According to the sequence of a 352bp fragment-M17 from the suppression subtractive hybridization library of the mutant line Cr3529 of Brassica napus,primers were designed and a 1000bp upstream fragment was cloned by 5’-RACE,which was joined with the original piece and composed a 1343bp sequence.According to the 3’ and 5’ ends sequences,a pair of primers were designed and the encoding region sequence of this unknown protein was amplified and cloned.Sequence analyzing showed that it had 80.1% identity with an unknown-protein-encoding gene from Arabidopsis thaliana.Swiss-model was used to predict the structure of the induced protein,and a potential transmembrane helix from site 46 Leu to 66 Phe was identified.GLOBE prediction of globularity showed that this protein possessed a compact globular domain.
A 1400bp DNA fragment in 5’ region of Toc33 Brassica napus was cloned by an improved single primer PCR method.The result of DNA sequence analysis showed that the fragment consisted of two regions.One of 491bp was partial coding sequence of Toc33 gene,the other of 909bp was the promoter of Toc33 gene.Besides TATA-box and CAAT-box,the promoter sequence included several cis-acting elements which had relation to light-regulation of plant.The cis-acting elements were G-box,GATA-box,I-box,SORLIP1AT motif,CIACADIANLELHC motif and so on.As a result,it was presumed that the transcription activity of promoter of Toc33 gene from Brassica napus may be regulated by light.
Two primers, designed according to the Arabidoposis thaliana Toc33 sequence, were used to amplify the full coding region of the Toc33 cDNAs in leaves of a chlorophyll-reduced (Cr) mutant Cr3529 and its wild type 3529, Brassica napus, by RT-PCR technique. The RT-PCR results showed that the fragment homologous to Toc33 was expressed in Cr3529 as well as 3529 seedlings. PCR fragments were inserted into the pMD18-T vector and transferred into E. coli, then two cDNA clones, BnToc33-c and BnToc33, were obtained. Sequence analysis showed that the two sequences were 894 bp and the nucleotide and the deduced amino acid sequences were highly homologous to those of A. thaliana. There were three diverged nucleotides between the Cr3529 and the 3529 Toc33 cDNAs, i.e., GGT/AGT, TTG/TTT, AGG/AGT, all of which belonged to missense mutation. The amino acid replacement ((Leu/Phe) caused by TTG/TTT mutation located in the membrane anchor domain may result in chlorophyll-reduced character in Cr3529.
