不同鲜食品质橄榄果实转录组测序及酚类代谢途径相关调控基因挖掘

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

摘要/Abstract

摘要：

【目的】 果实酚类物质类别、含量是与橄榄营养及风味密切相关的重要品质性状，探究橄榄酚类物质生物合成的分子调控机制。【方法】 以总酚含量差异显著的橄榄（低酚/高酚）为试材，分别取花后80-160 d的果实进行转录组分析，对莽草酸-水解单宁/苯丙烷-类黄酮生物合成通路差异基因进行表征，并对差异表达基因进行WGCNA分析，从中挖掘与酚类代谢途径相关的转录因子。【结果】 转录组测序共获得296 314条Unigene，其中73%的Unigene被注释到数据库；4个品种（系）橄榄成熟果共鉴定到1 628个差异表达基因（DEGs），KEGG分析显示，DEGs在酚类代谢途径的“类黄酮生物合成”通路上显著富集；进一步对果实成熟过程莽草酸-水解单宁/苯丙烷-类黄酮生物合成途径的DEGs进行表征，结合WGCNA分析中每个模块与性状的R²和P值，筛选到4个关键模块，根据模块内基因的调控关系利用MCC拓扑分析法以度值≥1挖掘模块内关键转录因子，挖掘到137个转录因子Unigene与30个酚类合成结构基因Unigene共表达，转录因子Unigene功能注释来自35个基因家族，最多的为锌指蛋白（C2C2、C3H、C2H2、PHD），其次为B3、HB、MYB、NAC基因家族。【结论】 从转录组角度初步解析了橄榄鲜食品质在酚类代谢途径的差异，同时挖掘了调控酚类代谢的差异基因，对进一步探究橄榄鲜食品质差异的分子机制提供了重要依据。

关键词: 橄榄, 转录组, 酚类代谢途径, 转录因子

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

谢倩, 江来, 贺进, 刘玲玲, 丁明月, 陈清西. 不同鲜食品质橄榄果实转录组测序及酚类代谢途径相关调控基因挖掘[J]. 生物技术通报, 2024, 40(3): 215-228.

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.

图/表 12

表1 RT-qPCR引物序列

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

图1 橄榄果实成熟过程及成熟期总酚含量 *表示0.05水平差异显著，**表示0.01水平差异显著，***表示0.001水平差异显著

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

表2 橄榄转录组序列拼接结果

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

图2 橄榄Unigene基于NR数据库比对物种分布

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

图3 橄榄各样本转录组数据相关性热图

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

图4 不同品种（系）橄榄差异基因与KEGG富集分析 A：上下调差异基因，基因上/下调表达均是指低酚橄榄（DQc、TQc）相对于高酚橄榄（SPc、HPc）；B：共有或独特差异基因；C：共有差异基因KEGG富集分析

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

图5 不同品种（系）橄榄成熟过程差异基因与KEGG富集分析 A：‘子阳1号’（SP）与‘东山长穗’（DQ）成熟过程差异基因，B：不同成熟时期差异基因的韦恩图，C：差异基因KEGG富集分析

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

图6 不同品种（系）橄榄成熟过程莽草酸-水解单宁生物合成途径差异基因分析 DPS：3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸合酶，DHQS：3-脱氢奎尼酸合酶，SKDH：莽草酸脱氢酶，UGTs：糖基转移酶，SK：莽草酸激酶，EPSPS：5-烯醇丙酮酰莽草酸-3-磷酸合酶，CS：分支酸合酶，CM：分支酸变位酶，pheA2：预苯酸脱水酶，GOT1：天冬氨酸氨基转移酶，TAT：酪氨酸氨基转移酶，hisC：磷酸组氨酸氨基转移酶；红色或蓝色的颜色块表示Log2FC（DQ/SP）的值，红色和蓝色分别表示低酚橄榄（DQ）相较于高酚橄榄（SP）上调和下调的基因，*表示P.adjust<0.05，下同

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

图7 不同品种（系）橄榄成熟过程苯丙烷-类黄酮生物合成途径差异基因分析 PAL：苯丙氨酸解氨酶，C4H：肉桂酸-4-羟化酶，4CL：4-香豆酰辅酶A连接酶，CHS：查尔酮合成酶，CHI：查尔酮异构酶，IFS：异黄酮合酶，FNS：黄酮合酶，F3H：黄烷酮-3-羟化酶，F3'H：类黄酮 3'-羟化酶，F3'5'H：类黄酮 3'5'-羟化酶，FLS：黄酮醇合成酶，DFR：二氢黄酮醇还原酶，LAR：无色花色素还原酶，ANR：花青素还原酶；ANS：花青素合成酶

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

图8 基因共表达网络的可视化 A：确定最佳软阈值，B：共表达模块的集群树状图，C：共表达模块之间的相关性

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

图9 酚类化合物模块鉴定及其相关转录因子挖掘 A：模块与总酚相关性热图，每一行对应一个模块，每个格子的值分别表示相关系数R和P值；格子颜色表示相关性，红色表示正相关，蓝色表示负相关；B：酚类物质合成基因与转录因子的共表达网络分析，颜色越深表示基因在互作网络中越重要；C：酚类物质合成相关转录因子类别统计

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

图10 RNA-Seq与RT-qPCR基因表达水平间的相关性

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

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