摘要:

目的

綜合評(píng)價(jià)番石榴Psidium guajava不同品種間的果實(shí)品質(zhì)差異并挖掘黃酮類化合物合成的關(guān)鍵基因。

方法

對(duì)番石榴6個(gè)品種的11個(gè)果實(shí)品質(zhì)指標(biāo)進(jìn)行測(cè)定，并結(jié)合主成分分析方法綜合評(píng)價(jià)其品質(zhì)差異，運(yùn)用轉(zhuǎn)錄組測(cè)序技術(shù)比較各品種間的差異表達(dá)基因(Differentially expressed gene, DEG)，通過(guò) GO 和 KEGG 富集分析，挖掘黃酮類化合物合成的DEG，利用實(shí)時(shí)熒光定量 PCR (Quantitative real-time PCR，qRT-PCR)研究DEG在不同品種間的特異性表達(dá)。

結(jié)果

6種番石榴試材中‘金斗香’和‘胭脂紅’品質(zhì)最優(yōu)，得分較高，‘水晶’和‘西瓜紅’較低，‘珍珠’和‘紅寶石’居中；‘金斗香’和‘胭脂紅’的類黃酮質(zhì)量分?jǐn)?shù)較高，分別為9.76和10.05 mg/g，是‘水晶’(5.74 mg/g)的1.5倍以上，顯著高于其他品種(P>0.05)。轉(zhuǎn)錄組測(cè)序分析顯示，‘金斗香’和‘胭脂紅’的DEG聚為一類，其余4種的DEG聚為一類。黃酮類化合物的生物合成途徑中CHS、FLS、CYP73A、CYP98A3、DFR、E2.1.1.104、E1.14.11.19和CYP75A基因在‘金斗香’和‘胭脂紅’中表達(dá)量顯著上調(diào)。qRT-PCR驗(yàn)證結(jié)果表明，F(xiàn)LS基因在‘胭脂紅’中表達(dá)量最高，是‘西瓜紅’的10倍以上；CYP73A、CYP75A、E2.1.1.104和CHS基因在‘金斗香’中表達(dá)量最高，‘珍珠’中表達(dá)量最低，其中CYP73A和CYP75A基因在‘金斗香’中的表達(dá)量是‘珍珠’的30倍以上；而DFR基因在‘胭脂紅’中表達(dá)量較高，‘金斗香’中表達(dá)量較低。qRT-PCR檢測(cè)到DEG的表達(dá)水平與轉(zhuǎn)錄組測(cè)序結(jié)果一致，證明番石榴6個(gè)品種的轉(zhuǎn)錄組測(cè)序結(jié)果可靠。

結(jié)論

本研究系統(tǒng)評(píng)價(jià)了6種番石榴果實(shí)品質(zhì)差異，并挖掘到8個(gè)與番石榴黃酮類化合物合成相關(guān)的關(guān)鍵基因，為后期番石榴的品種選育、功能基因挖掘和黃酮類化合物的生物合成途徑等研究提供科學(xué)依據(jù)。

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.

圖  1   6個(gè)品種番石榴果實(shí)差異基因(DEG)的表達(dá)情況

a：差異基因表達(dá)量熱圖；b：類黃酮合成信號(hào)通路中的DEG； G1：紅寶石，G2：西瓜紅，G3：胭脂紅，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

圖  2   差異表達(dá)基因的qRT-PCR分析

G1：紅寶石；G2：西瓜紅；G3：胭脂紅；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   差異表達(dá)基因的qRT-PCR引物序列

Table  1   Primer sequences of differentially expressed genes for qRT-PCR

基因名稱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   番石榴果實(shí)的營(yíng)養(yǎng)成分比較1)

Table  2   Comparison of the nutritional components of guava fruits

品種Cultivarw/%w/(mg·g?1)可溶性固形物Soluble solid總酸Total acid總糖Totalsugar還原糖Reducingsugar蔗糖Sucrose總酚Totalphenol紅寶石 Hongbaoshi9.40±1.05c0.14±0.02ab69.09±7.52c38.31±1.18a30.91±1.54d8.61±0.26b西瓜紅 Xiguahong8.60±0.92b0.18±0.04b60.70±7.08ab45.15±4.36b13.38±0.44a7.83±0.80a胭脂紅 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變異系數(shù)Variation coefficient0.250.050.120.180.280.37品種Cultivarw/(mg·g?1)w/(μmol·g?1)類黃酮Flavonoids抗壞血酸Ascorbic acid單寧TanninABTSDPPH紅寶石 Hongbaoshi7.73±0.20c2.42±0.17c5.22±0.02b33.70±1.14bc545.40±32.48b西瓜紅 Xiguahong6.54±0.18b1.40±0.10ab6.17±0.40c23.98±0.71a337.77±32.50a胭脂紅 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變異系數(shù)Variation coefficient0.460.300.230.190.33　1)同列數(shù)據(jù)后的不同小寫字母表示差異顯著(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、方差貢獻(xiàn)率和累計(jì)方差貢獻(xiàn)率

Table  3   Eigenvalue, variance contribution rate and cumulative variance contribution rate of principal components

