In this study, we used five different periods of colour trait changes in the fruit of Panax japonicus as test material, and determined the effects of chlorophyll, carotenoids, anthocyanosides, flavonoids and total phenols on the changes of fruit colour traits during the growth and development of Panax japonicus by using enzyme labeling assay. The results of the physiological indexes of Panax japonicus fruits indicated that the pattern of chlorophyll content in the pericarp decreased with the colour change of the fruits, which was verified in the process of colour trait changes of fruits of chilli peppers [11], yellow-fleshed kiwifruit [12], and cherry tomatoes [13], suggesting that in the maturation of the fruits of the plants, their chlorophyll content would gradually decrease and be converted into other pigments to show different colours; and the effect of chlorophyll content on the colour trait changes of Drosera fruits during growth and development of Drosera fruits was determined by carotenoids and total phenols. This conclusion is the same as that of Guo Yongmei [11], Huang Chunhui [12] and Ruan Meiying [13] in the study of chilli, kiwifruit and tomato; the changes in the content of anthocyanin in the fruits of Drosera showed a trend of gradual increase, while the synthesis of flavonoids and total phenolics showed a trend of increasing, then decreasing, and then increasing again. This phenomenon was also confirmed in the changes of fruit colour traits of Viburnum [14], Capsicum annuum [15], blueberry [16–18], apple [19] and mulberry [20]. The above analysis pointed out that the gradual degradation of chlorophyll and the gradual accumulation of carotenoids in the ripening process of Drosera fruits, and the simultaneous synthesis of anthocyanin, flavonoids and total phenolic substances are conducive to the change of fruit colour traits.
High-throughput sequencing technology was used to sequence the transcriptome of Panax japonicus fruit for the first time, and a total of 93.94 Gbp of transcriptome data was obtained from 15 samples of Panax japonicus fruit colour change, and 462117 functional annotations were obtained after clustering by Corset hierarchy. Transcriptome sequencing analyses of Pentaphyllaceae plants revealed that Total Unigenes 135,235 single genes were obtained by transcriptome sequencing of flowers, leaves, roots and rhizomes of Panax japonicus (Rai et al, 2016) [21]; and Panax notoginseng roots, leaves and flowers were transcribed and sequenced. Total Unigenes 107,340 single genes (Liuet al, 2015) [22]; Panax quinquefolius root transcript sequencing annotated a total of 21,684 (Sun et al, 2010) [23], which shows that the same genus of Pentaphyllum is differently organised and the results of transcript sequencing are different. The total number of DETs generated by comparing the five periods of G, Y, R, B, and P in the fruit of Duchess with each other was 17,895 (9435 up-regulated and 8460 down-regulated) differentially expressed Unigenes (DETs), respectively. Transcriptome sequencing of ‘Ningqi No.1’ and ‘Ningqi No. 7’ fruits at the green fruit stage, colour change stage and ripening stage obtained 2827, 2552 and 2311 differentially expressed genes at the green fruit stage, colour change stage and ripening stage (Liu Xuexia et al., 2023) [24]; Sequencing of black fruit Lycium barbarum and white fruit Lycium barbarum obtained 25,279 differentially expressed genes of black fruit Lycium barbarum and white fruit Lycium barbarum (Bao Xuemei et al., 2023) [25]. It can be seen that the total number of DETs obtained by sequencing the transcriptome of the fruits of Panax japonicus is larger than that of ‘Ningqi No. 1’ and ‘Ningqi No.7’, but smaller than that of the black-fruit goji berry and white-fruit goji berry.
Weighted co-expression network analysis of colour trait variations (DETs) in Panax japonicus fruits was carried out by WGCNA method to reveal candidate hub gene modules regulating colour trait variations in Panax japonicus fruits. The results indicated that a total of 10 co-expression modules were obtained in the co-expression network analysis, and 448 hub genes related to carotenoids, anthocyanins, flavonoids and total phenols in DETs were identified in the MEblack module. In the study of Capsicum annuum fruit colour traits, 17 modules were obtained for co-expression network analysis, of which 397 genes were involved in the accumulation of carotenoids in the ‘Darkgreen’ module (Li Quanhui, 2022) [26], and 14 co-expression modules were identified in nine developmental stages of Luffa aegyptiaca fruit, of which the Turquoise module was associated with fruit length and length. The Turquoise module was significantly correlated with fruit length and fruit diameter, and 10 candidate genes for the potential control of fruit length in lucerne were obtained (Chen Min-Ron et al., 2023) [27]. Four of the 23 modules in the analysis of the gene coexpression network related to quality traits in okra were involved in regulation (Tian Rumeng, 2023) [28], 15 modules in the gene coexpression network related to drought tolerance in the root system of potato, and a one-guinea gene coexpression module for root drought tolerance was obtained Qin Tianyuan et al. 2020)[29], and 15 modules were obtained from the Genetic Basis of Biological Functions of Maize Plant Height and Spike Height (Ma Juan et al. 2020) [29]. co-expression modules (Ma Juan, 2020) [30]. Six modules of gene co-expression were obtained in transcriptional regulators of banana fruit ripening (Kuang et al, 2021) [31]. It can be seen that different plant fruit traits (DETs) can be obtained as candidate genes for trait-related modules by the WGCNA method.
