| Walnut(Juglans regia L.)is a kind of fruit tree with high economic value.The peel and seed coat of red walnut,a wild walnut germplasm resource in China,are red because they are rich in anthocyanin,which can improve the appearance quality and nutritional value of walnut.Therefore,it is of great significance to analyze the mechanism of its color formation.In this paper,the biological characteristics of red walnut were observed;By measuring the p H value and mineral element content of different tissues at different development stages,the physical and chemical factors of red walnut coloring were systematically analyzed;Further,through the combined analysis of transcriptome and metabolome on the red leaves and green leaves of the natural hybrid progeny of red walnut,the key differentially expressed genes related to anthocyanin synthesis were screened.The key genes of anthocyanin synthesis,chalcone synthase(CHS)and UDP glucose-flavoid-3-oglycosyltransferase(UFGT),were analyzed for sequence and structure,tissue specificity and subcellular localization.The main results are as follows:1、Observation on biological characteristics and determination of physicochemical factors related to color of red walnut(1)The phenological observation results showed that the growth period of red walnut was 221 days,the germination period was in the middle of March,and the fruit matured in the middle of September,other phenological periods were 1 ~ 9 days later than common walnut,indicating that the red walnut was a late-fruiting strain.At the same time,the red walnut is a male-first strain,with a difference of about 1 week between male and female flowers.(2)The p H of red walnut red leaves and peel was higher than that of common walnut,and the p H value of seed coat was lower than that of common walnut seed coat.The p H value of red walnut leaves gradually increased with the greening of leaves,the p H value of peel gradually decreased with the deepening of red fruit,and the p H value in seed coat did not change significantly.(3)The analysis of mineral elements showed that the contents of Fe and Ca in red leaves and red peel were significantly higher than those in green leaves and green peel;Except that the Ca content of red seed coat was significantly lower than that of green seed coat in the early stage of development,it was significantly higher than that of green seed coat in other stages,and the content of other elements had little difference.2.Screening and analysis of genes related to anthocyanin synthesis of red walnut(1)Combined transcriptome and metabolome analysis red leaf(SR)and green leaf(SG)of natural hybrid progeny of red walnut in three periods,the pattern map of anthocyanin synthesis pathway of red walnut was constructed.A total of 17 differentially expressed genes were screened on the anthocyanin synthesis pathway related to color,there were significant differences in the expression of these differential genes in red and green leaves.Combined with the pathway map of anthocyanin synthesis,it was found that chalcone synthase(CHS)and flavonoid glycosyltransferase(UFGT)played a key role in the synthesis of anthocyanin.(2)The analysis of chalcone synthase(CHS)showed that its catalytic activity was significantly higher than that of common walnut in each developmental stage of red walnut.Combined with the four differentially expressed Jr CHSs genes screened by transcriptome,through the structural analysis,it was found that the four Jr CHSs genes contained an intron,and the number and location of protein conserved domains are the same,the protein structure analysis found that the four Jr CHSs genes contained typical CHS functional domain.Phylogenetic analysis showed that Jr CHSs were closely related to CHSs related to anthocyanin synthesis in other species.Subcellular localization analysis showed that the four Jr CHSs encoded proteins were mainly distributed in the cytoplasm and participated in the synthesis of substances.The results of cis-acting element analysis of the promoter sequences of Jr CHSs genes showed that all four Jr CHSs genes contained multiple MYB、MYC transcription factor binding sites.(3)The analysis of flavonoid glycosyltransferase(UFGT)showed that its catalytic activity was significantly higher than that of common walnut in each developmental stage of red walnut.Structural analysis of the two differentially expressed Jr UFGTs genes screened and found that they have no introns,the gene structure is basically similar,and both contain glycosyltransferase-GTB family domains,and the number and position of protein conserved motifs are the same.Analysis of cis-acting elements in the promoters of Jr UFGTs revealed that Jr UFGTs also contained multiple MYB and MYC transcription factor binding sites.In conclusion,the color formation of red walnut has a very important relationship with the p H of cells and the content of mineral elements.Both Jr CHSs and Jr UFGTs genes promote the synthesis of anthocyanin in red walnut. |
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