A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from geno...A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.展开更多
ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(...ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.展开更多
文摘A pair of primers was designed according to the reported conserved sequence of the defective in anther dehiscencel （DAD1） gene ofArabidopsis thaliana and Brassica rapa. A 558 bp long fragment was amplified from genomic DNA of Chinese kale, showing more than 88% identity with the known DAD1 nucleotide sequence and no intron. The reverse of the amplified fragment was ligated to the downstream of the CaMV35S promoter in the plant expression vector pBIl21. Antisense expression vector pBII21-DAD1F was constructed with DAD1 fragment of Chinese kale, and was transferred into Agrobacterium tumefaciens, which will be used in the transformation to create male sterile materials of Chinese kale.
基金supported by National Natural Science Foundation of China(31500247)Key Project of Department of Education of Sichuan Province(14ZA0016)Natural Science Foundation of Zhejiang Province(LZ15C150001)
文摘ζ-Carotene desaturase(ZDS)is an important enzyme in carotenoid biosynthesis.Here,the Brassica oleracea var.alboglabra ZDS(Boa ZDS)gene was cloned from Chinese kale via reverse transcription-polymerase chain reaction(RT-PCR)and deposited in Gen Bank(accession number KY662297).The Boa ZDS gene contains an open reading frame of 1 686 bp that encodes a 561-amino acid protein.Sequence analysis indicates that the ZDS protein is apparently conserved during plant evolution and is most closely related to B.oleracea var.capitata and B.rapa.The promoter sequence of the Boa ZDS gene was predicted to harbor several cis-acting elements that are related to light and phytohormone responses.Semiquantitative RT-PCR analysis showed that Boa ZDS expression varied among different developmental stages and organs.Relative ZDS expression remained stable during germination and seedling stages and rapidly increased at the mature leaf stage.The leaves showed the highest ZDS expression levels compared to the other organs.ZDS expression decreased in all flower tissues during blooming.The fused protein of Boa ZDS was obtained by prokaryotic expression.Heterologous expression of Boa ZDS in Escherichia coli confirmed that Boa ZDS encodes a functionalζ-carotene desaturase that increases β-carotene accumulation in E.coli cells harboring a β-carotene-producing plasmid.The findings of the present study provide a molecular basis for the elucidation of ZDS gene function in Chinese kale.
