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[1]马磊,张翔,田永强,等.拟南芥基因组中注释为异胡豆苷合成酶的基因克隆及异源表达[J].应用与环境生物学报,2013,19(02):224-230.[doi:10.3724/SP.J.1145.2013.00224]
 MA Lei,ZHANG Xiang,TIAN Yongqiang,et al.Molecular Cloning and Heterologous Expression of Putative Strictosidine Synthases from Arabidopsis thaliana[J].Chinese Journal of Applied & Environmental Biology,2013,19(02):224-230.[doi:10.3724/SP.J.1145.2013.00224]
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拟南芥基因组中注释为异胡豆苷合成酶的基因克隆及异源表达()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年02期
页码:
224-230
栏目:
研究论文
出版日期:
2013-04-25

文章信息/Info

Title:
Molecular Cloning and Heterologous Expression of Putative Strictosidine Synthases from Arabidopsis thaliana
作者:
马磊张翔田永强张国林罗应刚
(1四川大学化学工程学院 成都 610065)
(2中国科学院成都生物研究所 成都 610041)
Author(s):
MA Lei ZHANG Xiang TIAN Yongqiang ZHANG Guolin LUO Yinggang
(1College of Chemical Engineering, Sichuan University, Chengdu, 610065, China)
(2Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)
关键词:
萜类吲哚生物碱异胡豆苷合成酶拟南芥RT-PCRPCR异源表达
Keywords:
terpenoid indole alkaloids strictosidine synthase Arabidopsis thaliana RT-PCR PCR heterologous expression
分类号:
Q946.88 : Q78
DOI:
10.3724/SP.J.1145.2013.00224
文献标志码:
A
摘要:
拟南芥(Arabidopsis thaliana)是生物学研究中广泛使用的模式生物. 到目前为止,没有证据表明拟南芥中存在复杂生物碱,但对其基因组数据的生物信息学分析表明,有很多基因与萜类吲哚生物碱(TIAs)生物合成途径中的相关基因有高度的同源性. 为了从分子水平研究拟南芥是否具有合成该类复杂生物碱的潜力,首先对拟南芥中可能编码异胡豆苷合成酶(STR)的15个基因及其编码的酶进行广泛的生物信息学分析和计算;在此基础上,选择其中5个AtSTR基因为目的基因,设计特异性引物;从拟南芥中提取总RNA,一步法RT-PCR克隆得到上述基因;通过TA克隆将上述基因转入pGM-T载体,筛选、酶切及PCR鉴定,DNA测序验证它们的核酸序列;将测序验证的AtSTR基因经过PCR扩增、质粒构建、转入Escherichia coli BL21 (DE3)中进行异源表达和蛋白纯化;在整体细胞、细胞裂解液、细胞裂解液上清和纯化蛋白共4个层次进行AtSTR酶功能表征,未发现目标产物或新产物的生成;上述基因的具体功能还有待进一步研究. 图8 表3 参22
Abstract:
No complex alkaloids were proved to accumulate in Arabidopsis thaliana. However, many genes in the genome of A. thaliana were annotated as homologues to the genes involved in the biosynthetic pathway of terpenoid indole alkaloids (TIAs), for instance, 15 genes were annotated as strictosidine synthase (STR). To clarify the function of these genes, we selected 5 putative AtSTRs out of 15 genes on the basis of bioinformatic analysis of known functional RsSTR and AtSTRs. The five putative AtSTR genes were obtained by RT-PCR using total RNA as template. The plasmid containing AtSTRs were constructed, screened by the combination of X-gal and IPTG, enzymtic digestion and PCR. The sequences of these AtSTRs were confirmed by DNA sequencing. The expression plasmids were constructed, sequenced, and transformed into Escherichia coli BL21 (DE3) for protein overexpression. The fusion proteins were soluble and purified by nickel ion affinity chromatography. The putative catalytic activity of AtSTRs was assayed at four levels, including whole cells, cell lysates, supernatant, and purified enzyme, by adding the native substrates tryptamine and secologanin to the reaction mixture. No desired or new products were observed in the reaction mixture by HPLC-DAD detection, which suggested that more work is needed to clarify the possible function of these genes. Fig 8, Tab 3, Ref 22

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备注/Memo

备注/Memo:
中国科学院知识创新工程领域前沿项目、“西部之光”联合学者项目、生命科学领域优秀青年科技专项(KSCX2-EW-Q-6)和国家自然科学基金面上项目(21172216)联合资助
更新日期/Last Update: 2013-05-03