| Brassica napus L is one of the most important oil plants in the world, and it is also the most popular rapeseed species in China. Besides the increase of yield, the enhancement of oil content and improvement of oil composition are core goals of oilseed breeding. Combining modern biotechology and traditional breeding method to create yellow-seeded B. napus cultivars with excellent quality is the hotspot of modem oilseed breeding. In Arabidopsis thaliana, it is revealed that in the molecular and biochemical pathways to the formation of testa pigments, any function-losing mutation will lead to the block or feebleness of sediment of testa pigments and the color of seed coat will turn yellow even transparent from brown. 21 Transparent Testa (77) loci have been identified in A. thalicana to date. Both B. napus and A. thaliana belongs to Cruciferae and the manner of testa pigment phenotype formation in these two species is much similar, so the research results about A. thaliana transparent testa are helpful to elucidate the molecular mechanism of yellow seed formation in 3. napus. In natural, B. napus are all black-seeded and has no yellow seed coat Yellow seed coat resulted from artificial composition of tetraploid B. napus or from distant hybridization with relative species of B. napu is instable in seed color phenotype. Currently the most important task for the breeding of yellow-seeded B.napus is the clarification of the molecular mechanism of yellow seed phenotype, clone yellow-seed genes and adopt create artificial yellow-seeded B. napus using genetic engineering.Chalcone synthase is the key enzyme in the formation pathway of plant pigments such as flavonoids and anthocyanin, so it is one of the most important enzymes relative to the pigment in seed coat, petal, and so on. In A. thaliana, functional defection of CHS is identified as TT4 locus leading to transparent testa. WRKY44, encoding a WRKY transcription factor, plays an important role in the production of tannin in A. thaliana endothelium. Its functionally defective mutation is identified as TTG2 locus leading to transparent testaConsidering the importance of these two loci for the production of seed pigments in Cruciferae, the homologous gene fragments of 7T4 and WRKY44 were cloned in this research. According to the reported CHS gene sequence in Genbank, we designed primers to amplify CHSA and CHSB, members of CHS gene family, from genomic DNA of black-seeded and yellow-seeded B. napus materials simultaneously. Through comparative analysis of CHS gene seqences, the relation between the fromation of yellow seed coat and CHS genes in our yellow-seeded cultivar will be discussed. The homologous gene of WRKY44 was also cloned from black-seeded B. napus using the primers designed according to conserved regions of A. thaliana WRKY44.The black-seeded and yellow-seeded B. napus genomic DNA samples were PCR amplified using FCHSA/RCHS pair pair and the both resulted in a 1300bp bands. After fragment recovering, subcloning, sequencing and bioinfamatics analysis, it is found that both the black-seeded and yellow-seeded lines have a 1287bp sequence which is almost the same as CHSA2 gene that has sedimented in Genbank. CHSA2 is a functional gene encoding chalcone synthase with intact open reading frame, so for the CHSA2 gene locus, there are no differences in black-seeded and yellow-seeded B. napus, suggesting that the formation mechanism of yellow seed coat in our yellow-seeded B. napus line has nothing to do with CHSA2 gene.Though the amplification of CHSB gene in both black-seeded and yellow-seeded B. napus genomic DNA samples using FCHSB/RCHS primer pair resulted a 1500bp band, sequencing results showed that the 1514bp sequence cloned from black-seeded material is almost the same as CHSB\ gene in Genbank, while the 1500bp fragment from yellow-seeded material is quite different from both CHSB\ and CHSB2 gene sequences. BLAST analysis demonstrated that in this research black-seeded material have the functional gene completely identical to CHSBl, but...
|
PDF Full Text Request