Expression characteristics of ascorbic acid synthase gene in guava fruit during fruit development
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摘要: 【目的】测定番石榴果实发育过程中的抗坏血酸含量,并分析番石榴抗坏血酸合成酶基因的表达特性,为后续深入挖掘果实抗坏血酸合成调控机理及选育优质番石榴品种提供理论参考。【方法】以番石榴品种珍珠果实为研究对象,测定不同果实发育(花后10~130 d)过程中果肉(鲜重)中抗坏血酸含量及单果抗坏血酸总量,并从番石榴基因组数据中挖掘其抗坏血酸合成途径酶基因,通过实时荧光定量PCR检测其在果实发育过程中的表达特性。【结果】珍珠番石榴品种果实发育成熟周期为130 d,生长速率出现2个高峰期,分别出现在幼果膨大期和大果膨大期,果实果皮颜色从深绿色向浅绿转变,在花后110 d果皮颜色转变最明显。番石榴果肉抗坏血酸含量在果实发育成熟前期(花后10~40d)保持在较高水平,特别是在花后30d,果肉抗坏血酸含量达132mg/100g,在花后40~100d抗坏血酸含量稍微下降但较稳定,保持在102.73 mg/100 g以上,成熟后期随果实迅速膨大而抗坏血酸含量略有下降,为84.39 mg/100 g,但单果抗坏血酸总量持续上升。从番石榴基因组数据共鉴定获得13个基因编码果实抗坏血酸合成途径相关酶的基因,这些基因编码完整L-半乳糖途径的相关酶,其中有6个基因集中于3号染色体,推测3号染色体是番石榴果实维生素合成的关键染色体。实时荧光定量PCR检测结果显示,除磷酸甘露糖异构酶基因(PgPMI1)和甘露糖磷酸变位酶基因(PgPMM1)在果实发育成熟过程中表达不规律外,其余11个L-半乳糖途径相关酶基因均在果实发育中期上调表达,后期表达又下调。果实抗坏血酸含量与GDP-甘露糖焦磷酸化酶基因(PgGMP1)和GDP-甘露糖-3',5'-表型异构酶基因(gGME2)表达显著正相关(P<0.05)。【结论】番石榴果实抗坏血酸积累与其合成途径基因的完整性及表达水平密切相关,推测由9类酶组成的L-半乳糖途径存在复杂的分子调控网络,挖掘获得的13个L-半乳糖途径成员,可用于后续番石榴优良品种的选育及转录调控机理研究。Abstract: 【Objective】The content of ascorbic acid during the development of guava fruit was determined and the expression characteristics of ascorbic acid synthase gene in guava were analyzed, which provided theoretical reference for further exploring the regulation mechanism of fruit ascorbic acid synthesis and breeding guava varieties with high quality.【Method】The fruit of guava variety Pearl was used as the research object to determine the content of ascorbic acid in the flesh(fresh weight) and the total amount of ascorbic acid in single fruit during the development of different fruits(10-130 d after anthesis). The ascorbate synthesis pathway enzyme genes were obtained from the genomic data of guava, and their expression characteristics during the development of fruit were detected by real-time fluorescence quantitative PCR.【Result】The ripening period of Pearl guava variety was 130 d, and the growth rate showed two peaks, which appeared in the young fruit expansion stage and the large fruit expansion stage, respectively. The color of the fruit peel changed from dark green to light green, and the color change was the most obvious at 110 d after flowering. The content of ascorbic acid in guava flesh remained at a high level in the early stage of fruit development and ripening(10-40 d after flowering), especially at 30 d after flowering, the content of ascorbic acid in guava flesh reached 132 mg/100 g, and slightly decreased but remained stable at more than 102.73 mg/100 g at 40-100 d after flowering. The ascorbic acid content decreased slightly with the rapid expansion of fruit in the late ripening period, to 84.39 mg/100 g, but the total ascorbic acid content of single fruit continued to increase. A total of 13 genes encoding enzymes related to fruit ascorbate synthesis pathway were obtained from guava genome data, and these genes encoded complete L-galactose pathway related enzymes, among which 6 genes were concentrated in chromosome 3, suggesting that chromosome 3 was the key chromosome for vitamin synthesis in guava fruit. Real-time fluorescence quantitative PCR results showed that except for the irregular expression of phosphomannose isomerase gene(PgPMI1) and phosphomannomutase gene(PgPMM1) during fruit development and maturation, the expression of the other 11 L-galactose pathway related enzyme genes was up-regulated in the middle stage of fruit development, and down-regulated in the later stage. The content of ascorbic acid in fruit was significantly positively correlated with the expression of GDP-mannose pyrophosphorylase gene(PgGMP1) and GDP-mannose-3',5'-epimerase gene(PgGME2)(P<0.05). 【Conclusion】The accumulation of ascorbic acid in guava fruits is closely related to the integrity and expression level of the synthesis pathway genes. It is speculated that the L-galactose pathway composed of 9enzymes has a complex molecular regulatory network. The 13 members of L-galactose pathway obtained by mining can be used for subsequent breeding of excellent guava varieties and transcriptional regulation mechanism research.
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Keywords:
- guava /
- ascorbic acid /
- fruit development /
- gene expression
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