Regulation Of Anthocyanin Synthesis Functional Analysis Of BnaPAP2.C2 In Brassica Napus

Anthocyanins are a group of pigments with strong antioxidant activity and widely found in plants.At PAP2 regulates anthocyanin synthesis in Arabidopsis thaliana,and belongs to the MYB family of transcription factors.There are 9paralogues of BnaPAP2 in B.napus（designated as BnaPAP2.A2,BnaPAP2.A3,BnaPAP2.A7.a,BnaPAP2.A7.b,BnaPAP2.C2,BnaPAP2.C3,BnaPAP2.C6.a,BnaPAP2.C6.b,BnaPAP2.C6.c）.However,BnaPAP2.C2 was expressed in green leaf and purple leaf of B.napus.There showed the different function between BnaPAP2.C2 and At PAP2,but the function and regulation mechanism of BnaPAP2.C2 were not clear in B.napus.In this study,the biological functions of BnaPAP2.C2 in B.napus and its regulatory mechanism were preliminarily analyzed by expression pattern analysis,promoter analysis,creation of mutant with loss of function,and anthocyanin content determination,which provides a reference for the study of synergistic effect of multi-copy genes in polyploid.The main results are as follows:1.The 2.8 kb promoter and 1.7 kb gene sequences from PR,Zhongshuang 11 and Westar were compared,and the gene structure of BnaPAP2.C2 was the same as that of the At PAP2 gene,which contained 3 exons and 2 introns.Sequence alignment showed that the 2.8 kb promoter region and exon region of BnaPAP2.C2 in purple and green rape were identical,and only four bases,ATAT,were missing in the first intron of Westar（+439 bp）.2.The 2.8 kb promoter sequence of BnaPAP2.C2 was compared with the 1.9 kb promoter sequence of BnaPAP2.A2.It was found that there were two fragments of486 bp and 463 bp inserted in the BnaPAP2.C2 promoter.Bioinformatics analysis of the motif of the two inserts revealed that multiple transacting factors could be bound.Expression vector validation and population analysis showed that the two inserts were enhancer elements and were ubiquitous in B.napus.3.The full-length c DNAs of BnaPAP2.C2 from PR were constructed on the PDOE-20 vector,and Bna FLC.A10 gene was used as the nuclear localization reference.The transient expression was carried out in the tobacco system,and the fluorescence signal was observed under FV1200 laser confocal microscope.It was found that BnaPAP2.C2 protein was localized in the nucleus.4.Based on transcriptome sequencing analysis,it was found that BnaPAP2.C2 was mainly expressed in the leaves of PR and Zhongshuang 11,while its expression was very low in other tissues.The dynamic expression patterns of BnaPAP2.C2 in leaves of 8 cultivars of spring type rape（Westar,NO2127）,winter type rape（Quinta,Tapidor）and semi-winter type rape（Gangan,Shengli,Zhongshuang11,Zheyou7）ecotypes at different developmental stages（24,54,82,115 and 147 days after sowing）were analyzed.It was found that the expression of BnaPAP2.C2 in leaves of different ecotypes of B.napus reached the peak at about 115 days after sowing.The dynamic expression trend of BnaPAP2.C2 among different ecotypes was similar.5.The exon of BnaPAP2.C2 gene was edited by CRISPR/Cas9 technology.Six mutant plants were obtained from T₀ generation with Westar as the transgenic receptor,among which 2 were undecoded,3 had 1bp base insertion,and 1 had 5 bp base deletion.Two mutant plants were obtained from T₀ generation with Tapidor as receptor,but none of them were decoded.A total of 160 mutant plants were obtained from transgenic T₀ generation using PR as the receptor,among which 155 plants were terminated prematurely due to base insertion and deletion.There were 18 with amino acid frameshift mutations due to base deletion.Mutations in the fourth and fifth amino acids due to base insertion and deletion occurred in 13 strains.Only one showed no change in amino acid translation due to base substitution.A total of 31overexpressed BnaPAP2.C2 T₀ generation positive plants were obtained using PR as transgenic receptor.6.The leaves of 10 plants with premature termination mutation,10 plants with overexpression of BnaPAP2.C2 in T₀ generation and 5 negative control plants were selected for the determination of anthocyanin content.The average content of anthocyanin in the leaves of the BnaPAP2.C2 mutant was 17.7μg/g and that of the negative control was 15.5μg/g.The average content of anthocyanin in the leaves of the mutant was higher than that of the negative control,but the difference was not significant.The average anthocyanin content of OE-BnaPAP2.C2 plants was 29.4μg/g,and that of the negative control plants was 33.4μg/g.The content of anthocyanin in the leaves of the overexpressed plants was significantly lower than that of the negative control plants.