Cloning And Identification Of Cytochrome P450 CYP75 Gene From Eupatorium Adenophorum

Two cDNA fragments were obtained from Eupatorium adenophorum by RT-PCR strategy using primers based on the homologus nucleotide sequences of CYP75B5 (GenEMBLAF313489) ,CYP75B6 (GenEMBLAF313488) in Callistephus chinensis and members of CYP75 in Arabidopsis and the conserved region of plant cytochrome P450 F× ×G×R×C×G. One is 378bp(GenBank accession number: AY954289) which encodes 125 amino acids, another is 644bp (GenBank accession number: AY954290) which encodes 214 amino acids. AY954289 and AY954290 shared a homology of 100%. The necleotide acid sequence of 378bp shared significant identity to CYP75B5 (79.5%) and CYP75B6 (85.6%). The results indicated that two sequences are structural gene fragment of one or two members of CYP75 family.The homologus analysis of 378bp sequences from seven different groups in geography showed that they shared over 99% homologus. Single nucleic acid exchange of three positions presented near the 5 ' sequence. The result indicated that there are more than two members of CYP75 in Eupatorium adenophorum. Based on the background of plant CYP75 genes, it was suggested that these members of CYP75 contacted with plant secondary metabolism during the course of evolution.The homologus analysis of 644bp sequence between original group and newly group indicated they shared 99.2% homologus. Either of them has 214 deduced amino acid sequence, And the sequences include the conserved region of plant cytochrome P450 F××G×R×C×G. This characteristic of P450 showed that the sequences obtained form Eupatorium adenophorum were part of the member of CYP75, and there are not less than two alleles of CYP75 in Eupatorium adenophorum.