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J. Hefei Univ. Tech. (Nat. Sci.)

合肥工业大学学报(自然科学版)

JOURNAL OF HEFEI UNIVERSITY OF TECHNOLOGY (NATURAL SCIENCE)

Competent Authorities: Ministry of Education of the People's Republic of China

Sponsor by: Hefei University of Technology

ISSN 1000-1646, CN 34-1001/N

Cloning and expression analysis of phytoene synthase gene in Tagetes erecta L.

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万寿菊八氢番茄红素合成酶基因的克隆与表达分析

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Nov.2024, Vol.47 No.11: 1547-1552

DOI: 10.3969/j.issn.1003-5060.2024.11.017

Authors

QIU Yaqiong, ZHANG Enqi, LU Yican, TIAN Bin, LU Chuanfu, NIU Xiangli

(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

Abstract and Keywords

Abstract: Phytoene synthase(PSY) is the rate-limiting enzyme in the carotenoid biosynthetic pathway of plants, catalyzing the biosynthesis of lycopene. Marigold(Tagetes erecta L.) is rich in carotenoids in its flowers to imply possible high activity of the related catalase genes in biosynthesis pathway. In this paper, TePSY, a gene encoding for PSY of marigold, was cloned based on the transcriptomic data and ligated into the cloning vector pEASY-Blunt. The sequencing results showed that TePSY has a full-length frame of 1 272 bp for a hydrophilic protein with a relative molecular mass of 48 kDa. Evolutionary analysis suggested that PSY gene is conserved in plants, and TePSY has the highest similarity to the homologous gene from the same family plant of sunflower. Real-time fluorescent quantitative polymerase chain reaction(PCR) indicated the expression of TePSY in marigold flowers, the abundance increases with flower development and decreases at maturity. TePSY was furtherly used for plant expression vector pBTEX-TePSY construction and Agrobacterium-mediated transient expression in tobacco leaves and then subjected to western blot(WB). The results showed the expected protein product of TePSY expressed in plant system for further functional analysis of marigold TePSY.

Keywords: marigold(Tagetes erecta L.); phytoene synthase(PSY); gene cloning; expression analysis; western blot(WB)

作者信息

邱雅琼，张恩启，卢熠灿，田彬，鲁传富，牛向丽

（合肥工业大学食品与生物工程学院，安徽合肥230601）

摘要和关键词

摘要: 八氢番茄红素合成酶(PSY)是植物类胡萝卜素生物合成途径中的主要限速酶,催化番茄红素的合成。万寿菊(Tagetes erecta L.)花中富含类胡萝卜素,提示其合成途径催化酶基因可能具有较高活性。文章利用转录组数据克隆获得编码万寿菊PSY的基因TePSY,连接于克隆载体pEASY-Blunt。测序结果显示,TePSY全长读码框为1272 bp,编码相对分子质量约48 kDa的亲水性蛋白;进化分析表明,PSY基因在植物中保守存在,TePSY与同科植物向日葵同源基因相似性最高;实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)显示,TePSY在万寿菊花组织中表达,丰度先升高,至成熟时降低。将TePSY用于构建植物表达载体pBTEX-TePSY,进一步利用农杆菌介导法在烟草叶片中瞬时表达,并进行蛋白印迹(western blot,WB)检测,结果表明,在植物体内表达预期大小TePSY蛋白产物,可用于后续TePSY功能分析。

关键词: 万寿菊；八氢番茄红素合成酶(PSY)；基因克隆；表达分析；蛋白印迹(WB)

Funding

安徽省科技创新平台重大科技资助项目（202305a12020030）；合肥工业大学产学研校企合作资助项目（W2022JSKF0793；W2023JSKF1185）

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