The Construction Of The Suppression Subtractive Library Of The Brassica Napus Mutant Cr3529 And The Cloning Of Toc33 CDNA In The Line Cr3529

To research the differential expressed gene of the etiolation mutation, a subtracted library of Cr3529 was constructed by using the suppression subtractive hybridization (SSH). The secondary PCR products of forward subtraction were cloned, insert into T/A vector and transferred into E.coli. In forward subtraction, the cDNA from the Cr3529 seedlings leaf was used as Tester and the cDNA from 3529 was Driver, screened the forward subtraction library with colony PCR and dot hybridization labeled by DIG, 125 clones was identified as differential expressed cDNA fragments in 3529 line. After sequencing the 38 randomly chosen clones, there are two redundant clones and four novel sequence fragments, other clones were involved in the protein metabolism, photosysthesis process, the nucleotide metabolism, fatty acid metabolism, the energy metabolism, the stress-response and hormone function, the express level of these genes were changed greatly after the suppression subtractive hybridization, that is, they were obviously enriched by SSH.Two primer, 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, Brassica napus, by RT-PCR technique. The RT-PCR results showed that the fragment homologous to Toc33 was expressedin Cr3529. PCR fragments were inserted into the pMD18-D vector and transferred into E. coli, then the cDNA clone, BnToc33-c, was obtained. Sequence analysis showed that the sequence was 894bp and the nucleotide and the deduced amino acid sequences was highly homologous to those of A. thaliana. There were three diverged nucleotides between the Cr3529 and the wild type 3529 Toc33 cDNAs, i.e, GGT/AGT, TTG/TTT, AGG/AGT, all which belonged to missense mutation. The amino acid replacement ((Leu/Phe) caused by TTG/TTT mutation located in the membrane anchor domain may affect the targeting into the chloroplast, reduce the insert of TOC33, also maybe influence the GTPase enzyme of the protein, thereby interrupt the protein translocation of the chloroplast outer membrane and cause the transport obstruction, the change may result in chlorophyll-reduced character in Cr3529.