成分Component特征值Eigenvalue方差貢獻(xiàn)率/%Variancecontribution rate累計(jì)方差貢獻(xiàn)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   番石榴果實(shí)主要質(zhì)地參數(shù)相關(guān)矩陣的規(guī)格化特征向量

Table  4   Normalized eigenvectors of correlation matrix of main textural parameters in guava fruits

品質(zhì)指標(biāo)Quality index主成分 Principal component123可溶性固形物含量Soluble solid content0.736?0.290?0.509總酸含量Total acid content0.5200.6540.464總糖含量Total sugar content0.417?0.477?0.598蔗糖含量Sucrose content0.913?0.0550.347還原糖含量Reducing sugar content0.8200.409?0.195類黃酮含量Flavonoids content0.2060.9320.136抗壞血酸含量Ascorbic acid content?0.6850.3820.004總酚含量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單寧含量Tannin content0.184?0.4630.830

表  5   不同番石榴品種的主成分得分與綜合評(píng)價(jià)指數(shù)

Table  5   Scores of principal components and synthetic analysis indexes for different guava cultivars

品種Cultivar主成分得分 Score ofprincipal component綜合評(píng)價(jià)指數(shù)Syntheticanalysis indexY1Y2Y3紅寶石Hongbaoshi1.424?1.1330.9430.450西瓜紅Xiguahong0.110?0.829?1.939?0.500胭脂紅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   番石榴果實(shí)差異表達(dá)基因(DEG)的KEGG 信號(hào)通路富集分析

Table  6   KEGG enrichment analysis of differentially expressed genes (DEGs) in guava fruit

類別TermDEG個(gè)數(shù)DEG number基因總數(shù)Total gene numberP苯丙烷的生物合成 Phenylpropanoid biosynthesis421110.000角質(zhì)、亞硫酸和蠟的生物合成Cutin, suberine and wax biosynthesis11180.004淀粉和蔗糖的代謝 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謝Glyoxylate and dicarboxylate metabolism27870.026ABC轉(zhuǎn)運(yùn)蛋白 ABC transporters13370.054類黃酮生物合成 Flavonoid biosynthesis10260.057半乳糖代謝 Galactose metabolism20660.059不飽和脂肪酸的生物合成Biosynthesis of unsaturated fatty acids10270.068脂肪酸降解 Fatty acid degradation14430.071氰基氨基酸代謝 Cyanoamino acid metabolism14430.071二苯乙烯類、二芳基庚烷類和姜辣酚的生物合成Stilbenoid, diarylheptanoid and gingerol biosynthesis6130.075植物激素信號(hào)轉(zhuǎn)導(dǎo) Plant hormone signal transduction481970.096色氨酸代謝 Tryptophan metabolism9260.105檸檬烯和蒎烯的降解Limonene and pinene degradation5110.105油菜素內(nèi)酯生物合成 Brassinosteroid biosynthesis480.118α?亞麻酸代謝 α-Linolenic acid metabolism11350.118苯丙氨酸代謝 Phenylalanine metabolism14480.124脂肪酸生物合成 Fatty acid biosynthesis12400.131戊糖和葡萄糖醛酸鹽的相互轉(zhuǎn)化Pentose and glucuronate interconversions18660.132

表  7   番石榴果實(shí)差異表達(dá)基因(DEG)的GO 生物功能分析

Table  7   GO biofunctional analysis of differentially expressed genes (DEG) in guava fruit

類型TypeGO 名稱GO nameDEG個(gè)數(shù)DEG number校正PCorrected P生物學(xué)過(guò)程Biologicalprocess多細(xì)胞生物過(guò)程 Multi-cellular organism process340.0008授粉 Pollination300.0016花粉?雌蕊相互作用 Pollen-pistil interaction300.0016花粉識(shí)別 Recognition of pollen300.0016防御反應(yīng) Defense response1000.0019細(xì)胞識(shí)別 Cell recognition300.0019生物刺激反應(yīng) Response to biotic stimulus670.0019細(xì)胞學(xué)組分Cellular component細(xì)胞外基質(zhì) Extracellular matrix390.0029蛋白質(zhì)的細(xì)胞外基質(zhì) Proteinaceous extracellular matrix350.0184分子功能Molecularfunction血紅素的結(jié)合 Heme binding920.0003四吡咯的結(jié)合 Tetrapyrrole binding930.0008催化活性 Catalytic activity20730.0008氧化還原酶活性、作用于配對(duì)供體、合并或還原分子氧Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen870.0037水解酶活性、水解鄰糖基化合物Hydrolase activity, hydrolyzing O-glycosyl compounds1300.0037作用于糖基鍵的水解酶活性 Hydrolase activity, acting on glycosyl bonds1390.0047碳水化合物結(jié)合 Carbohydrate binding530.0278核酸結(jié)合轉(zhuǎn)錄因子活性 Nucleic acid binding transcription factor activity1940.0335轉(zhuǎn)錄因子活性、序列特異性DNA結(jié)合Transcription factor activity, sequence-specific DNA binding1940.0335 [1] 寧琳, 陳豪君, 潘祖健, 等. 我國(guó)南亞熱帶地區(qū)番石榴種質(zhì)資源保護(hù)現(xiàn)狀[J]. 中國(guó)南方果樹, 2015, 44(5): 147-149. doi: 10.13938/j.issn.1007-1431.20140650 [2]

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