Anthocyanins are one of the important parts of pigments produced by the flavonoid biosynthetic pathway, and the key enzymes and structural genes involved in the flavonoid biosynthetic pathway have been confirmed accordingly in fruits (Jaakola, 2013) [32]. GO and KEGG Pathway functional enrichment analyses of DETs for colour trait changes in Panax japonicus fruits yielded 56 genes for flavonoid metabolism in the GO database and 149 genes for flavonoid biosynthesis in the KEGG Pathway database, which may be involved in the regulation of colour trait changes in D. japonicus fruits during ripening;A total of 29 differentially expressed genes were significantly enriched in the isoflavonoid biosynthesis pathway by KEGG Pathway analysis of DETs at three stages of Capsicum annuum green ripening fruit colour development (Quanhui Li, 2022) [26]; Ficus carica pericarp transcriptome KEGG Pathway flavonoid biosynthesis has 74 differentially expressed genes enriched (Li Jing, 2020) [33], 4 different varieties of chilli pepper fruit colour metabolome and transcriptome comprehensive analysis, KEGG Pathway based on the WGCNA method to identify 7 flavonoid synthesis candidate genes (Liu et al., 2020) [34]. Studying five aubergines with different fruit colour traits in KEGG Pathway obtained 144 DETs enriched in aubergines, including flavonoid metabolites (Zhou et al., 2022) [35]; the flavonoid pathway enriched DETs in the fruits of Panax japonicus were all more than that of chilli pepper green ripening [26, 34], figs [33] and aubergines [35], which is also the process colour trait variability. Secondly, KEGG Pathway enrichment of DETs for fruit colour trait changes in Panax japonicus yielded 19 genes in the photosynthesis-antenna protein pathway, which may be involved in the chlorophyll metabolism pathway; the green colour of aubergine pericarp is mainly affected by chlorophyll KEGG Pathway enrichment yielded 27 DETs related to chlorophyll metabolism (Zhou X et al. 2022) [35], and wheat breeders indicated that the up-regulation of photosynthesis-antenna protein genes could maintain the green colour of leaves for a long time. ) [35], wheat breeders indicated that the up-regulation of photosynthesis-antenna proteins genes can maintain the green colour of leaves for a long time, KEGG Pathway enrichment found 46 differentially expressed genes involved in photosynthesis-antenna eggs (Li Q et al., 2022) [36], in grape (Vitis vinifera) fruit study, 122 DETs were found in KEGG Pathway enriched photosynthetic antenna proteins (Wang Q et al.,2019) [37]. The candidate genes enriched in the KEGG Pathway for photosynthesis-antenna proteins for colour trait changes in the fruits of DETs were less than those of aubergine [35], wheat [36], and grapes [37], suggesting that DETs' unique growth environments of high altitude, high humidity, and high shade are inherently caused by the unique growth environment of DETs.
In order to further prove the reliability of the transcriptome data for the functional enrichment of key candidate genes for the colour trait changes in the fruits of Panax japonicus, six highly expressed candidate genes of the photosynthesis-antenna protein pathway were selected by real-time fluorescence quantitative PCR (qRT-PCR) for qRT-PCR validation. The results pointed out that with the same pattern of change of transcriptome data, the expression of green fruit stage (G) was the highest; the gene expression decreased gradually with the maturity of fruit. The information of the candidate genes was obtained and named as PjCABⅠ, PjCABⅡ, PjCABⅢ, PjCAB1, PjCAB2, PjCAB3 through NCBI database comparison. 9 highly expressed key candidate genes for colour trait changes were selected in KEGG Pathway flavonoid biosynthesis metabolism pathway for qRT-PCR verification. The results pointed out the same results with the transcriptome data, and the information of key candidate genes were obtained and named as PjF3‘H, PjF3’H1, PjCHI, PjCHI1, PjCHS, PjCHS1, PjDFR, PjANS, and PjC3'H through the comparison of NCBI database, and it is noteworthy that the PjDFR gene had the highest expression among the nine candidate genes. It was found that the chlorophyll content during kiwifruit fruit development was highly correlated with chlorophyll degradation-related genes, especially three chlorophyll a/b binding proteins (CABs) genes play an important role in chlorophyll degradation (Tu, Mei-yan et al, 2021) [38]. Chlorophyll a/b binding protein (CAB) genes were also obtained during peach fruit (Prunus persica ' Hujingmilu ') development as key degradation proteins for chlorophyll metabolism (Zhou et al, 2018) [39]. These studies are highly consistent with the decrease in chlorophyll content and expression of six CAB genes of the related photosynthesis - antenna protein pathway during fruit development in Panax japonicus. Flavonoid metabolites are important pigmentation factors that play important roles in leaf, flower, fruit and seed tissues [40, 41]. The key enzyme genes CHI, CHS and F3′H were found to be the main factors affecting pericarp color in the color formation mechanism of passion fruit (Xin M et al. 2021) [42]. The key flavonoid biosynthesis genes MiF3H and MiANS genes in the post-ripening process of green ripe fruits of the red mango variety Zill were also expressed in the variation of color traits (Zhu W et al. 2023) [43]. All of these findings indicated the same as the nine candidate genes screened in the flavonoid biosynthesis pathway in Zill fruits, further suggesting that these candidate genes are the key enzyme genes for color trait changes in Panax japonicus fruits.
The changes of color traits in the fruit of Panax japonicus showed an opposite trend in the dynamic pattern of chlorophyll content and carotenoids, anthocyanins, flavonoids and total phenolics in physiological and biochemical aspects. 19 candidate genes in the photosynthesis-antenna protein pathway related to chlorophyll degradation were also obtained in the transcriptome analysis, and 6 key candidate genes for high-expression were verified by qRT-PCR, and 81 candidate genes were obtained in the flavonoid biosynthesis metabolic pathway, and 9 key candidate genes for high-expression were selected. 81 candidate genes were obtained in flavonoid biosynthesis metabolism pathway, and 9 highly expressed key candidate genes were selected for qRT-PCR validation. Physiological, biochemical and transcriptomic analyses revealed the mechanism of fruit color trait changes in S. dubiae, and that the degradation of chlorophyll and the synthesis of carotenoids, anthocyanosides and flavonoids were synchronized in the fruit.