Regulatory Genes Mining Related to Transcriptome Sequencing and Phenolic Metabolism Pathway of Canarium album Fruit with Different Fresh Food Quality

doi:10.13560/j.cnki.biotech.bull.1985.2023-0747

Abstract

Abstract:

【Objective】 The category and content of phenolic compounds in fruits are important quality traits closely related to the nutrition and flavor of Chinese olives（Canarium album）. This work is to explore the molecular regulation mechanism of phenolic biosynthesis in Chinese olives.【Method】 The fruits of Chinese olives（low-phenol/high-phenol）with significant differences in total phenol content were taken as test materials, and the transcriptome analysis was carried out 80-160 d after flowering, and the differential genes of shikimic acid-hydrolyzed tannin/phenylpropane-flavonoid biosynthesis pathway were characterized, and the differentially expressed genes were analyzed by WGCNA, from which the transcription factors related to phenolic metabolism pathway were mined.【Result】 The 296 314 Unigenes were obtained, of which 73% were annotated to the database. A total of 1 628 differentially expressed genes（DEGs）were identified in the mature fruits of four varieties（lines）of Chinese olive. KEGG analysis showed that DEGs was significantly enriched in the “flavonoid biosynthesis” pathway of phenolic metabolism pathway. Furthermore, DEGs of shikimic acid-hydrolyzed tannins/phenylpropane-flavonoids biosynthetic pathway in fruit ripening process was characterized. Combining R² and P values of each module and character in WGCNA analysis, four key modules were screened out. According to the regulation relationship of genes in modules, MCC topological analysis was used to mine key transcription factors in modules with degree value ≥1. A total of 137 transcription factors Unigenes were found to be co-expressed with 30 phenolic synthetic structural genes Unigenes. The functional annotations of the transcription factors Unigenes were from 35 gene families, the most of which were zinc finger proteins（C2C2, C3H, C2H2 and PHD）, followed by B3, HB, MYB and NAC gene families.【Conclusion】 This study initially analyzed the differences in phenolic metabolic pathways in Chinese olive fresh food quality from a transcriptomic perspective, and concurrently excavated the differential genes regulating phenolic metabolism, which may provide an important basis for further investigation of the molecular mechanisms underlying the differences in Chinese olive fresh food quality.

Key words: Canarium album（Lour.）Rauesch., transcriptome, phenolic metabolic pathway, transcription factor

XIE Qian, JIANG Lai, HE Jin, LIU Ling-ling, DING Ming-yue, CHEN Qing-xi. Regulatory Genes Mining Related to Transcriptome Sequencing and Phenolic Metabolism Pathway of Canarium album Fruit with Different Fresh Food Quality[J]. Biotechnology Bulletin, 2024, 40(3): 215-228.

Figures/Tables 12

Table 1 Primer sequences for RT-qPCR

基因ID Gene ID	引物序列Primer sequence（5'-3'）	扩增品种（系）Amplification variety（lines）
Cluster-0.119989-F	TGACTTCTGTGTCCTGCTCTT	DQc、TQc
Cluster-0.119989-R	GGTAGTGCCAACTGCTTCTTC
Cluster-0.121834-F	CTTGACATGAATACAGCAGAGGAT	DQc、TQc
Cluster-0.121834-R	GCACCATCAATTTCAGCAACTT
Cluster-0.121871-F	TCTGTGTCCTGCTCTTTGAAG	DQc、TQc
Cluster-0.121871-R	GCAACCTCAGAGATGGGAAG
Cluster-0.121919-F	TAACCATAACGCCTCCATCTCT	DQc、TQc
Cluster-0.121919-R	TCATCTCACGAGACACAATAGACT
Cluster-0.121949-F	TAACCATAACGCCTCCTTCTCT	DQc、TQc
Cluster-0.121949-R	GCTCCTACAATCACCACATCAG
Cluster-0.155662-F	AGACTATGACGAGCAAGACAACT	DQc、TQc
Cluster-0.155662-R	GTTACCACACCACCGACTCT
Cluster-0.119990-F	CTGTTGCCGTAACTTCACTGT	TQc
Cluster-0.119990-R	CTGAGAGATGGGAAGCGTTTC
Cluster-0.120915-F	ATCATTCTCCAGGCATTTCTCAG	TQc
Cluster-0.120915-R	GGAACAGCCACAGCTTGTAT
Cluster-0.121783-F	CAGGAAGTGTCAGCAACATCAA	TQc
Cluster-0.121783-R	CTGTTCTTCGCAAGGTTCATCT
Cluster-0.121896-F	AGGAGTGAAGTGGTTGAAGAGTA	DQc
Cluster-0.121896-R	ACAATCATCCCAGGCACAATC
Cluster-0.122437-F	TAACCATAACGCCTCCATCTCT	DQc
Cluster-0.122437-R	ATCAGTGTCCGCATGTGTAATC
Cluster-0.122522-F	CCTCCATCTCTGCTTCTTCCT	DQc
Cluster-0.122522-R	TGTACCTGCGTGTCATCTCA
18S rRNA-F（内参基因）	CCTGAGAAACGGCTACCACA	DQc、TQc
18S rRNA-R（内参基因）	CACCAGACTT GCCCTCCA

