Evaluation of guava fruit quality and mining of genes related to flavonoid synthesis
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摘要:目的
综合评价番石榴Psidium guajava不同品种间的果实品质差异并挖掘黄酮类化合物合成的关键基因。
方法对番石榴6个品种的11个果实品质指标进行测定，并结合主成分分析方法综合评价其品质差异，运用转录组测序技术比较各品种间的差异表达基因(Differentially expressed gene, DEG)，通过 GO 和 KEGG 富集分析，挖掘黄酮类化合物合成的DEG，利用实时荧光定量 PCR (Quantitative real-time PCR，qRT-PCR)研究DEG在不同品种间的特异性表达。
结果6种番石榴试材中‘金斗香’和‘胭脂红’品质最优，得分较高，‘水晶’和‘西瓜红’较低，‘珍珠’和‘红宝石’居中；‘金斗香’和‘胭脂红’的类黄酮质量分数较高，分别为9.76和10.05 mg/g，是‘水晶’(5.74 mg/g)的1.5倍以上，显著高于其他品种(P>0.05)。转录组测序分析显示，‘金斗香’和‘胭脂红’的DEG聚为一类，其余4种的DEG聚为一类。黄酮类化合物的生物合成途径中CHS、FLS、CYP73A、CYP98A3、DFR、E2.1.1.104、E1.14.11.19和CYP75A基因在‘金斗香’和‘胭脂红’中表达量显著上调。qRT-PCR验证结果表明，FLS基因在‘胭脂红’中表达量最高，是‘西瓜红’的10倍以上；CYP73A、CYP75A、E2.1.1.104和CHS基因在‘金斗香’中表达量最高，‘珍珠’中表达量最低，其中CYP73A和CYP75A基因在‘金斗香’中的表达量是‘珍珠’的30倍以上；而DFR基因在‘胭脂红’中表达量较高，‘金斗香’中表达量较低。qRT-PCR检测到DEG的表达水平与转录组测序结果一致，证明番石榴6个品种的转录组测序结果可靠。
结论本研究系统评价了6种番石榴果实品质差异，并挖掘到8个与番石榴黄酮类化合物合成相关的关键基因，为后期番石榴的品种选育、功能基因挖掘和黄酮类化合物的生物合成途径等研究提供科学依据。
Abstract:ObjectiveThe purpose of this study was to comprehensively evaluate the differences in fruit quality among different guava (Psidium guajava) cultivars and explore key genes for flavonoid synthesis.
MethodA total of 11 fruit quality indexes of six guava cultivars were measured and principal component analysis was carried out. Transcriptome sequencing technology was used to compare the differentially expressed genes (DEGs) among the cultivars, and GO and KEGG enrichment analyses were carried out to mine the DEGs of flavonoid synthesis. Quantitative real-time PCR (qRT-PCR) was used to study the specific expression of differential genes in different cultivars.
ResultAmong the six guava cultivars, ‘Jindouxiang’ and ‘Yanzhihong’ scored higher, ‘Shuijing’ and ‘Xiguahong’ scored lower, and ‘Zhenzhu’ and ‘Hongbaoshi’ scored in the middle. The flavonoid contents of ‘Jindouxiang’ and ‘Yanzhihong’ were significantly higher compared to other cultivars (P>0.05), which were 9.76 and 10.05 mg/g, respectively, more than 1.5 times that of ‘Shuijing’ (5.74 mg/g). Transcriptome sequencing analysis showed that the DEGs of ‘Jindouxiang’ and ‘Yanzhihong’ were clustered into one category, and the DEGs of the other four cultivars were clustered into one category.CHS, FLS, CYP73A, CYP98A3, DFR, E2.1.1.104, E1.14.11.19 and CYP75A genes in the biosynthetic pathway of flavonoids were significantly up-regulated in ‘Jindouxiang’ and ‘Yanzhihong’. qRT-PCR verification showed that the expression of FLS gene was the highest in ‘Yanzhihong’, which was more than 10 times of that in ‘Xiguahong’. The expression levels of CYP73A, CYP75A, E2.1.1.104 and CHS genes were the highest in ‘Jindouxiang’ and the lowest in ‘Zhenzhu’. Among them, the expression levels ofCYP73A and CYP75A genes in ‘Jindouxiang’ were more than 30 times of those in ‘Zhenzhu’, while the expression level of DFR gene was higher in ‘Yanzhihong’ and lower in ‘Jindouxiang’. The expression levels of DEGs were consistent comparing the qRT-PCR and transcriptome sequencing results, indicating the transcriptome sequencing results of six guava cultivars were reliable.
