Phytoene Synthase Gene Cloning from Citrus sinensis Osbeck cv. Cara Cara and Its Prokaryotic Expression

作     者：ZHANG Jian-cheng TAO Neng-guo TONG Zhu DENG Xiu-xin 

作者机构：National Key Laboratory of Crop Genetic Improvement Huazhong Agricultural University Wuhan 430070 RR.China 

出 版 物：《Agricultural Sciences in China》 (中国农业科学（英文版）)

年 卷 期：2008年第7卷第2期

页      面：148-156页

学科分类：09[农学] 0902[农学-园艺学] 090201[农学-果树学] 

基　　金：National 973 of China, (2006CB708202) Ministry of Education of the People's Republic of China, MOE, (IRT0548) 

主　　题：Citrus sinensis Osbeck cv. Cara Cara phytoene synthase prokaryotic expression 

摘      要：Using the mRNA from the fruit of Cara Cara as the template, the cDNA of phytoene synthase （PSY） gene was amplified by reverse transcription polymerse chain reaction （RT-PCR）. Sequence analysis indicated that the cDNA was of 1 520 bp, which had an open reading frame of 1 308 bp and encoded a protein of 436 amino acids. The homology analysis showed that PSY of Cara Cara shared high similarities of nucleotides and deduced amino acids with those in other plants up to more than 75 and 70%, respectively. A putative signal transit peptide for plastid targeting was found in the N-terminal region of PSY. The mature forms of PSY included a transmembrane （TM） domain. The recombinant plasmid pET-CitPSY was constructed by subcloning the full coding sequence of PSY cDNA into pET-28 （＋）. After transformation of E. coli BL21 and induced by 1 mmol L^-1 isopropyl-β-D-thiogalacropyranoside （IPTG）, the fusion protein （6× His-PSY） with 52 kD was produced at a high level by prokaryotic expression system. The results of Western blot demonstrated that the fusion protein （6× His-PSY） could be recognized by anti-6 × His monoclonal antibody. The study could establish a basis for molecular improvement of Citrus fruit colors.