Fig. 1 Total phenol content in Chinese olive fruit ripening process and maturity period * indicates significant difference at 0.05 level, ** indicates significant difference at 0.01 level, and *** indicates significant difference at 0.001 level

Table 2 Sequence splicing results of Chinese olive transcriptome

类型Type	序列条数Number of sequences	序列平均长度Mean length/bp	N50/bp	N90/bp	序列总碱基Total bases/bp
Unigene	296 314	1 163	1 764	522	344 722 448

Fig. 2 Species distribution of Chinese olive Unigene based on NR database alignment

Fig. 3 Correlation heatmap of transcriptome data of Chinese olive samples

Fig. 4 Differential genes and KEGG enrichment analysis of Chinese olive in different varieties（lines） A : Up-regulated and down-regulated differential genes, gene up/down-regulated expression refers to low phenolic Chinese olive（DQc, TQc）relative to high phenolic Chinese olive（SPc, HPc）; B : common or unique differential genes ;C : KEGG enrichment analysis of common differential genes

Fig. 5 Differential genes and KEGG enrichment analysis of different varieties（lines）of Chinese olive ripening process A: Differential genes in the maturation process of ‘Ziyang 1’ （SP）and ‘Dongshanchangsui’ （DQ）. B : Venn of differential genes in different maturation periods. C : KEGG enrichment analysis of differential genes

Fig. 6 Differential gene analysis of shikimic acid and hydrolyzed tannin biosynthesis pathway in different varieties（lines）of Chinese olive during maturation DPS : 3-deoxy-D-arabinoheptulose-7-phosphate synthase; DHQS : 3-dehydroquinic acid synthase; SKDH : shikimate dehydrogenase; UGTs : glycosyltransferase; SK : shikimate kinase; EPSPS : 5-enolpyruvylshikimate-3-phosphate synthase; CS : chorismate synthase; CM : chorismate mutase; pheA2 : prebenzoic acid dehydratase; GOT1 : aspartate aminotransferase; TAT : tyrosine aminotransferase; hisC : phosphohistidine aminotransferase ; Red or blue color blocks represent the value of Log2FC（DQ / SP）, red and blue represent the up-regulated and down-regulated genes of low-phenol olive（DQ）compared to high-phenol olive（SP）, respectively, and * indicates P.adjust < 0.05, the same below

Fig. 7 Differential gene analysis in phenylpropanes and flavonoids biosynthesis pathways in different varieties（lines）of Chinese olive during maturation PAL : Phenylalanine ammonia lyase; C4H : cinnamic acid-4-hydroxylase; 4CL : 4-coumaroyl-CoA ligase; CHS : chalcone synthase; CHI : chalcone isomerase; IFS : isoflavone synthase; FNS : flavonoid synthase; F3H : flavanone-3-hydroxylase; F3'H : flavonoid 3'-hydroxylase; F3' 5'H : flavonoid 3' 5' -hydroxylase; FLS : flavonol synthase; DFR : dihydroflavonol reductase; LAR : achromatic anthocyanin reductase; ANR : anthocyanin reductase ; aNS : anthocyanin synthase

Fig. 8 Visualization of gene co-expression network A: Determination of the optimal soft threshold. B: Cluster dendrogram of co-expression modules. C: Correlation between co-expression modules

Fig. 9 Module identification of phenolic compounds and mining of their associated transcription factors A：Module and trait correlation heat map，each row corresponds to a module. The values of each lattice indicate the correlation coefficients R and P, respectively. The lattice color indicates correlation, red indicates positive correlation, and blue indicates negative correlation. B：Co-expression network analysis of phenolic synthesis genes and transcription factors. Darker colors indicate that the gene is more important in the interaction network. C：Category statistics of transcription factors related to phenol synthesis

Fig. 10 Correlation of the gene expression obtained from RNA-Seq and RT-qPCR

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