Scientia Agricultura Sinica ›› 2011, Vol. 44 ›› Issue (23): 4848-4857.doi: 10.3864/j.issn.0578-1752.2011.23.011

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning，Sequence Analysis of Phytoene Synthase Gene from Cerasus humilis (Bge) Sok. and Its Functional Expression in E.coli

 ZHANG  Jian-Cheng, DU  Jun-Jie, LIU  He  , WANG  Peng-Fei, XUE  Xiao-Fang, MU  Xiao-Peng, CHEN  Jun-Qi   

  1. 1.山西农业大学园艺学院，山西太谷 030801
  • Received:2011-08-17 Online:2011-12-01 Published:2011-09-26

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Abstract: 【Objective】The present study aimed at cloning and analyzing the phytoene synthase (PSY) gene from Cerasus humilis (Bge) Sok. and verifying its catalytic function by heterologous expression in E. coli containing a β-carotene producing plasmid. 【Method】Using the total RNA from the fruit of C. humilis (Bge) Sok. as the template, the cDNA specific fragment of PSY gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then the full-length cDNA sequence was obtained through rapid amplification of cDNA ends (RACE) techniques and its sequence was analyzed. The full coding sequence of PSY cDNA was amplified using PCR and further subcloned into vector pET-28a (+). The recombinant plasmid pET-ChPSY was expressed in a prokaryotic expression system after its transformation into BL21 (DE3). The catalytic activity of the fusion protein (6×His-PSY) was investigated in E. coli strain engineered to accumulate β-carotene. 【Result】 Sequence analysis indicated that the full-length cDNA was 1 559 bp, with an open reading frame of 1 194 bp and encoded a protein of 398 amino acids. The cloned cDNA exhibited a homology of the nucleotides and amino acid sequences of over 70% and 65%, respectively, aligned with PSY genes from other plants. The deduced protein has a signal transit peptide consists of 55 amino acid residues in the N-terminal region and two predicted transmembrane domain in the sites of 37-58 and 219-240 amino acids. The analysis of SDS-PAGE demonstrated that the recombinant fusion proteins (6×His-PSY) was produced at a higher level by prokaryotic expression and migrated at a size of about 45.8 kD. The heterogenous expression in E. coli system confirmed that ChPSY could encode a functional phytoene synthase which could enhance β-carotene content of E. coli engineered to produce β-carotene. 【Conclusion】 The PSY gene from Cerasus humilis (Bge) Sok. was successfully cloned and fucntioally expressed in E. coli, which has established a basis for studying the protein characterization and biosynthetic mechanism of carotenoids in fruits of Cerasus humilis (Bge) Sok.

Key words: Cerasus humilis (Bge) Sok, phytoene synthase, cloning, functional expression

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