Cloning And Analysis Of Partial Enzyme Members CDNA In Papaya (Carica Papaya L.) Of Carotenoid Biosynthetic Pathway

Papaya(Carica papaya L.) is a fruit crop cultivated in tropical and sub-tropical regions worldwide and rich in carotenoids. Carotenoids are a large class of plant pigments,which have a wide range of biological functions. In this study, the cDNA sequence of phytoene desaturase(PDS),ζ-carotene desaturase(ZDS) and lycopene-β-cyclase(LCY-B) of carotenoid biosynthetic pathway in red flesh of'NO.10 ma lai′papaya were cloned by RACE and RT-PCR, and the expresstion patterns of phytoene synthase(PSY),PDS,ZDS and LCY-B in different developmental stages of papaya fruits were analyzed by semi quantitative RT-PCR.This study could provid the the theoretical basis for the molecular mechanism of carotenoids biosynthesis and of differnial flesh color in papaya.1 Cloning and analysis of the full length cDNA of CpPDS by RACE and RT-PCRObtained the 5′and 3′end sequence by RACE,using CpPDS fragment from GenBank.And the full length cDNA of CpPDS(Genbank accession number DQ666830) is 2202bp, it contains a largest ORF that is 1752bp encoding 583 amino acids (Mw= 65110.0, pI=6.26),a 5′untranslated region of 223 bp and a 3′flanking sequence of 227 bp. The deduced amino acid sequence of CpPDS was compared with the sequences of other higher plants such as Solanum lycopersicum, Citrus unshiu, Zea mays,and so on.2 Cloning and analysis of the full length cDNA of CpZDS by RACE and RT-PCRObtained the 5′nucleotide sequence by RACE, using CpZDS 3′nucleotide seqence from GenBank. And the full length cDNA of CpZDS(Genbank accession number DQ666829) is 1951bp, it contains a largest ORF that is 1659bp encoding 552 amino acids (Mw=60888.8, pI=7.52),a 5′untranslated region of 25 bp and a 3′flanking sequence of 267 bp. The deduced amino acid sequence of CpZDS was compared the sequences of other higher plants such as Citrus unshiu, Arabidopsis thaliana, Zea mays, and so on.3 Cloning and analysis of the ORF of CpLCY-B by RT-PCRObtained the ORF(Genbank accession number FJ599643) by RT-PCR, using the full length DNA of CpLCY-B from GenBank.And the nucleotide sequence is 1613bp, it contains a largest ORF that is 1512bp encoding 503 amino acids (Mw=56736.5, pI=6.62),a 5′untranslated region of 76 bp and a 3′flanking sequence of 25 bp. The deduced amino acid sequence of CpLCY-B was compared with the sequences of other higher plants such as Citrus unshiu, Solanum lycopersicum, Narcissus pseudonarcissus, and so on.4 Analysis the expression of CpPSY, CpPDS, CpZDS and CpLCY-B by sqRT-PCRThe expression patterns of CpPSY, CpPDS, CpZDS and CpLCY-B in the expansion stage, color break stage, 50% ripening stage and full ripening stage were carried out and CpActin was used as positive control. sqRT-PCR indicated that all of the four enzyme genes expressed in different developmental stages of papaya fruit with higher expression level the intermediate stages. So it shows that these enzyme genes are indispensable in carotenoids metabolism, and this is the manifestation of their importance. The levels of the four genes′expression reach the maximum during the same period and all in the downward trend in full ripening stage .It shows that they have close collaborative relationship.