Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (21): 4515-4522.doi: 10.3864/j.issn.0578-1752.2013.21.014

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Expression and Analysis of Candida cloacae Long-Chain Fatty Alcohol Oxidase FAO1 by Nuclear Expression Vector in Chlamydomonas reinhardtii

 LI  Ming-Ze, CHENG  Qi   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2013-04-19 Online:2013-11-01 Published:2013-07-19

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Abstract: 【Objective】 fao1 encods a iron-containing long-chain fatty alcohol oxidase in C.cloacae. By constructing the nuclear expression vector, according to the biological activity of protein we can judge whether fao1 can take full advantage of iron in Chlamydomonas reinhardtii chloroplast. Meanwhile it will provide a new direction for researches on the gene of iron-containing proteins.【Method】The PsaD transit peptide gene(TP), C. cloacae fatty-alcohol oxidase (fao1) gene and His-tag were cloned separately by polymerase chain reaction (PCR). The 3-piece fusion gene TFHis was inserted into pDBle. This construct was transformed into C. reinhardtii CW15 by glass beads method. The transgenic Chlamydomonas was obtained under the selection of zeocin and they were confirmed positive by PCR amplification and RT-PCR. The activity of the purified recombinant protein was tested by a color-linked assay. 【Result】 The correctness of the constructed pDBle-TFHis was confirmed by DNA sequencing. PCR and RT-PCR analysis of the genomic DNA and RNA from transgenic Chlamydomonas showed that the recombinant plasmid has been integrated into C. reinhardtii CW15 genome and transcribed successfully. Protein purification elution made substrate reaction turn blue.【Conclusion】In this study, fao1-bearing nuclear expression vector pDBle-TFHis was correctly constructed and integrated into C. reinhardtii CW15 genome and fao1 transcript was detected successfully. The recombinant protein was purified by an affinity column and the activity was tested.

Key words: long-chain fatty alcohol oxidase , transit peptide , His-tag , Chlamydomonas nuclear transformation , RT-PCR

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