Cloning and analysis of the cDNA of a gene encoding an unknown protein in Brassica napus

Two full-length cDNA sequences were amplified and cloned by RT-PCR and RACE-PCR according to a differentially expressed fragment of the backward subtraction library of young leaves in chlorophyll-reduced mutant Cr3529 of Brassica napus,named BnCr4 and BnCr4-1 respectively.They encoded 465 and 437 amino acids respectively.Results of BLAST in GenBank showed they shared 87 % and 85 % identity to the cDNA of an unknown function gene(accession number: At5g19540)in Arabidopsis thaliana,respectively.Prediction of protein function showed they contain multiple types of functional sites.Northern blots showed that the expression of the unknown gene presented more strongly in cotyledon and young leaves of chlorophyll-reduced mutant than that of wild type.The open reading frame of BnCr4 was cloned into expression vector pET-32a(+),and then transformed into host bacterium,BL21(DE3).The host cells were induced by IPTG.The molecular weight of the expressed protein in E.coli was identified with the deduced protein.