| High yield is a main goal of rapeseed breeding, so many researchers tried to find methods to increase the seed number per silique and seed yield, and one of the effective methods is to create multilocular rapeseed. Researchs on multilocular trait mainly focused on its inheritance and morphological observation at present. However, the cloning of the multilocular genes and the molecular mechanism of multilocular silique development haven’t been reported. In our previous work, the genomic region containing the multilocular gene, ml4, was fine- mapped to an approximately 38.6-kb DNA fragment through the map-based cloning. In the present study, we tried to confirm the function of the multilocular candidate gene Br CLV3 as well as the molecular mechanism analysis of the gene in the multilocular silique development. The main results of this study are as follows:1. The comparison of the floral organs, siliques and SAMs between multilocular mutants and wild type showed that the multilocular mutant contains more stamens and carpels, and most of its siliques have 4 locules with a shorter, rounder and thicker shape, and extra gynoecium inside. Histological analysis of the SAMs and gynoecium in the wild type and mutants during different development showed that the enlarged SAMs in mutants increased gradually with plant development, which might relate to the generation of multilocular siliques; Observation of the gynoecia indicated that the carpel numbers were initiated from early stages of the developing gynoecium in the multilocular mutant.2. The analysis of gene structure revealed that the Br CLV3 gene is consisted of three exons and two introns and encodes a small peptide of 94 amino acids with a secretion signal peptide at its N terminus and a conserved CLE motif at its C terminus. Comparative sequence analysis revealed a C-to-T nucleotide substitution in the third exon in the Brclv3 allele and the missense mutation resulted in the 9t h Pro substituded by Leu in the core C LE motif.3. The overexpression vector and complementary expression vectors of Br CLV3 allele were transformed into mutant(clv3-2) of Arabidopsis and the multilocular mutant of B. rapa. Part of the transgenic lines recovered to wild type siliques in several independent transgenic lines which dicated that Brclv3 is the gene that controls the formation of multilocular siliques. In addition, We also generated transgenic plants constitutively overexpressing the Brclv3 allele both in the clv3-2 and wild type Arabidopsis genetic backgrounds, none of the transgenic plants exhibited any obvious changes compared with their respective controls, which indicated that Br CLV3 don’t have an antagonistic effect in wild type.4. We conducted in vitro assays using the synthetic dodecapeptides Br C LV3 and Br CLV3Leu9, which represent the wild type and mutated version of the CLE motifs, respectively. And found Br CLV3 peptide severely suppressed the growth of root, root apical meristem and shoot apical meristem of Arabidopsis and B. rapa; while Br CLV3Leu9 lose this kind of the function. These results indicate that the Br C LV3 peptide can function in both Arabidopsis and B. rapa, whereas the Pro-to-Leu substitution completely disrupts the function of Br CLV3 and might be responsible for the formation of multilocular siliques in the P12 mutant.5. The multilocular mutant of B. rapa was used as the recurrent parent, and crossed with wild-type to build a nearly isogenic line through backcrossing and molecular marker-assisted selection. The analysis in BC4F2 population showed that the plants with recessive homozygous genotype have multilocular silique(silique locule number greater than two) and compared with the plants of other genotypes, the length of siliques decreased, but silique seed number per silique and thousand seed weight increased significantly. Thus, as a newly founded gene, Brclv3 in B. rapa will be a valuable resource and have great potential for the yield breeding of B. rapa in the future. |
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