| Comparing with brown-seeded rapeseed, yellow-seeded rapeseed has a high oil content,high protein content, thin seed coat, low hull and low fiber content, therefore the yellow seed has been paied more attention in rapeseed breeding. In Brassica juncea L. the yellow seed color gene has been mapped on A09 chromosome of Brassica rapa, and its homologous region was located on chromosome 3 of Arabidopsis in previous study.TT6 and TT5 genes are located on chromosome 3 of Arabidopsis thaliana, these two genes were hence identified as the canadiate genes of yellow seed in B. juncea. Therefore,these two two genes will be focused in our study, they were cloned in yellow and brown seed color meterials of three kinds of rapeseed, respectively. And the sequence and structural differences between the three types of rapeseed in two genes were analyzed. The open reading frame(ORF), upstream and downstream sequence, animo acid residues were analyzed using the bio-software to investigate the molecular mechanism of formation of yellow seeds, which will lay the foundation for yellow seed rapeseed transgenic breeding. We analyzed expression of TT6 and TT5 in different tissues and the different developmental stages of seed in B. juncea(Wuqi yellow-seeded mustard and Wugong mustard), the result will further determine TT6,TT5 whether a candidate gene for the yellow seeds. The main results obtained in this study are as follows:1. Cloning and sequence analysis of TT6 and TT5 in B. junceaTT6 and TT5 were isolated from gDNA and cDNA of Wuqi yellow-seeded mustard and Wugong mustard, respectively.(1) TT6 gene was isolated from gDNA of Wuqi yellow-seeded mustard and Wugong mustard, named BjTT6-y1 and BjTT6-b1, their coding regions have 1457 bp and 1513 bp,respectively. And those of TT5 were named BjTT5-y1 and BjTT5-b1, and the coding regions were 1621 bp and 1623 bp, respectively.(2) Sequence analysis showed TT6 genes isolated from Wuqi yellow-seeded mustard and Wugong mustard containd three exons and two introns. Bj TT6-b1 sequence was consistent with Bra036828 exactly. The ORF of TT6 from Wuqi yellow-seeded mustard and Wugong mustard were 1077 bp, encoding 358 amino acids. And there were seven single nucleotidedifferences between BjTT6-b1 and BjTT6-y1, where the 760 th nucleotides were different(Wugong mustard as T, Wuqi yellow-seeded mustard as G)which resulted in differences in the 254 th amino acids(Wuqi yellow-seeded mustard as phenylalanine(F), Wugong mustard as valine(V)).(3) Sequence analysis showed TT5 cloned from Wuqi yellow-seeded mustard and Wugong mustard genes containd four exons and three introns, consistent with that of Bra007142. Sequences of BjTT5-y1 and BjTT5-b1.1 were identical. The ORF of BjTT5-y1 and BjTT5-b1 were 759 bp, encoding 252 amino acids. There were four single nucleotide differences between BjTT5-b1.1 and BjTT5-b1.2, in which the 346 th nucleotides(G/T) were different, the stop codon appearance caused termination of translation. The ORF of BjTT5-b1.2 was 348 bp, encoding 115 amino acids, resulting in differences between protein structure and other aspects of the physical and chemical properties.2. Sequence analysis of gene TT6 and TT5 in three types of rapeseedsCloning of cDNA sequence from B.juncea, B.rapa and B.napus was conducted by homology-based cloning, and sequences of three types of oilseed rape TT6 and TT5 genes were analyzed. The results were as follows:(1) There were 2 different sites among TT6 sequences of the three types of rapeseed,respectively, the 510 th and 897 th nucleotide. All of B.napus brown-seed materials were A, but all of B.rapa brown-seed materials were G at 510 th site. All of B.juncea yellow-seed and brown-seed materials were G at 897 th site. The 510 th, 760 th, 834 th in B.rapa and the 30 th,126th, 378 th in B.napus were also found in B.juncea, in which the differences at 760 th nucleotides caused a change in the amino acid. In addition, in the three types of rapeseed, TT6 gene in B.juncea was highest homology, followed by B.rapa, and the lowest one was B.napus.(2) 2 different sites among TT5 gene sequences of the three types of rapeseed were identified respectively, the 202 th and 345 th nucleotides. In both sites, B.rapa yellow-seed materials were A and G, respectively, while B. juncea brown-seed materials were C and A.The difference at 202th(A/C) nucleotide resulted in an amino acid change(threonine T/proline P). In addition, in the three types of rapeseed, TT5 gene in B.rapa was highest homology, followed by B.napus, and B.juncea was lowest.3. Phylogenetic analysisThe results on sequence analysis and phylogenetic were as follows:(1) For TT6, the genetic relationship between rapeseed and other species from near to far distance were followed by Arabidopsis, corn and grapes, tomatoes, soybeans. The near genetic distance between B.juncea and B.rapa TT6 showed yellow- and brown- seeded genes in B. juncea may come from B.rapa. But TT6 genes from different yellow- and brown- seedmaterials were complex in B.napus, and their origin could not be determined.