Isolation Of The CDNA Of A Gene Encoding An Unknown Protein In Brassica Napus Mutant Cr3529 And Construction Of Expression Vector

Two full-length cDNAs were amplified and cloned by RT-PCR according to 5' and 3' sequence of BnCr4 which was isolated from the backward subtraction libraries (the Cr3529 seedlings leaf was used as Tester and the cDNA from 3529 as Driver) of young leaves in chlorophyll-reduced mutant Cr3529 of Brassica napus, named BnCr4-l and BnCr4-2 respectively. They encoded 437 and 462 amino acids respectively. BLAST results of BnCr4, BnCr4-l and BnCr4-2 showed they shared 87 %, 80% and 85 % identity to the cDNA of an unknown function gene (accession number: At5gl9540 ) in Arabidopsis thaliana, respectively. Their secondary structure and 3D structure were predicted. The results indicated that there were three domains: about 110 amino acids closed to the N-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 C-end contained lots of Alpha helix. There were lots of alpha helix, beta-turn and random coil, so the 3D structure was complex. Otherwise, their proteins might be a transmembrane protein because they possessed two transmembrane domains. Prediction of protein function showed they contain multiple types of functional sites such as cAMP-dependent protein kinase phosphorylation site, Protein kinase C phosphorylation site, Casein kinase II phosphorylation site and Tyrosine kinase phosphorylation site etc., which showed that the three proteins could be involved in cAMP-inducible protein phosphorylation. The open reading frame of BnCr4 was recombinated into the expression vector pET-32a(+), and then transferred into the 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 by SDS-PAGE. A cDNA fragment of 582 bp in BnCr4 was subcloneed and inserted into RNAi vector pFGC5941 in reverse direction to consdructe plant antisense expression vector. The recombinated expression vector confirmed by PCR and DNA sequencing was transferred into Agrobacterium tumefaciens. Some factors which affected the transformation of hypocotyle of B. napus were investigated.