Primary Analysis Of The Differentially Expressed Genes In The Shoot Apical Meristem At The Beginning Of The Stem Elongation In The Dwarf Mutant '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 height is one third of that of the wild type . 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 as 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 were labeled by DIG. The lengths of the inserted fragments were between 100bp and 500bp. By the result of dot hybridization, 30 clones, which showed differential expression between dwarf mutant "NDF-1" and its wild type 3529 were sequenced and Blasted in GenBank. The results showed that thirteen clones have explicit results; five clones were related to signaling, fatty acid metabolism, hormone, the protein decrease and metabolism, etc. On the other hand, eight clones shared homology to unknown protein genes.Based on a known fragment from the backward SSH library,RACE was applied to amplify the 3' -end of G6 clone for the purpose of the further studying .After two rounds of PCR amplification, a 3' -end cDNA about 350 bp was obtained. BLAST research showed it was homologous with a gene encoding a chitinase from Arabidopsis thaliana at the ratio of 86%.Then the differential expressed gene , was semi-quantitied by reverse transcription cDNA synthesis and RT-PCR technique.The result showed that G6 gene was only expressed in stem shoot and not expressed in the roots ,the stems ,the cotyledons and the leaves in mutant "NDF-1".In the related parts,G6 was not expressed in wild type. These results showed that G6 clone was likely related to the dwarf mutant of the Brassica napus.It's necessary to the further studying.