Cloning And Regulation Mechanism Of Carotenoid-related Genes Of Different Fruit Colors Of Pepper

Pepper(Capsicum annuum L.)is one of the widely cultivated vegetable crops in the world,which has important economic value and dietary health care effects.As an important component of commodity quality,pepper fruit color is increasingly valued by breeders.In this study,three pairs of pepper mutants with different ripe fruit color were used as research materials to clone,bioinformatically and expressly analyze the genes related to carotenoid biosynthesis in pepper fruit.The NAC transcription factors closely related to fruit carotenoid metabolism were screened out by comparative transcriptomic analysis of peppers with different fruit colors at 25 d,40 d and 55 d after flowering.The functions of NAC transcription factors were verified by q RT-PCR and VIGS.The main findings of this paper are as follows:(1)In this study,three pairs of pepper mutants with different fruit colors were used as research materials,and the key genes GGPS,PSY,LCYB,CRTZ,ZEP and CCS in the carotenoid biosynthesis pathway were cloned,bioinformatics and expression analysis.The results showed that the complete sequences of GGPS,PSY,CRTZ,LCYB and ZEP genes can be amplified in three pairs of different mutant materials;the fulllength CCS genes can be amplified in the three red wild-type and yellow mutant H0809,and in other The two mutants(SP02 and PC02)were not amplified,indicating that the formation of yellow mutant fruit color in SP02 and PC02 is closely related to the deletion of CCS gene,while the formation of fruit color in H0809 may be related to the differential expression regulation pattern of CCS gene.(2)Based on the comparative transcriptome study of red(XHB)and yellow(H0809)pepper fruits at 25,40 and 55 days after flowering,12 differentially expressed genes related to carotenoid biosynthesis and 96 NAC transcription factors were screened out,and pearson correlation analysis was performed between them.The results showed that the CaNAC008 transcription factor was significantly positively correlated with CCS genes in red fruits,and significantly negatively correlated with genes such as PSY1 and PSY2 in yellow fruits,indicating that CaNAC008 genes may be involved in the regulation of carotenoid synthesis and metabolism in pepper fruits.(3)In this study,a total of 109 full-length NAC genes were identified in the pepper genome,and comprehensive analysis of their gene structure,chromosomal distribution,gene duplication,conserved motifs and cis-elements was carried out.A total of 109 NAC genes were identified in the pepper genome,divided into 11 major subgroups,of which 98 were distributed on 12 chromosomes in pepper,and 8 tandem repeat sequences were found to involve 15 NAC genes.The CaNAC008 gene is located at the position of Chr1:182349891-182352960,encoding 248 amino acids;the expression of the CaNAC008 gene was significantly different in different developmental stages of the red material XHB and the yellow mutant material H0809.(4)The function of CaNAC008 gene was verified by q RT-PCR and VIGS.The results showed that after the silencing of the CaNAC008 gene,the expression of the CaNAC008 gene was significantly down-regulated in the in vitro fruits of the red material XHB and the yellow mutant H0809,and the silencing efficiency reached 82%and 84.9%,the phenotype changed significantly;after silencing the CaNAC008 gene,the expression levels of CCS,LCYB and CRTZ were significantly changed,indicating that the CaNAC008 gene may be involved in the regulation of carotenoid biosynthesis in pepper fruit.,thereby affecting the formation of pepper fruit color.The research results preliminarily clarified the structural information and differential expression of genes related to carotenoid biosynthesis in pepper fruit in different mutant materials,and preliminarily revealed the regulatory mechanism of NAC transcription factors in the process of carotenoid biosynthesis in pepper fruit.This provides a reference for further research on the molecular regulation mechanism of pepper fruit color formation in the future.