Three highly identical cDNA clones of APETALA3 (AP3) gene, BnAP3-2, BnAP3-3 and BnAP3-4 were isolated from Brassica napus L. by RT-PCR. The sequence analysis showed that all the three AP3 cDNAs contained a complete open reading frame. Their nucleotide sequences had 91 -97 % similarity and their predicted amino acid sequences shared 93 -98 % identity. Real-time quantitative RT-PCR result showed that all the three BnAP3 genes were expressed at the transcriptional level in petals as well as stamens. Among the three BnAP3 genes, BnAP3-3 was expressed at the highest level and BnAP3-2 was expressed at the lowest level in petals. The transcription level of BnAP3-3 was 1.59 times than that of BnAP3-2. The transcription levels of BnAP3-2, BnAP3-3 and BnAP3-4 in stamen were 7.75, 5.11 and 3.88 times than those in petal, respectively. The yeast two-hybrid assays results showed that all the three BnAP3 proteins could form strong heterodimers with BnPI, and obviously different dimerization affinities among the three proteins to BnPI were observed. The ratio of the affinity of BnAP3-2, BnAP3-3 and BnAP3-4 to BnPI-1 was 1.27:1:1.62. Although the three BnAP3 genes were highly identical, the differences of their expression and affinity of protein interaction might reflect some functional divergence.
The floral organ morphogenesis of the apetalous flower mutant Apet33-10 in Brassica napus was investigated and the result showed that all the floral organ morphogenesis was normal except that petal primordium was not observed during flower development. Eighteen genes were found to be down regulated in early floral buds (less than 200 mum in length) of Apet33-10 at the stage of floral organ initiation by means of suppressive subtraction hybridization (SSH) and RT-PCR. These genes were involved in petal identity, calcium iron signal transduction, mRNA processing, protein synthesis and degradation, construction of cytoskeleton, hydrogen transportation, nucleic acid binding, alkaloid biosynthesis and unknown function. Three overall coding region cDNAs of APETALA3 (AP3) gene, BnAP3-2, BnAP3-3 and BnAP3-4 were obtained by RT-PCR, respectively. Real-time quantitative PCR analysis showed that the expression ratio among BnAP3-2, BnAP3-3 and BnAP3-4 was 3.67:3.68:1 in early floral buds of wild type Pet33-10. The expression level of BnAP3-2, BnAP3-3 and BnAP3-4 in early floral buds of Apet33-10 was down-regulated to 36.6, 28.3 and 66.8% with the comparison of that of wild type, respectively, and the overall expression level of AP3 genes in apetalous mutant amounted to 45.0% of that in wild type. The difference in the expression level of each AP3 gene in stamen between apetalous and wild type lines was not significant. It is suggested that lower abundant expression of AP3 genes during the early flower development might be enough for stamen primordium initiation, but not enough for petal primordium initiation in the apetalous line Apet33-10.
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