ConclusionThe quality differences of six guava cultivars were systematically evaluated, and eight key genes related to the synthesis of guava flavonoids were discovered. This study provides a scientific basis for the research of guava cultivar breeding, functional gene mining and biosynthetic pathway of flavonoids.
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Keywords:
- Psidium guajava /
- Fruit quality /
- Transcriptome /
- Flavonoid /
- Differentially expressed gene
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图 1 6个品种番石榴果实差异基因(DEG)的表达情况
a：差异基因表达量热图；b：类黄酮合成信号通路中的DEG； G1：红宝石，G2：西瓜红，G3：胭脂红，G4：珍珠，G5：水晶，G6：金斗香
Figure 1. The expressions of differential genes (DEGs) in fruits of six guava cultivars
a：Heat map of differential gene expression; b: The DEGs in flavonoid synthesis signaling pathway; G1: Hongbaoshi, G2: Xiguahong, G3: Yanzhihong, G4: Zhenzhu, G5: Shuijing, G6: Jindouxiang
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图 2 差异表达基因的qRT-PCR分析
G1：红宝石；G2：西瓜红；G3：胭脂红；G4：珍珠；G5：水晶；G6：金斗香
Figure 2. The qRT-PCR analysis of differentially expressed genes
G1: Hongbaoshi; G2: Xiguahong; G3: Yanzhihong; G4: Zhenzhu; G5: Shuijing; G6: Jindouxiang
表 1 差异表达基因的qRT-PCR引物序列
Table 1 Primer sequences of differentially expressed genes for qRT-PCR
基因名称 Gene name 上游引物序列(5′→3′) Forward primer sequence 下游引物序列(5′→3′) Reverse primer sequence FLS ATGGAGGTGGAGAGAGTTCAAGC CTTAGCATATTCCTTGTTGGCCT CYP73A CAATTGAGACAACACTATGGTCGAT TTCTTCAGGGTTTTTCCAGTGG CYP75A TATGGTGTTTGCTCATTACGGATC AGCAACATGCTCCTCAATCATG CHS GTCCCTAAGCTAGGCAAAGAAGC CGAAGATGGGTTTCTCTCCGA DFR CTGACTCTCTGGAAGGCCGA GGATTCGCAGAGATCGTCGG E2.1.1.104 ATGGAAGAGAAAATGAAAGCAGCA TTTCCATCATCAGGAATTGCTAGG GAPDH TTGCTGGACGCGTCGCAC GGAGCAGCGGAAGTCGACG 
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表 2 番石榴果实的营养成分比较1)
Table 2 Comparison of the nutritional components of guava fruits
品种 Cultivar w/% w/(mg·g−1) 可溶性 固形物 Soluble solid 总酸 Total acid 总糖 Total sugar 还原糖 Reducing sugar 蔗糖 Sucrose 总酚 Total phenol 红宝石 Hongbaoshi 9.40±1.05c 0.14±0.02ab 69.09±7.52c 38.31±1.18a 30.91±1.54d 8.61±0.26b 西瓜红 Xiguahong 8.60±0.92b 0.18±0.04b 60.70±7.08ab 45.15±4.36b 13.38±0.44a 7.83±0.80a 胭脂红 Yanzhihong 8.23±0.31b 0.27±0.02d 65.84±5.12b 40.50±2.21ab 21.06±1.69c 11.25±0.70c 珍珠 Zhenzhu 9.87±0.80c 0.11±0.02a 70.25±2.38c 52.93±1.51c 17.48±0.85b 8.48±1.03b 水晶 Shuijing 8.99±0.79bc 0.23±0.02c 65.55±2.59b 42.41±2.15b 22.74±1.05c 7.67±1.11a 金斗香 Jindouxiang 7.34±0.66a 0.29±0.02d 58.91±2.79a 39.40±1.83a 16.97±0.40b 11.51±0.54c 平均值 Mean 8.74±1.84 0.20±0.01 65.06±7.35 43.12±8.01 18.26±5.89 9.23±3.44 变异系数 Variation coefficient 0.25 0.05 0.12 0.18 0.28 0.37
