番石榴果实品质评价及黄酮类化合物合成相关基因挖掘

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:

Objective

The 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.

Method

A 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.

Result

Among 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.

Conclusion

The 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.

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

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 sequenceFLSATGGAGGTGGAGAGAGTTCAAGCCTTAGCATATTCCTTGTTGGCCTCYP73ACAATTGAGACAACACTATGGTCGATTTCTTCAGGGTTTTTCCAGTGGCYP75ATATGGTGTTTGCTCATTACGGATCAGCAACATGCTCCTCAATCATGCHSGTCCCTAAGCTAGGCAAAGAAGCCGAAGATGGGTTTCTCTCCGADFRCTGACTCTCTGGAAGGCCGAGGATTCGCAGAGATCGTCGGE2.1.1.104ATGGAAGAGAAAATGAAAGCAGCATTTCCATCATCAGGAATTGCTAGGGAPDHTTGCTGGACGCGTCGCACGGAGCAGCGGAAGTCGACG

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Cultivarw/%w/(mg·g−1)可溶性固形物Soluble solid总酸Total acid总糖Totalsugar还原糖Reducingsugar蔗糖Sucrose总酚Totalphenol红宝石 Hongbaoshi9.40±1.05c0.14±0.02ab69.09±7.52c38.31±1.18a30.91±1.54d8.61±0.26b西瓜红 Xiguahong8.60±0.92b0.18±0.04b60.70±7.08ab45.15±4.36b13.38±0.44a7.83±0.80a胭脂红 Yanzhihong8.23±0.31b0.27±0.02d65.84±5.12b40.50±2.21ab21.06±1.69c11.25±0.70c珍珠 Zhenzhu9.87±0.80c0.11±0.02a70.25±2.38c52.93±1.51c17.48±0.85b8.48±1.03b水晶 Shuijing8.99±0.79bc0.23±0.02c65.55±2.59b42.41±2.15b22.74±1.05c7.67±1.11a金斗香 Jindouxiang7.34±0.66a0.29±0.02d58.91±2.79a39.40±1.83a16.97±0.40b11.51±0.54c平均值 Mean8.74±1.840.20±0.0165.06±7.3543.12±8.0118.26±5.899.23±3.44变异系数Variation coefficient0.250.050.120.180.280.37品种Cultivarw/(mg·g−1)w/(μmol·g−1)类黄酮Flavonoids抗坏血酸Ascorbic acid单宁TanninABTSDPPH红宝石 Hongbaoshi7.73±0.20c2.42±0.17c5.22±0.02b33.70±1.14bc545.40±32.48b西瓜红 Xiguahong6.54±0.18b1.40±0.10ab6.17±0.40c23.98±0.71a337.77±32.50a胭脂红 Yanzhihong10.05±1.90d1.53±0.15b4.70±0.35b36.79±1.60cd1004.30±95.67e珍珠 Zhenzhu6.68±0.36b1.27±0.31ab3.80±0.96a32.17±1.27b726.83±47.06c水晶 Shuijing5.74±0.49a1.09±0.02a7.22±0.28d25.02±3.15a639.83±34.40c金斗香 Jindouxiang9.76±1.78d1.54±0.14b4.72±0.32b38.65±1.81d875.90±40.25d平均值 Mean7.75±4.011.54±0.465.31±1.2131.72±5.87688.34±227.40变异系数Variation coefficient0.460.300.230.190.33　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)

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/%Variancecontribution rate累计方差贡献率/%Cumulative variancecontribution rate14.53141.19441.19422.85425.95067.14431.91317.38984.53340.8838.02692.56050.3583.25995.81960.1551.41397.23270.1211.10498.33680.0760.69399.02990.0480.43799.466100.0450.41299.878110.0130.122100.000

