| Anthocyanins,the main water-soluble pigments that accumulate in leaves,petals,sepals and fruits of plants,endow color,attract insects for pollination,and protect against biotic and abiotic stresses.Moreover,anthocyanins are well known for their applications in human health to protect against cardiovascular disease,diabetes,cancer and vision loss due to their free radical-scavenging abilities.Therefore,the food rich in anthocyanin is increasingly popular to consumers and the market.Heading Chinese cabbage[Brassica campestris L.ssp.pekinensis?Lour?Olsson],a crop native to China,is the largest cultivated vegetable with the highest consumption in China.Since the purple cabbage is not found in nature,the cabbage rich in anthocyanin has both important nutritional value and universal health significance.Thus,it's very urgent to study the mechanism of anthocyanin accumulation in purple-head cabbage,which not only fulfils the knowledge of anthocyanin metabolism in plant but also provides theoretical basis and technical support for the improvement of purple-head cabbage.In this study,Chinese cabbage with purple heading leaves supplied by Chinese cabbage research group in Northwest A&F University was selected as the test material,and the original female parent Chinese cabbage with white-head and the male parent flowering Chinese cabbage supporting genetic background of purple trait were treated as two controls.We identified and cloned the key gene that control the character of purple-head Chinese cabbage through combination of the expression results about anthocyanin biosynthesis genes and related accumulation characters in different leaves position.By comparing the sequence structure of the gene among the original parents and the new purple-head Chinese cabbage,we found that there are significant differences among the three lines.In addition,we developed codominant function markers of the gene based on the DNA sequence difference in three Chinses cabbages,and validated the codominant markers in F2 group acquired from crossing between the purple-head Chinese cabbage and white-head Chinese cabbage,which further illustrated the gene was the key factor in controling the purple trait in purple-head Chinese cabbage.Meaningfully,we validated the gene function through genetic transformation the key gene into wild-type Arabidopsis with related complementary test.We further investigated effect of the gene to anthocyanin biosynthesis pathway in Arabidopsis.These results clearified the mechanism of purple trait formation in Chinese cabbage.This research not only enriches the anthocyanin metabolism regulation theory in cruciferous plant,also provides theoretical basis and technical support for the genetic improvement about leaf-head of Chinese cabbage.The main results of this paper were listed as following:1.Microscopic observation of sections from the leaves of purple-head Chinese cabbage revealed that the purple pigment mainly resided in three cell layers under the upper epidermis and two cell layers under the lower epidermis respectively,whereas the the number of purple layers decreased in both the upper and lower epidermises in purple trait donor flowering Chinese cabbage.However,the number and the accumulation area of anthocyanin cell layers of these two materials were identical in stalk,and young silique in these two purple plants.Total anthocyanin content of purple-head Chinese cabbage dropped dramatically from the internal heading leaves of external heading leaves,which showed extremely high correlation coefficient with total antioxidant ability,whereas total antioxidant ability was poorly correlated with the contents of carotenoid and chlorophyll.2.The anthocyanins of purple-head Chinese cabbage were more stable at pHs below 3.0and temperatures below 45?.The absorption peak of anthocyanin appears redshift accompanied with increase of pH,and the absorption peak of anthocyanin could disappear after the redshift when the pH and tempeareture were both increased.The purple pigments of purple-head Chinese cabbage were anthocyanins,and the main components of them were highly glycosylated and acylated cyanidins.3.A regulatory R2R3-MYB gene named‘BrMYB2'that controlled the purple trait of purple-head Chinese cabbage was isolated according to the results from combination of the expression results about anthocyanin biosynthesis genes and related accumulation characters in different leaves position.This novel gene was located in the candidate area which was reported in previous report about the map-based cloning of the purple gene in our purple-head Chinese cabbage,which combied map-based cloning and homology cloning.4.We acquired the sequence of the gene by using homology-based cloning technique in three Chinese cabbages and analyzed using sequence alignment analysis:the promoter sequence of the three materials is different with several differences in a single CT-repeat microsatellite sequence area;in the gene sequence,the gDNA and CDS of purple-head cabbage was identical with that of the purple flowering Chinese cababge,whereas there was a significant difference with the white leaf cabbage.In the gDNA area of the gene,there was a big 3773 bp deletion and several base differences in the first intron of the gene in purple-head Chinese cabbage;in the CDS of the gene,there were two SNP between purple-head Chinese cabbage and the white-head Chinese cabbage,which lead to the change of two amino acids.Tissue specificity of the gene expression was that BrMYB2 showed higher expression in tissues close to flower buds,flower stalk,and stalk leaves in purple-head Chinese cabbage and its purple trait donor.5.According to the large segment difference in the first intron region of BrMYB2,the function codominant markers‘Marker1'and‘Marker2'were developed,these functional markers can accurately distinguish the purple and white-head materials at seedling stage.On one hand,it improved the breeding efficiency and assistant selection of the purple-head cabbages.On the other hand,it was further explained that BrMYB2 was the key gene to control the purple trait of Chinese cabbage from the genetic view.6.Genetic transformation of promoter area of BrMYB2 about three Chinese cabbages indicated that the number of CT-repeat would not affect the expression of downstream gene.Promoter with the loss of the CT-repeat still remained activity of high expression in downstream gene.These results indirectly showed that the different expression of BrMYB2was not caused by the difference of promoter.7.According to the genetic transformation of BrMYB2 CDS into the Colombia wild-type Arabidopsis,we found that the introduction of CDS of BrMYB2 from both purple-head Chinese cabbage?35S-Brmyb2?and white-head Chinese cabbage?35S-Brmyb2?can also lead to identical purple phenotype,similar expression of BrMYB2 and related anthocyanin biosynthesis genes in wild-type Arabidopsis though there were two SNPs.These results indicated that the two SNPs in CDS of BrMYB2 can not lead to loss of the gene function.Meanwhile,the CDS of BrMYB2 from both purple-head Chinese cabbage and white-head Chinese cabbage had identical function,and the difference of the leaf head of Chinese cabbages was not generated by the SNP of BrMYB2.8.We further investigated complementary analysis of the genetic transformation about the gDNA of BrMYB2.Over-expression of gDNA of BrMYB2?35S-gBrMYB2,with 3773 bp deletion in the first intron?about purple-head Chinese cabbage lead to purple/red color in wild-type Arabidopsis and which was deeper than the transformation of only CDS of BrMYB2,whereas the insertion with 3773bp in the first intron of gDNA of BrMYB2?35S-gBrmyb2?about white-head Chinese cabbage can not lead to purple/red phenotype.These results illustrated that the deletion mutation in the first intron of BrMYB2 can promote gene expression of BrMYB2 in both purple-head Chinese cabbage and its purple dornation.The large insertion in the first intron of BrMYB2 was the reason to repress expression of the BrMYB2 in white-head Chinese cabbage,which was without anthocyanins accumulation. |
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