肿瘤等方面得到广泛应用。雷公藤甲素(triptolide)是中药雷公藤公认的主要活性成分之一，自1972年Kupchan[2]等首次分离并发现雷公藤甲素具有明显抗白血病作用以来，研究发现雷公藤甲素具有明显的抗炎、免疫抑制、抗肿瘤等重要生物活性[3]，最近其抗肿瘤研究取得了突破性进展[4-6]，成为国内外研究人员的关注热点，并进入临床评价研究阶段。, 百拇医药(赵瑜君 张萌 刘雨佳 苏平 童宇茹 胡添源 陈忻 高伟 黄璐琦)