Transcriptomic Analysis Of Orange-red Petals And Genetic Research On The Anthocyanins Metabolic Pathway In Brassica Napus L.

Brassica napus(AACC,2n = 38)is the third largest oil crop around the world after soybeans and palms.It has good ornamental value,as a result of beautiful flowers and the long flowering period.At present,the bright yellow is the conventional flower color of rapeseed,thus it's slightly monotonous to enjoy the flower color.This has caused vigorous development the tourism of rapeseed flower to be limited.During rape breeding,some flower color mutants have been found successively,such as golden yellow,lemon yellow,orange-red,milky yellow,milky white,white,white flowers with light purple silk and yellow-white chimeras.If these mutants can be used to develop new varieties of colorful rapeseed with stable inheritance without affecting yield,it will effectively increase the value of rapeseed tourism and increase the income of farmers in rapeseed producing areas.At present,the coloring mechanism of rapeseed petals is not clear.In this study,transcriptomics was used to analyze the orange-red flower line R03 and yellow flower line ZS11.At last 13 candidate genes related to the anthocyanin metabolism pathway were screened.Seven candidate genes were molecularly cloned and the resulting sequences were analyzed.Bioinformatics analysis was performed;six overexpression vectors for these candidate genes were finally constructed,and the basic biological functions of candidate genes were verified by transient expression of tobacco leaves and transformation of Arabidopsis thaliana.The main conclusions are as follows:1.Transcriptomic Analysis of Brassica napus with Orange-red / Yellow FlowersTranscriptome sequencing was performed between the petals of the orange-red flower line R03 and the yellow flower line ZS11 at the stage of before color changed(S1)and after color changed(S2).1368 and 1235 differentially expressed genes were found between R03-S1 and ZS11-S1,R03-S2 and ZS11-S2,respectively,by the standard of the threshold parameter P-value ?0.001,the multiple of the difference | log2(FoldChange)| ?8,and the error detection rate FDR ?0.001.And there are 476 common differential expressed genes(DEGs)between those two parts of DEGs.GO enrichment analysis was performed on these three parts of DEGs,which were all enriched to binding,cells,cell components,cellular processes,and metabolic processes.The topGO enrichment analysis of these DEGs was further performed.In the "Molecular Function" annotation,entries related to anthocyanin synthesis were proliferated,including proanthocyanidin biosynthesis process,anthocyanin-containing complex biosynthesis process,phenylpropane,biosynthesis process,flavonoid biosynthesis process,anthocyanin-containing compound metabolism process,cyanidin 3-O-glucoside metabolism process,etc.In the KEGG enrichment analysis,only 789 genes of 2127 DEGs had annotation entries.The enrichment results showed that these DEGs were significantly enriched to metabolic pathways,such as phenylpropane biosynthesis and flavonoid biosynthesis.Based on the key structural genes,transcription factors and transporter genes in the anthocyanin biosynthesis pathway of Arabidopsis thaliana,a total of 133 genes related to anthocyanin biosynthesis were identified in Brassica napus using colinearity analysis,including 69 Structural genes,55 regulatory genes and 9 genes encoding transporters.Combined with transcriptome data,13 key genes related to anthocyanin biosynthesis were selected,including 9 structural genes(BnC4H_A4,BnDFR_A9,BnDFR_C9,BnANS_A1,BnANS_C1,BnANS_A3,BnANS_C7,BnUGT79B1_A10,BnUGT79B1_C9),3 regulatory genes(BnPAP1_A7,BnTT8_A9,BnMYBL2_C6),and one transporter gene(BnTT19_C9),and all of them have high expression levels in orange-red material R03.The candidate genes were verified by qRT-PCR,and the results were basically consistent with the transcriptome data.2.Sequence analysis of candidate genesSeven genes(BnDFR_A9,BnDFR_C9,BnANS_A1,BnANS_C1,BnANS_A3,BnANS_C7,and BnPAP1_A7)were selected from 13 candidate genes,and their CDS sequences were cloned and 1,1,1,3,2,1 and 1 copy were sequentially obtained from the orange-red flower line R03.And 1,1,1,1,2,1 and 1 copy were sequentially obtained from the yellow flower line ZS11;homologous sequence alignment results showed that in the orange-red flower color line material there were no differences in the sequences of BnDFR_A9,BnDFR_C9,BnANS_C1,BnANS_C7,and BnPAP1_A7 compared with that cloned from the yellow flower line ZS11,but there were stable differences in the sequences of BnANS_A1 and BnANS_A3.3.Functional analysis of candidate genesFor the genes cloned from the orange-red flower line R03,six over-expression vectors were built successfully,and named pEarlygate101-BnANS_A1,pEarlygate101-BnANS_A3_R03.1,pEarlygate101-BnANS_C1_R03.1,pEarlygate101-BnANS_C1_R03.2,pEarlygate101-BnANS_C7 and pEarlygate101-BnPAP1_A7,respectively.The six over-expression vectors were subcellularly localized in tobacco leaves.Among them,BnANS_A1,BnANS_A3_R03.1,and BnPAP1_A7 were expressed only in the nucleus;BnANS_C1_R03.1 and BnANS_C1_R03.2 were expressed in both the cytoplasm and thenucleus,and the signal in the nucleus is significantly stronger than that in the cytoplasm;BnANS_C7 is only expressed in the cell membrane.These six over-expression vectors were transiently expressed in tobacco leaves by agrobacterium-mediated injection,and no anthocyanin accumulation was observed.Finally,T1 generation transgenic seeds of over-expressed BnANS_A1,BnANS_A3_R03.1,BnANS_C1_R03.1,BnANS_C1_R03.2,BnANS_C7 and BnPAP1_A7 were successfully transformed by inflorescence infection in Arabidopsis.Subsequent research will observe the change in petal color of these genetically modified materials.