The Construction Of The Suppression Backward Subtractive Library Of The Brassica Napus Mutant Cr3529 And The Cloning Of BnCr4 CDNA In The Line Cr3529

The backward subtraction libraries of young leaves in chlorophyll-reduced mutant Cr3529 and its wild type 3529 were constructed by suppression subtractive hybridization (SSH) . In backward subtraction, the cDNA from the Cr3529 seedlings leaf was used as Tester and the cDNA from 3529 as Driver. The secondary PCR products of the backward subtraction were cloned, inserted into the pMD18-T vector and transferred into E. coli. About 1000 white clones were obtained. These clones in the backward subtraction libraries were verified by colony PCR and dot hybridization labeled by DIG. The recombined efficiency of the backward subtraction libraries was 76.5% and the different efficiency of recombined clones was 75%. The inserted fragments were between 100bp and 1000bp. Among the positive clones, 36 clones were randomly selected to have their inserts sequenced and Blast searches were performed. The results showed that two reduplicate clones (clone 24 and clone 34) were found and the efficiency of different is 94.4%. Twenty-nine clones were related to the protein transportation, photosysthesis process, fatty acid metabolism and the stress-response, etc. Otherwise, six clones shared homology to unknown protein genes.The full length cDNA of differential expressed gene, named BnCrA, in chlorophyll-reduced mutant Cr3529 of Brassica napus was cloned by reverse transcription cDNA synthesis and Race-PCR technique. Sequence analysis indicated that it has an open reading frame of 1395 nucleotide acids for encoding 465 amino acids. Its components of amino acids, homolog, physical and chemic characters were analyzed. The results showed that its deduced amino acid sequence was highly homologous to an unknown protein from Arabidopsis thaliana (81%). Its bonding state of cysteines, hydrophilicity, as well as the secondary structure and 3D structure were predicted. The results showed that there were three domains: about 110 amino acids closed to the 5' end were random coil containing lots of beta-turn; about 240 amino acids in the middle had abundant secondary structure, maybe reacting important function; about 100 amino acids closed to the 3' end contained lots of Alpha helix. There were lots of alpha helix, beta-turn and random coil, so the 3D structure was complex. Otherwise, BnCrA protein might be a transmembrane protein because it possessed two transmembrane domains. Northern blot showed that there was a notable expression difference between mutant and the wild type in cotyledon and young leaves periods.