Map-based Cloning And Functional Study Of Seed Coat Color Gene In Barssica Rapa

With the sustainable development of national economy, more and more breeders pay attention to rapeseed quality. Yellow-seeded rapeseed is characterized with thinner seed coat and lower hull percentage, which in turn is correlated with higher oil and protein content compared to black-seeded rapeseed under the same genetic background. In addition, the rough oil color is shallow and clear of yellow-seeded rapeseed, which can reduce the manufacturing cost and easy to popular with consumers. So they have been the excellent germplasm resources for rapeseed quality breeding. But there were no natural yellow-seeded material in Brassica napus. This situation brings difficult problem in yellow seeded traits application. As the basic species of rapeseed, B.rapa has abuntant yellow-seeded germplasm, which used to create several artificial synthetic yellow-seeded varieties of B. napus. Through the method of cytology and molecular biology, the seed coat color gene has been cloned in B.rapa. The main points in this study as follow:1. The seed coat color gene was located on linkage group9corresponding with the two molecular markers Isr126and bsrl. In addition, we developed co-separation SSR molecular markers that linked to the seed coat color gene from the Scaffold sequence on A9from http://brassicadb.org/brao/.Blast analysis with the Arabidopsis genome showed that the sequence was similar to a region of chromosome4. There are22Arabidopsis genes in the region including at4g09820to at4g10620.2. BrTT8gene was seleced as candidate gene, we used tail-PCR technology and B.rapa genome sequences as reference for designing specific primer pair Tu1L and Tu1R to clone the full-length TT8ortholog gene by PCR amplification.3. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. The sequence analysis showed that the inserted fragment contained the structural characteristics of a recently discovered class of transposable elements in eukaryotes, termed Helitron. The insertion was4320bp, starting with5’TC, ending with3’CTAG, and containing two short palindromic sequences that were possibly formed by the17-bp hairpin that was located near its3’terminus.4. We determined the level of mRNA expression in the seeds of the yellow-seeded line in comparison with that of the black-seeded line, to detect the expression changes of the Brassica TT8orthologue gene due to the insertion. There was no mRNA expression detected in the immature seeds of yellow-seeded line. 5. The BrTT8gene encodes a putative bHLH protein consisting of520amino acids with a predicted molecular weight of59.5kD, and a pI of5.45(http://www.expasy.org/tools/protparam/). The BrTT8protein sequence contains a typical bHLH signature near the C terminus that corresponds with a putative binding domain (http://www.expasy.ch/tools/scanprosite/).6. Through the method of agrobacterium-mediated transformation, BrTT8gene was transported into Arabidopsis mutant tt8-1for function complementary experiments.The T2seed progeny originating from25independent T1kanamycin-resistant transformants exhibited phenotypic reversions in seed color compared with the wild type. Furthermore, analysis of PAs deposition during seed development was performed by staining PAs with vanillin in the wild type and T2. These data indicated BrTT8was also involved in the PA synthetize pathway.7. The expression level of five flavonoid genes, from two group EBGs and LBGs, were analyzed during the development of seeds in yellow-seed line and black-seed line, respectively. Quantitative Real-time PCR (QRT-PCR) analysis revealed that BrTT8was necessary for the expression of flavonoid LBGs, such as LDOX and BAN in the developing seeds, similar to TT8in Arabidopsis.8. Histological analysis showed that the black color of seeds was also due to the PAs accumulation in the plant endothelium layers of immature seeds. However, there was no pigment present in the seed coats of the yellow-seeded plants. Compared with the black-seeded plants, there was no accumulation of red granules in the endothelium layers of yellow seed coat after Safranine O and Fast Green treatments. But compare the seed coat structure of mutants tt8-1with wild-type in Arabidopsis, we found that only the pigment accumulation was different from the wild type, and no change was detected in structure of the seed coat cell Iyer in the tt8-1mutant. Which means BrTT8gene is similar, not completely the same, on the function with TT8gene that participate in the flavonoids synthesis. The BrTT8gene seems to have effect on the structure of seed coat.9. Yellow sarson is a major yellow-seeded germplasm, and used to create artificial synthetic yellow-seeded varieties of B. napus No.2127-17. BrTT8gene doesn’t show polymorphism in the nearly isogenic line population of No.2127-17. These data showed that the seed coat color main QTL locus of B. napus on N9linkage group is not derived from the BrTT8gene. In conclusion, we reported the isolation and functional characterization of the BrTT8in this study. The molecular mechanism of seed coat colore formation between the black and yellow seeded line in nearly isogenic line population as forllow:the alleles in the yellow seeded line showed that a transposable element could disturbed the BrTT8expression in B.rapa. BrTT8encodes bHLH proteins, which is important transcription factor in plant. Additional experiments also demonstrated that BrTT8modulated the expression of flavonoid "LBGs", and the BrBAN could not be normal transcripted, and the proanthocyanidin synthesis biological was blocked. So there was no pigment present in the seed coats of the yellow-seeded plants, and the seed coat showed the colorless, transparent. Therefore, the seed colore also became the yellow.