Clone And Sequence Analysis Of A Brassica Napus A09 Mapping Interval

Brassica napus mutants are important materials for functional genomics research.Based on the previous work on a dwarf-related mutated trait mapping in Brassica napus,present studies has cloned eight candidate genes for the trait,and analyzed the sequence characteristics and expression patterns of these genes.The main results are as follows:1.Auxin response factor gene(ARF):ARF is a transcription factor,it can binding with promoter region of auxin response elements,facilitating or inhibiting gene expression to affects plant growth and development,most of the Arabidopsis thaliana AtARFs class 1 genes can inhibit gene expression,and having closest relationship with BnARF we studied,so BnARF mutations may cause B.napus drawf.①The gene was obtained from mutant and wild-type’s GDNA,it contains 12 introns and 13 exonsin the two materials,there are 1 nucleotide difference in the introns and 2 nucleotides difference in the extrons,amino acids sequence also have 2 mutant sites,and they all encoding 540 amino acids,having three conserved domains,and the two amino acids mutant sites are in the B3-DNA binding and AUXIN-IAA conserved domains,it may affect gene function.②Phylogenetic analysis showed the gene is closest to ARF class 1.③Expression analysis showed that expression level of the gene in wild-type stems is extremely significantly higher than mutant,mutant seven-days roots and hypocotyls expression level are extremely significantly higher than wild-type,cotyledons expression level is also significantly higher than wild-type;there are no significant differences in other tissues.2.We also cloned Nucleotide sugar protein gene(NSTs)、Root growth factor gene(RGF)、18S pre-ribosomal assembly protein gar2-related gene,there are no difference in their GDNA,so it can not determine whether they cause B.napus drawf.3.Sulfotransferase genes(STs):Sulfotransferase can sulfonate plant hormones to regulate plant growth and development.In this work,three BnST genes in the mapping interval were cloned from mutant and wild-type’s cDNA.These three BnSTs all have sulfotransferase-1 domain,and are different in the conserved domain between the two materials,thus may possiblely lead to drawf trait in B.napus.1)BnaA09g53490D gene:①The open reading frame(ORF)of these two genes from the mutant and the wild are all 972 bp in length.These two genes encode 323 amino acids,but have 30 amino acids difference in the gene’s ST1 domain,and one amino acids difference in the gene’s 5’PSB and 3’PB.②Expression analysis showed that the expression level of the gene in seven-days roots of wild-type is significantly higher than the mutant;expression level in the flowers was also significantly higher than the mutant,however its expression level was also significantly lower than the mutant.③Overexpression vectors of these two genes were constructed,and used to transform oilseed rape by floral-dip method,resulting in 11 TO transgenic plants.2)gene:①The open reading frame(ORF)ofthesetwo genes from the mutant and the wild are all 978 bp in length.These two genes encode 325 amino acids,but have 32 amino acids difference in the gene’s ST1 domain,and one amino acids difference in the gene’s 3’PB.②Expression analysis shows that the expression level of the gene in wild-type flowers and buds is extremely significantly higher than the mutant,cotyledons expression level is also significantly higher than the mutant,mutant stems expression level was significantly higher than the wild type,there are no significant difference in other tissues.③Overexpression vectors of these two genes were constructed,and used to transform oilseed rape by floral-dip method.3)BnaA09g53480D gene:①The open reading frame(ORF)of these two genes from the mutant and the wild are all 807 bp in length.These two genes encode 268 amino acids,but have 29 amino acids difference in the gene’s ST1 domain,and one amino acids difference in the gene’s 5’PSB.②Expression analysis shows that the expression level of the gene in mutant flowers is extremely significantly higher than the wild-type,stems and pods expression level is also significantly higher than the mutant,there is no significant difference in other tissues.③Overexpression vectors of these two genes were constructed,and used to transform oilseed rape by floral-dip method,resulting in 8 TO transgenic plants.4)gene:① It was obtained from mutant and wild-type’s GDNA,it contains 7 introns and 8 exons in the two materials,there are 113 nucleotides difference in the introns and 6 nucleotides difference in the extrons,amino acids sequence also have 2 mutant sites,they all encoding 589 amino acidss,having two conserved domains,and the two amino acids mutant sites are not in the conserved domains,it may not affect gene function.②Expression analysis showed that expression level of the gene seven-days roots is extremely significantly higher than other tissues in the two materials,wild-type seven-days roots expression level was extremely significantly higher than mutant,stems and flowers expression level are also significantly higher than mutant;there are no significant differences in other tissues.