Cloning of a Full 3′ End cDNA Sequence of ACC Synthase from Fuyu Persimmon

Gene cloning is the bases to research gene function and expressing characterization.The popular used method is to partial clone gene coding sequence first by the gene specific prime desired from the gene conserve amino acid,then to get 5′ and 3′ end sequence which is much difficult than coding region cloning.A cDNA fragment with a 3′ end sequence of ACC synthase was cloned from Fuyu persimmon fruit by 3′ RACE PCR.The PCR product was linked into the pMD18-T Vector and recombinant plasmid was transformed to E.coli DH5α,which was selected with xgal/IPTG.Sequence analysis showed the PCR product was 912 bp,which encoded 251 amino acids with three highly conserved regions of ACC synthase.There was only one amino acid different from the sequence DK-ACS1 which entered in GenBank(AB073005).There was a 155 bp non-encoding sequence in 3′ end,in which only one nucleotide was different from DK-ACS1.The sequence included 5'GSP inner primer and 3'RACE inner primer,which indicated to get a full 3′ end sequence of Fuyu persimmon fruit.