Isolation of promoter for N-methyltransferase gene associated with caffeine biosynthesis in Coffea canephora.
N-Methyltransferases (NMTs) catalyze the three SAM dependent sequential methylation of xanthosine, producing caffeine in Coffea species. In the present work, a PCR based genome walking method was adopted to isolate and clone the promoter for the NMT gene. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The whole fragment was fused to the beta-glucuronidase (gus) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. The differences in the restriction pattern among the genomic clones were studied using PCR-RFLP. This is the first report of cloning of the promoter for a gene involved in caffeine biosynthetic pathway and it opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine.
Satyanarayana
K V
KV
Plant Cell Biotechnology Department, Central Food Technological Research Institute (CFTRI), Mysore 570020, Karnataka, India.
Kumar
Vinod
V
Chandrashekar
A
A
Ravishankar
G A
GA
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002110:Caffeine / Q000096:biosynthesis*; D003001:Cloning, Molecular; D018387:Codon, Initiator; D040503:Coffea / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005091:Exons; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007438:Introns; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
D018387:Codon, Initiator; D017931:DNA Primers; D002110:Caffeine; D008780:Methyltransferases; D005966:Glucuronidase