Positional Cloning And Functional Study Of Seed Coat Color Gene In Dahuang (a Landrace Of Brassica Rapa L.)

Brassica rapa(B.rapa L,2n = 20,AA)is a specie of Brassica genus,belonging to the basic species of cultivated rapeseed.China is the original center of Chinese cabbage and Brassica campestris.Compared with Brassica napus,it has a long history of origin and cultivation and rich genetic resources.It has natural and stable yellow seed resources.At present,a large number of studies have shown that,compared with the common black-and brown-seeded rapeseed,yellow-seeded rapeseed has lots of advantages,such as more clear oil,higher fat and protein content,lower hull rate,higher feed value,lower tannin and other toxic substances.Therefore,as a desirable indicative character,people paid a great attention on the yellow seed trait when breeding.In this study,the gene responsible for the yellow seed trait in Dahuang was fine-mapped by whole genome re-sequencing and genetic linkage map.Cloning and functional analysis of the candidate gene was carried out to identify the target gene.In addition,the dynamic changes of seed coat color during seed formation and the differential expression of candidate gene and other related transcription factors and structural genes in flavonoid pathway were studied.The accumulation of pigment in the seed coat of B.rapa was revealed during the seed development period,which provided preliminary clues for probing the regulatory mechanism of candidate gene and other genes of flavonoid pathway in Brassica rapa.The main results are listed as follows:1.Fine mapping of the seed coat color gene Brsc1 in B.rapa,On the basis of previous studies on the chromosomal loci of Dahuang,8 specific SSR markers and 7 other markers which have been developed before were used to increase the density of the genetic linkage map and the integrated physical map was also obtained,The target gene was located in a 1.08 Mb(A9: 18.26 Mb-19.34 Mb)interval on the A9 chromosome.The results showed that three candidate segments for Brsc1 were located on the chromosome A9(17.83 Mb-18.93 Mb;20.53 Mb-20.99 Mb;22.57 Mb-26.09 Mb)by whole genome re-sequencing.One of the candidate segment(17.83 Mb-18.93 Mb)overlapped with the interval located by the genetic linkage map,and the overlapped segment contained a large number of linked markers that were co-segregated with the target gene.The target region was shortened to 678 Kb(A9: 18.26 Mb-18.93 Mb)according to the result of genetic linkage map and the location of whole genome re-sequencing.2.Cloning and sequencing analysis of Brtt1,a candidate gene of seed color in Dahuang.A total of 46 candidate genes were found in the candidate interval in the BRAD database and the function of these 46 candidate genes were analyzed by homology comparison with whole Arabidopsis thaliana genome sequences.One of the candidate gene BrTT1(Bra028067)is the homologous gene of At1G34790 in Arabidopsis thaliana,which is known as the key gene of flavonoid pathway and mutants of this genes showed significantly reducing of pigment in the seed coat,suggesting that the gene BrTT1 is a key candidate gene for the seed color trait in B.rapa.4586 bp genomic sequence containing BrTT1 was amplified,which contained 1796 bp upstream sequence,986 bp downstream sequence and 1804 bp coding sequence.The gene contained two exons and one intron.In contrast to the reference sequence of BrTT1 in BRAD database,this sequence encodes at 39 bp upstream of the initiation codon,resulting in the first exon of the brown-seeded sequence in B.rapa having 39 bases greater than the reference sequence,but which did not cause frameshift mutation.The results showed that the upstream sequences of BrTT1 and Brtt1 were completely identical between the yellow-and brown-seeded sequence.Compared with the brown-seeded sequence,yellow-seeded sequence had 7 base substitutions in the exon region and 13 SNPs in the intron regions,and there were also 2bp,9bp,114 bp deletion in the intron regions.Further analysis of the amino acid sequence showed that the Brtt1 encoded protein sequence had four amino acid synonymous mutations and three amino acid sense mutations,namely N45 H,S294Y and H299 L.The amino acid sequence of BrTT1 was submitted to the Ex PASy database(http://www.expasy.org).The results showed that BrTT1 encodes WIP zinc finger transcription factor protein,which had the molecular formula C145C148H161H165 and belonged to C2H2 type zinc finger protein.3.Genetic transformation of BrTT1,a candidate gene for seed color in B.rapa.The full length of BrTT1 gene from brown seeded plant was transferred into tt1 mutant CS82 by highlights mediated Agrobacterium tumefaciens.Twenty T1-positive plants were obtained,and the seed coat color was completely restored.The recovery rate was 100%.The T1-positive plants were sub-cultured to T3 generation,and the trait was stable.The gene of BrTT1 had similar function with homologous gene TT1 in Arabidopsis thaliana,involved in flavonoid metabolic pathway.The over-expression vector p35 S :: TT1 and p35 S :: tt1 were constructed and were transformed into the tt1 mutant CS82 of Arabidopsis thaliana by Agrobacterium tumefaciens-mediated trapping method.31 positive plants of T1 generation was obtained by the vector of p35 S :: TT1,and the seed color was completely restored to the wild type.39 positive plants of T1 generation was obtained by the p35 S :: tt1 vector.The seed coat color was close to the mutant CS82.The expression of BrTT1:GFP fusion protein was detected by Agrobacterium-mediated transformation in tobacco leaf epidermal cells and onion endothelial cells.BrTT1 was localized in the nucleus,which was in accordance with the characteristics of transcription factor.4.Differential expression of BrTT1 and other flavonoid metabolism-related genes in the seed developmental stages.The seed coat color changes during the seed formation of yellow-and brown-seed were observed and pictured by stereo microscope.The seed coat color of yellow-and brown-seeds were different at middle stage of seed development(28 days and 35 days after pollination).In the early yellow-seeded stage(21 days,28 days and 35 days after pollination),the seeds showed green color,and the transparent seed coat showed the color of the embryo until the seed germination of the yellow seed material did not change.Seed development and maturity of seeds also showed seed color yellow seed traits.The results of qRT-PCR showed that BrTT1 was mainly expressed in the seed-forming stage,and in the early and middle stages of seed formation(7 days,14 days,21 days,28 days and 35 days after pollination).The expression of BrTT1 was significantly higher in brown seeds than in yellow seeds.The expression level of BrTT1 in brown seeds reached the peak at 21 days after pollination,but the expression level of BrTT1 between yellow-and brown-seeds was not significant at the later stage of seed formation(49 days after pollination).The expression patterns of 8 structural genes and 6 transcription factors related to flavonoid metabolic pathways in rapeseed at different stages of seed formation were analyzed by heat map and expression profile.The expressions of Br TT3,BrTT18 and Br BAN were detected in brown seed material,but not in yellow seed material.Based on the results of TT1 gene's function in other plants,it was concluded that the differential expression of flavonoid metabolism related genes may be the primary cause of the different seed coat colors of yellow-and brown-seeds.The aberrant expression of the regulatory factor BrTT1 in yellow-and brown-seeded material resulted in the aberrant expression or non-expression of the target gene BAN and other structural genes of the flavonoid metabolism pathway,blocked the accumulation of proanthocyanidins,and led to yellow seeds.