Study On Flower Color Formation Metabolism Mechanism And Key Genes Function Of Camellia Nitidissima

Known as the"Queen of Camellia"and the"Panda in the plant world",Camellia nitidissima has a special golden flower color,which increases its ornamental value and makes it a rare genetic resource for the breeding of yellow camellias.In order to known the main color substances and metabolic process of its flower color,and to explore the key genes and regulatory mechanism,this study did a systematic study used C.nitidissima as the main material.We did the analysis on the connection between flower color and main cellular environment factors,measured the main component contents related to flower color and performed transcriptome sequencing to find the key genes and verify their function,etc.This study showed the metabolic mechanism and molecular regulation mechanism of the flower color formation in C.nitidissima,which made a foundation for molecular precise breeding of yellow camellias.(1)Nine species in Chrysantha sect.were used to measure their petal colors,total flavonoid content,water content,cell pH and contents of seven kinds of metal ion.The results showed that the mean value of lightness L*,hue a*,hue b*,saturation C*,hue angle h was80.82,-2.88,53.97,54.10,93.19,respectively.The petal color of yellow camellias was regarded as bright yellow and hue b*is the main index to describe the color.The content of total flavonoid and water in petals was measured to 20.17%and 88.14%separately,both of them were significant among different species and not closely related to petal color.The pH in petal cells was 6.19 in average and significant among the species.The pH in petal cells was positively correlated with petal color.There were significantly differences for seven kinds of metal ions among these species.Besides the content of K⁺was the highest,with an average of12.61 mg·g^-1,and that of Cu²⁺was the lowest,with an average of 0.0038 mg·g^-1.The contents of Al³⁺,Fe³⁺and Ca²⁺were negatively correlated with the flower colors.The medium weak acid cell environment and low contents of Al³⁺,Fe³⁺and Ca²⁺were conducive to the yellow flower color of C.nitidissima.(2)The contents of 12 flavonoids,9 polyphenols,5 carotenoids and 2 anthocyanins in 3flowers organs(petal,stamen and sepal)of 23 species in Chrysantha sect.and 6 species with 5bud developments were determined by HPLC.The flower color was measured by the Color Testers,23 species could be divided into 5 categories:gold yellow,orange yellow,yellow,light yellow,red and yellow.In the flowering process,the yellow color index(hue b*)increased initially and then decreased.The main components of flowers color are flavonoids and polyphenols.The main components of flavonoids were quercetin 7-O-glucoside(Qu7G),quercetin 3-O-glucoside(Qu3G)and rutin(Ru)and the main components of polyphenols were epicatechin(EC),epigallocatechin(EGC)and catechin(C).Anthocyanin was only found in the petals or sepals of a few species,and the main component was cyanidin 3-glucoside(Cy3G).The content of carotenoids was very low,and the main components were zeaxanthin(Zea),xanthophyll(Xan)and?-carotene(?-Ca).The hue b*was mainly proportional to the contents of Qu7G,Qu3G and total flavonoid(TF).The main metabolites of flower color were Qu7G and Qu3G.(3)Transcriptome sequencing was carried out by Pac Bio RS II and Illumina Hi Seq Xten.A total of 19.13 Gb of clean data was obtained by Pac Bio RS II sequencing.Illumina Hi Seq Xten sequencing got a total of 264.40 Gb of Clean Data,with Q30 reached 93.17%.A total of 67018 differentially expressed genes were obtained.The correlation analysis showed that FLS gene was highly correlated with the major components of flower color(Qu7G/Qu3G/TF),which was an important candidate functional gene.MYB transcription factor had a large number of genes that were significantly correlated with the main color indicators(Qu7G/Qu3G/TF),which was speculated to be an important regulatory transcription factor.WGCNA analysis found that FLS was the main Hub gene,closely related to F3'H and CHS that were co-expressed genes and MYB111 was a transcription factor in key modules.(4)PCR amplification obtained a CnFLS gene,a CnDFR gene,a CnF3'H gene,a CnUFGT15 gene,a CnUFGT14 gene and a CnMYB111 gene.The real-time quantitative PCR was used to measure the genes expression levels in different tissues of C.nitidissima.And the subcellular localization of genes was observed and the function of genes was verified by agrobacteria-mediated leaf disk transformation in tobacco.In the petals of C.nitidissima,the expression of CnFLS was positively correlated with the yellowness of flower color(hue b*),while the expression of CnF3'H and CnDFR was negatively correlated with the yellowness of flower color(hue b*).After the agrobacterium-mediated leaf to transform tobacco,it showed that in the flowers of positive lines of CnFLS,the content of polyphenols was not significantly different from that of wild-type tobacco while the contents of Qu3R,Qu7G and Qu3G increased significantly in the highly expressed strains.However,the content of flavonol was very low,which did not change the flower color of transgenic tobacco.In the positive flowers of overexpression CnF3'H,the contents of polyphenols and flavonoids in the positive lines with high expression levels were significantly higher than that of wild-type tobacco.In the positive flowers of CnDFR,the contents of polyphenol in the positive lines were significantly higher than that in the wild-type tobacco,while anthocyanin was not detected in flowers.It indicated that CnFLS mainly promoted the synthesis of flavanols,CnF3'H gene mainly promoted the synthesis of polyphenols and CnDFR gene promoted the synthesis of polyphenols.However,the overexpression of CnUFGT14,CnUFGT15 and CnMYB111 did not promote the synthesis of flavonols,which were no significant regulatory effect on yellow flowers.(5)The functional verification of tobacco was carried out by constructing double genes expression vector.After overexpressed CnFLS and antisense expressed CnF3'H,the content of polyphenols decreased and the content of flavonoid increased,which reduced the effect of CnF3'H gene on promoting the synthesis of polyphenols and increased the accumulation of flavonoids.Overexpression FLS and antisense expression DFR significantly inhibited the formation of polyphenols and promoted the synthesis of flavonoids,but the promotion was not significant due to its low expression.Compared with overexpression CnFLS gene and CnUFGT14 gene alone,the effect on the synthesis of polyphenols of overexpression CnFLS and overexpression CnUFGT14 was not obvious,but the effect on the synthesis of flavonoids was greater than that of overexpression of CnFLS gene alone.In all,the formation of golden flower color of C.nitidissima was caused by the synthesis of some flavonols,such as Qu7G and Qu3G,while the cell pH with neutral weak acid and low concentration of Al³⁺,Fe³⁺and Ca²⁺provided favorable conditions for the formation of golden color.In terms of molecular regulatory mechanisms,it was mainly positively regulated by CnFLS gene and negatively regulated by CnF3'H,CnDFR.High expression of FLS gene and low expression of F3'H and DFR were more conducive to the formation of yellow flowers.