The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with...The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp(GenBank Accession No. DQ243892) was de-duced from RACE results,including untranslated 21 bp 5'-and 520 bp 3'-flanking regions and an open reading frame of 582 amino acids,coding a protein of 64.196 kDa. The DNA sequence of 2908 bp(GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex,including two bases' perfect repeats(GT) 10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species,and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance of pds was discussed.展开更多
Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the id...Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.展开更多
A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading...A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.展开更多
文摘The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp(GenBank Accession No. DQ243892) was de-duced from RACE results,including untranslated 21 bp 5'-and 520 bp 3'-flanking regions and an open reading frame of 582 amino acids,coding a protein of 64.196 kDa. The DNA sequence of 2908 bp(GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex,including two bases' perfect repeats(GT) 10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species,and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance of pds was discussed.
基金the National Natural Science Foundation of China （Grant No. 30470172）.
文摘Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.
文摘A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Key words saffron - carotenoids - phytoene desaturase - gene expression - antiserum - Western blot CLC number Q 781 - Q 786 Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012)Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.
