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.
Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.Additional key words: oilseed rape, RNA gel blot analysis, sequence analysis, subtractive library.
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