Cloning And Identification Of Cytochrome P450 Cyp75 Gene From Cdna Of Eupatorium Adoratum

The effect of guanidine isothiocyanate method,CTAB method,and Trizol method on total RNA extraction from Eupatorium adoratum were compared,three methods had good effect but the first one was the best.Three cDNA fragments were obtained from Eupatorium adoratum,Lycopuslucidus turcz,and Eupatorium fortunei by RT-PCR stragtegy using primers based on the homologus nucleotide sequences of CYP75(AY954290)in Eupatorium adenophorum and members of CYP75 in Arabidopsis thliana.One was 467bp from Eupatorium adoratum named FJC.CYP.M,the second was 467bp from Lycopuslucidus turcz named ZL.CYP.M,the third was 470bp from Eupatorium fortunei named PL.CYP.M.Three sequences shared a homology of 99%with each other and 90%with CYP75 in Eupatorium adenophorum (AY954290)and were demonstrated to come from p450 super familiy.Two cDNA nucleotide sequences were gotten from Eupatorium adoratum by RACE-PCR using primers designed from FJC.CYP.M. FJC.CYP.3 was 309bp with ployA end obtained by 3'RACE-PCR,and FJC.CYP.5 is 702bp obtained by 5'RACE-PCR.Compared with the 5'end of Eupatorium adenophorum CYP75 gene,FJC.CYP.5 lacked 364bp.A 384bp cDNA fragments named FJC.CYP.5e was obtained by RT-PCR using primers designed from FJC.CYP.5 and the 5'end of CYP75 gene in Eupatorium adenophorum.One full-length cDNA nucleotide sequence named FJC.CYP.COM was gotten by combining all the fragments above in Eupatorium adoratum.It had 1653bp encoding 509 amino acids and had a ORF from 31bp-1560bp.The sequence included three conserved regions of P450 such as F××G×R×C×G, and a promotor sequence TATA box could be found in 27bp before the transcriptional initiation site of ATG.FJC.CYP.COM is a stable, hydropathicity protein with 9 transmembrate helices,which were located in mitochondria.Two cDNA fragments were obtained by 3'RACE PCR using primers designed from ZL.CYP.M and PL.CYP.M.One was 340bp from Lycopuslucidus turcz named ZL.CYP.3 and the other was 625bp from Eupatorium fortunei named PL.CYP.3.ZL.CYP.3 was one of Chlorophyll A-B binding protein in P450 super family whose function was not clearly. PL.CYP.3 was GASA superfamily protein in P450 super family which took part in the synthesine of GASA.