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 component123可溶性固形物含量Soluble solid content0.736−0.290−0.509总酸含量Total acid content0.5200.6540.464总糖含量Total sugar content0.417−0.477−0.598蔗糖含量Sucrose content0.913−0.0550.347还原糖含量Reducing sugar content0.8200.409−0.195类黄酮含量Flavonoids content0.2060.9320.136抗坏血酸含量Ascorbic acid content−0.6850.3820.004总酚含量Total phenol content0.853−0.1520.167ABTS清除能力ABTS removing capacity0.0660.833−0.427DPPH清除能力DPPH removing capacity0.8730.019−0.077单宁含量Tannin content0.184−0.4630.830

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 ofprincipal component综合评价指数Syntheticanalysis indexY1Y2Y3红宝石Hongbaoshi1.424−1.1330.9430.450西瓜红Xiguahong0.110−0.829−1.939−0.500胭脂红Yanzhihong0.3691.2300.0780.484珍珠Zhenzhu−1.079−0.6710.852−0.472水晶Shuijing−1.3970.1400.135−0.513金斗香Jindouxiang0.5721.262−0.0680.551

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TermDEG个数DEG number基因总数Total gene numberP苯丙烷的生物合成 Phenylpropanoid biosynthesis421110.000角质、亚硫酸和蜡的生物合成Cutin, suberine and wax biosynthesis11180.004淀粉和蔗糖的代谢 Starch and sucrose metabolism461580.011花青素生物合成 Anthocyanin biosynthesis7120.025乙醛酸酯和二羧酸酯代谢Glyoxylate and dicarboxylate metabolism27870.026ABC转运蛋白 ABC transporters13370.054类黄酮生物合成 Flavonoid biosynthesis10260.057半乳糖代谢 Galactose metabolism20660.059不饱和脂肪酸的生物合成Biosynthesis of unsaturated fatty acids10270.068脂肪酸降解 Fatty acid degradation14430.071氰基氨基酸代谢 Cyanoamino acid metabolism14430.071二苯乙烯类、二芳基庚烷类和姜辣酚的生物合成Stilbenoid, diarylheptanoid and gingerol biosynthesis6130.075植物激素信号转导 Plant hormone signal transduction481970.096色氨酸代谢 Tryptophan metabolism9260.105柠檬烯和蒎烯的降解Limonene and pinene degradation5110.105油菜素内酯生物合成 Brassinosteroid biosynthesis480.118α−亚麻酸代谢 α-Linolenic acid metabolism11350.118苯丙氨酸代谢 Phenylalanine metabolism14480.124脂肪酸生物合成 Fatty acid biosynthesis12400.131戊糖和葡萄糖醛酸盐的相互转化Pentose and glucuronate interconversions18660.132

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TypeGO 名称GO nameDEG个数DEG number校正PCorrected P生物学过程Biologicalprocess多细胞生物过程 Multi-cellular organism process340.0008授粉 Pollination300.0016花粉−雌蕊相互作用 Pollen-pistil interaction300.0016花粉识别 Recognition of pollen300.0016防御反应 Defense response1000.0019细胞识别 Cell recognition300.0019生物刺激反应 Response to biotic stimulus670.0019细胞学组分Cellular component细胞外基质 Extracellular matrix390.0029蛋白质的细胞外基质 Proteinaceous extracellular matrix350.0184分子功能Molecularfunction血红素的结合 Heme binding920.0003四吡咯的结合 Tetrapyrrole binding930.0008催化活性 Catalytic activity20730.0008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 oxygen870.0037水解酶活性、水解邻糖基化合物Hydrolase activity, hydrolyzing O-glycosyl compounds1300.0037作用于糖基键的水解酶活性 Hydrolase activity, acting on glycosyl bonds1390.0047碳水化合物结合 Carbohydrate binding530.0278核酸结合转录因子活性 Nucleic acid binding transcription factor activity1940.0335转录因子活性、序列特异性DNA结合Transcription factor activity, sequence-specific DNA binding1940.0335 [1] 宁琳, 陈豪君, 潘祖健, 等. 我国南亚热带地区番石榴种质资源保护现状[J]. 中国南方果树, 2015, 44(5): 147-149. doi: 10.13938/j.issn.1007-1431.20140650 [2]

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