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品种 Cultivar w/(mg·g−1) w/(μmol·g−1) 类黄酮 Flavonoids 抗坏血酸 Ascorbic acid 单宁 Tannin ABTS DPPH 红宝石 Hongbaoshi 7.73±0.20c 2.42±0.17c 5.22±0.02b 33.70±1.14bc 545.40±32.48b 西瓜红 Xiguahong 6.54±0.18b 1.40±0.10ab 6.17±0.40c 23.98±0.71a 337.77±32.50a 胭脂红 Yanzhihong 10.05±1.90d 1.53±0.15b 4.70±0.35b 36.79±1.60cd 1004.30±95.67e 珍珠 Zhenzhu 6.68±0.36b 1.27±0.31ab 3.80±0.96a 32.17±1.27b 726.83±47.06c 水晶 Shuijing 5.74±0.49a 1.09±0.02a 7.22±0.28d 25.02±3.15a 639.83±34.40c 金斗香 Jindouxiang 9.76±1.78d 1.54±0.14b 4.72±0.32b 38.65±1.81d 875.90±40.25d 平均值 Mean 7.75±4.01 1.54±0.46 5.31±1.21 31.72±5.87 688.34±227.40 变异系数 Variation coefficient 0.46 0.30 0.23 0.19 0.33 1)同列数据后的不同小写字母表示差异显著(P < 0.05， LSD法) 1) Different lowercase letters in the same column indicate significant differences(P < 0.05, LSD method)
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表 3 主成分的特征值、方差贡献率和累计方差贡献率
Table 3 Eigenvalue, variance contribution rate and cumulative variance contribution rate of principal components
成分 Component 特征值 Eigenvalue 方差贡献率/% Variance contribution rate 累计方差贡献率/% Cumulative variance contribution rate 1 4.531 41.194 41.194 2 2.854 25.950 67.144 3 1.913 17.389 84.533 4 0.883 8.026 92.560 5 0.358 3.259 95.819 6 0.155 1.413 97.232 7 0.121 1.104 98.336 8 0.076 0.693 99.029 9 0.048 0.437 99.466 10 0.045 0.412 99.878 11 0.013 0.122 100.000
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表 4 番石榴果实主要质地参数相关矩阵的规格化特征向量
Table 4 Normalized eigenvectors of correlation matrix of main textural parameters in guava fruits
品质指标 Quality index 主成分 Principal component 1 2 3 可溶性固形物含量 Soluble solid content 0.736 −0.290 −0.509 总酸含量 Total acid content 0.520 0.654 0.464 总糖含量 Total sugar content 0.417 −0.477 −0.598 蔗糖含量 Sucrose content 0.913 −0.055 0.347 还原糖含量 Reducing sugar content 0.820 0.409 −0.195 类黄酮含量 Flavonoids content 0.206 0.932 0.136 抗坏血酸含量 Ascorbic acid content −0.685 0.382 0.004 总酚含量 Total phenol content 0.853 −0.152 0.167 ABTS清除能力 ABTS removing capacity 0.066 0.833 −0.427 DPPH清除能力 DPPH removing capacity 0.873 0.019 −0.077 单宁含量 Tannin content 0.184 −0.463 0.830
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表 5 不同番石榴品种的主成分得分与综合评价指数
Table 5 Scores of principal components and synthetic analysis indexes for different guava cultivars
品种 Cultivar 主成分得分 Score of principal component 综合评价指数 Synthetic analysis index Y1 Y2 Y3 红宝石 Hongbaoshi 1.424 −1.133 0.943 0.450 西瓜红 Xiguahong 0.110 −0.829 −1.939 −0.500 胭脂红 Yanzhihong 0.369 1.230 0.078 0.484 珍珠 Zhenzhu −1.079 −0.671 0.852 −0.472 水晶 Shuijing −1.397 0.140 0.135 −0.513 金斗香 Jindouxiang 0.572 1.262 −0.068 0.551
