Analysis On The Coloring Mechanism Of Ornamental Pepper Fruit

Fruit color is one of the main factors affecting the ornamental value of ornamental peppers.The formation of ornamental pepper fruit color is related to the accumulation of carotenoids,anthocyanins and chlorophyll and the transcription level of related genes.In this study,the extraction and determination conditions of anthocyanins in ornamental peppers were optimized,and the changes in fruit color and pigment content during fruit growth and development were tracked and determined.The gene expression patterns of carotenoid synthesis pathways were analyzed.We analyzed the gene expression pattern of carotenoid synthesis pathway,screened the important genes that affect carotenoid synthesis in ornamental pepper fruits,and identified the function of this gene.In addition,the role of CCS gene in the coloring of mature fruits was clarified,and the reason for the appearance of orange in mature fruits of ornamental peppers was revealed,which laid the foundation for the research on the coloring mechanism of ornamental peppers.The main findings are as follows:1.The extraction conditions of anthocyanin from ornamental pepper were optimized by single factor method and response surface method.The optimum p H,equilibrium time and equilibrium temperature of p H differential method for anthocyanin determination were screened,and the recovery test and stability investigation were carried out to verify the accuracy of the extraction and determination conditions.The single factor test and response surface methodology determined the best extraction process was 90 %methanol(1% HCl)as extraction agent,solid-liquid ratio of 1:25 g·m L-1 and ultrasonic-assisted extraction for 40 min.When measured by p H differential method,the best p H is 1 and 4.5,and the best equilibrium temperature and time is 40℃ for 90 min.2.According to the changes in the color chromaticity parameters(L*,a*,b*),carotenoids,anthocyanins,and chlorophyll content during the growth and development of the ornamental pepper fruits,12 ornamental peppers were divided into 3 categories.The first category included cultivars B3,J1,J2 and Q3,cultivars M3,M3’,W8 and WU belonged to the second category,and the third category included C2,S1,W3 and W4.During the growth and development of the ornamental pepper fruit,carotenoids begin to accumulate with the gradual degradation of chlorophyll and anthocyanins.Capsanthin,zeaxanthin,andβ-carotene are gradually increasing.Our paper showed that there were significant correlation between important genes of carotenoid biosynthetic pathway,among which CCS and CRTZ-2 was highly correlated with,PSY gene CRTZ-2 was highly correlated with CCS.Analysis of the expression patterns of genes related to the carotenoid synthesis pathway shows that the GGPS gene is an important gene for inducing carotenoid precursor substances,and the PSY gene is the first rate-limiting enzyme in the carotenoid synthesis pathway.In addition,the expression levels of CRTZ-2 and CCS genes reached peak in the red ripening stage.And CCS genes were considered to be important genes that affect the carotenoid synthesis of ornamental peppers.To further verify the important role of CCS gene,TRV-mediated VIGS gene silencing technology was used to silence CCS gene after transcription.Infected red cultivars B3 and C2 failed to turning color successfully,showing orange color.3.Based on previous studies,the chromaticity values,total carotenoid content and relative expression of CCS gene of orange,yellow and red ornamental pepper fruits were analyzed and compared.The results showed that the a*,total carotenoid content and CCS conversion abundance of red cultivars were significantly higher than those of orange and yellow.In addition,we successfully cloned full-length CCS genes of different cultivars,it was found that red cultivars(B3,C2,W6)could amplify the full-length CDS of CCS genes with 1497 bp,encoding 498 amino acids;Orange cultivars G1,W9,G3,W4-3,W4-2 and W5 can amplified 1496 bp encoding 494 amino acids.Orange cultivars S1-1,B7,W4-1 and yellow cultivar G2 failed to amplify CDS fragments.There was a deletion of "C" base at 1265 bp in the CDS fragment of orange cultivars by sequencing,which leads to frameshift mutation,and then affects the coding of amino acids.The information will contribute to the research on the coloring mechanism of ornamental pepper fruit and provide a theoretical basis for the breeding of ornamental pepper fruit color.