The Construction Of The Suppression Subtractive Library Of The Brassica Napus Mutant "NDF-1" And The Cloning Of BnD5 CDNA And BnD11 CDNA In The Line "NDF-1"

The line "NDF-1" , which is a dwarf mutant , has been obtained from the doubled haploid Brassica napus inbred line 3529 induced by fast neutron and diethyl sulfate (DES), whose high is one third of that of the wildness line . The leaves of the "NDF-1" mutant are wrinkled and thick compared with those of the wild-type control, and wane earlier than those of the wildness.The subtraction library of dwarf mutant "NDF-1" and its wild type 3529 was constructed by suppression subtractive hybridization (SSH) . In the subtraction, the cDNAs from the "NDF-1" were used as Tester and those from 3529 Driver. The secondary PCR products of the backward subtraction were cloned, were inserted into the pMD18-T vector and transferred into E. coli. About 1000 clones were obtained. These clones in the backward subtraction library were verified by colony PCR and dot hybridization which was labeled by DIG. The lengths of the inserted fragments were between 100bp and 1000bp. By the result of dot hybridization, 32 clones which showed different expression between leaves in dwarf mutant "NDF-1" and its wild type 3529 were sequenced and Blasted in GenBank. The results showed that four reduplicate clones (clone 4 and clone 11, clone 6 and clone 12) were found and the efficiency of difference was 86.2%. Seventeen clones were related to secondary messenger, transformation factor, senescence, the protein decrease and fatty acid metabolism, etc. On the other hand, twelve clones shared homology to unknown protein genes.