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表 6 番石榴果实差异表达基因(DEG)的KEGG 信号通路富集分析
Table 6 KEGG enrichment analysis of differentially expressed genes (DEGs) in guava fruit
类别 Term DEG个数 DEG number 基因总数 Total gene number P 苯丙烷的生物合成 Phenylpropanoid biosynthesis 42 111 0.000 角质、亚硫酸和蜡的生物合成 Cutin, suberine and wax biosynthesis 11 18 0.004 淀粉和蔗糖的代谢 Starch and sucrose metabolism 46 158 0.011 花青素生物合成 Anthocyanin biosynthesis 7 12 0.025 乙醛酸酯和二羧酸酯代谢 Glyoxylate and dicarboxylate metabolism 27 87 0.026 ABC转运蛋白 ABC transporters 13 37 0.054 类黄酮生物合成 Flavonoid biosynthesis 10 26 0.057 半乳糖代谢 Galactose metabolism 20 66 0.059 不饱和脂肪酸的生物合成 Biosynthesis of unsaturated fatty acids 10 27 0.068 脂肪酸降解 Fatty acid degradation 14 43 0.071 氰基氨基酸代谢 Cyanoamino acid metabolism 14 43 0.071 二苯乙烯类、二芳基庚烷类和姜辣酚的生物合成 Stilbenoid, diarylheptanoid and gingerol biosynthesis 6 13 0.075 植物激素信号转导 Plant hormone signal transduction 48 197 0.096 色氨酸代谢 Tryptophan metabolism 9 26 0.105 柠檬烯和蒎烯的降解 Limonene and pinene degradation 5 11 0.105 油菜素内酯生物合成 Brassinosteroid biosynthesis 4 8 0.118 α−亚麻酸代谢 α-Linolenic acid metabolism 11 35 0.118 苯丙氨酸代谢 Phenylalanine metabolism 14 48 0.124 脂肪酸生物合成 Fatty acid biosynthesis 12 40 0.131 戊糖和葡萄糖醛酸盐的相互转化 Pentose and glucuronate interconversions 18 66 0.132
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表 7 番石榴果实差异表达基因(DEG)的GO 生物功能分析
Table 7 GO biofunctional analysis of differentially expressed genes (DEG) in guava fruit
类型 Type GO 名称 GO name DEG个数 DEG number 校正P Corrected P 生物学过程 Biological process 多细胞生物过程 Multi-cellular organism process 34 0.0008 授粉 Pollination 30 0.0016 花粉−雌蕊相互作用 Pollen-pistil interaction 30 0.0016 花粉识别 Recognition of pollen 30 0.0016 防御反应 Defense response 100 0.0019 细胞识别 Cell recognition 30 0.0019 生物刺激反应 Response to biotic stimulus 67 0.0019 细胞学组分 Cellular component 细胞外基质 Extracellular matrix 39 0.0029 蛋白质的细胞外基质 Proteinaceous extracellular matrix 35 0.0184 分子功能 Molecular function 血红素的结合 Heme binding 92 0.0003 四吡咯的结合 Tetrapyrrole binding 93 0.0008 催化活性 Catalytic activity 2073 0.0008 氧化还原酶活性、作用于配对供体、合并或还原分子氧 Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 87 0.0037 水解酶活性、水解邻糖基化合物 Hydrolase activity, hydrolyzing O-glycosyl compounds 130 0.0037 作用于糖基键的水解酶活性 Hydrolase activity, acting on glycosyl bonds 139 0.0047 碳水化合物结合 Carbohydrate binding 53 0.0278 核酸结合转录因子活性 Nucleic acid binding transcription factor activity 194 0.0335 转录因子活性、序列特异性DNA结合 Transcription factor activity, sequence-specific DNA binding 194 0.0335
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