(2) For TT5, genetic relationship between rapeseed and other species from near to far distance were followed by cabbage, rice, Arabidopsis, grapes, oranges, soybeans and green beans. B.rapa and B.napus showed near genetic distance. TT5 genes were centralized in B.napus brown seeds, and nearest with BrB2-TT5, therefore, TT5 in B.napus brown-seed may be derived from BrB2(Jingyang rapeseed)4. Expression AnalysisExpression of genes TT6 and TT5 in different tissues and the different developmental stages of seed in B. juncea(Wuqi yellow-seeded mustard and Wugong mustard) was analyzed,the results showed that:(1) In Wuqi yellow-seeded mustard and Wugong mustard, BjTT6 were expressed at leaves, buds, flowers, pericarps and seeds at different times. The expression had significant tissue specificity, and that of Wuqi yellow-seeded mustard and Wugong mustard was inconsistent. In the buds of Wuqi yellow-seeded mustard showed highest expression level(89.216), followed by 23 DAP seeds(40.078), and the leaves were lowest(1.325). In 9DAP seeds of Wugong mustard showed highest expression level(176.013), followed by 16 DAP seeds(87.862), and pericarp were lowest(2.116). For BjTT6 gene, the expression in leaves,9DAP, and 16 DAP seeds in Wugong mustard was significantly higher than the expression of the same tissue in Wuqi yellow-seeded mustard. And the expression of BjTT6 gene in buds,flowers, 23 DAP and 30 DAP seeds in Wuqi yellow-seeded mustard was significantly higher than the same tissue in Wugong mustard.(2) Both Wuqi yellow-seeded mustard and Wugong mustard, For BjTT6, expression in the early seed development was higher than the expression of the late seed development. In the early seed development, the expression of BjTT6 gene in Wugong mustard was rapidly decreased from the peak(176.013) to a minimum 23DAP(2.435), after then a slight increase with seed maturation was followed. In Wuqi yellow-seeded mustard, expression of BjTT6 increased with a slight expression in 23 DAP peak period(40.078), and then gradually reduced to 45 DAP minimum(3.255) in the early seed development.(3) In Wuqi yellow mustard, BjTT5 expressed lower in leaves, buds, flowers, pericarps and seeds at different times without tissue specificity. In Wugong mustard, BjTT5 could express and had obvious expression of tissue-specific in the leaves, buds, flowers, pericarp and seeds. The gene showed highest expression level(6.922) in buds, followed by flowers(1.620), and 9DAP seed expression level was the lowest(0.095).(4) Both Wuqi yellow-seeded mustard and Wugong mustard, BjTT5 expression in the early seed development was lower than the expression of the middle and late seeddevelopment. In the early stage of seed development, expression of BjTT5 in Wuqi yellow-seeded mustard was higher than that in Wugong mustard. In the late stage of seed development, expression the gene in Wuqi yellow-seeded mustard was lower than Wugong mustard. Expression of BjTT5 in Wugong mustard increased to a peak rapidly with seed development(1.123) in the early seed development, andafter then maintained in a stable level for seed maturation. In Wuqi yellow-seeded mustard, BjTT6 showed low expression in whole development of seed. In the early and middle seed development, expression of BjTT5 increased to 30 DAP peak period(0.678), then gradually decreased with the expression of mature seeds.(5) In the development of seed formation, expression of BjTT5 in the late stage was higher than that of early, indicating that BjTT5 possiblely worked in flavonoid pathway of seed late development, but in the whole development of the seed, the expression of genes BjTT5 was low. In the development of seed formation, the expression of BjTT6 in the late stage was lower than that of early, indicating that BjTT5 also possiblly worked in the flavonoid pathway of seed early development.In addition, in the seed early development, expression of BjTT6 showed obvious differences between Wuqi yellow-seeded mustard and Wugong mustardit, which was inferred BjTT6 may play a role in yellow- ane brown- seed formation in B.juncea.5. Development of yellow seed color gene makerCompared with BjF3H-b1, a deleted 55 bp fragment was identified in the first intron of BjF3H-y1. Based on this discovery, a characteristic molecular maker of TT6 gene was developed successfully, which was designated as IP1 by using PCR and electrophoresis techniques.6. The application of SSR markers in purity identification of seedsIn this study, SSR technology was used to detect the hybridseed purity in Shanyou 16(a Brassica napus hybrided variety.). The results showed that 3 pairs of SSR primers, SSR313,SSR354 and SSR359, showed polymorphysm, and they perfomed co-dominant nature,therefore these three markers could be used in detection of hybridseed purity in Shanyou 16. |
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