利用转基因番茄验证柚转录因子CgDREB对果实成熟性状的影响
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摘要
柑橘是多年生木本作物,在全世界内都具有广泛的经济价值。然而目前关于柑橘果实成熟的研究还较少,其调控机制也了解不多。为了研究转录因子对果实成熟的调节作用,在HB柚中克隆CgDREB基因并且将该基因在番茄(Lycopersicon esculentum)中超量表达进行功能验证。亚细胞定位表明CgDREB蛋白定位在细胞核内。通过实时荧光PCR检测23个转基因系和野生型植株的表达模式,发现其中35S-CgDREBline5,35S-CgDREBlinel5和35S-CgDREBlinel9这三个株系该基因表达量较高。因此,本研究以35S-CgDREBline5和35S-CgDREBlinel9转基因株系为材料,研究番茄果实成熟过程中果肉和果皮中代谢物质的变化。利用GC-MS气质联用仪对转基因株系和野生型番茄的果肉和果皮中的初生代谢物进行了分析,结果表明两个转基因株系果肉和果皮的有机酸、糖类和脂肪酸含量相比野生型番茄显著升高。这些代谢物的显著变化表明CgDREB基因在果实成熟过程中起到了重要作用。相对野生型果实,在两个转CgDREB基因株系果实乙烯释放量也显著增加。本研究发现果实成熟与代谢物具有密切的关系,这些代谢物包括有机酸、糖类、脂肪酸以及乙烯释放,这一发现强调了果实成熟过程中这些代谢物的变化以及乙烯的释放的重要作用。以上结果进一步揭示了CgDREB基因与果实成熟过程的关系。
Citrus is a perennial woody crop with a world-widely economic importance. However, the regulatory mechanism of fruit ripening in citrus has been less investigated. To explore the regulation role of transcription factors in fruit ripening, CgDREB gene was characterized in Citrus grandis and functionally over-expressed in tomato (Lycopersicon esculentum). Subcellular localization showed that CgDREB protein was localized in the nucleus. Expression patterns of23transgenic lines comparing with wild type were examined by real time-PCR.35S-CgDREBline5,35S-CgDREBlinel5and35S-CgDREBlinel9showed higher expression level among the23transgenic lines. Based on realtime-PCR results,35S-CgDREBline5and35S-CgDREBlinel9lines were used to study the metabolites profile in flesh and peel of tomato fruit during ripening. Primary metabolites in tomato flesh and peel fruits for the transgenic lines and wild type were measured by gas chromatography-mass spectrometry. The levels of organic acids, sugars and fatty acids were dramatically increased in tomato flesh and peel of the two CgDREB lines than wild type. Notable changes of the metabolic profiling response to CgDREB during fruit ripening suggest that CgDREB are involved in fruit ripening regulation. Ethylene production was significantly increased in both CgDREB lines than wild type. A sturdy correlation between fruit ripening and primary metabolite groups, such as organic acids, sugars, and fatty acids and ethylene releasing were observed, which emphasizes the significance of these metabolic changes and ethylene emancipation during fruit ripening. These results shed a new light into the relation between CgDREB and ripening process.
Citrus is a perennial woody crop with a world-widely economic importance. However, the regulatory mechanism of fruit ripening in citrus has been less investigated. To explore the regulation role of transcription factors in fruit ripening, CgDREB gene was characterized in Citrus grandis and functionally over-expressed in tomato (Lycopersicon esculentum). Subcellular localization showed that CgDREB protein was localized in the nucleus. Expression patterns of23transgenic lines comparing with wild type were examined by real time-PCR.35S-CgDREBline5,35S-CgDREBlinel5and35S-CgDREBlinel9showed higher expression level among the23transgenic lines. Based on realtime-PCR results,35S-CgDREBline5and35S-CgDREBlinel9lines were used to study the metabolites profile in flesh and peel of tomato fruit during ripening. Primary metabolites in tomato flesh and peel fruits for the transgenic lines and wild type were measured by gas chromatography-mass spectrometry. The levels of organic acids, sugars and fatty acids were dramatically increased in tomato flesh and peel of the two CgDREB lines than wild type. Notable changes of the metabolic profiling response to CgDREB during fruit ripening suggest that CgDREB are involved in fruit ripening regulation. Ethylene production was significantly increased in both CgDREB lines than wild type. A sturdy correlation between fruit ripening and primary metabolite groups, such as organic acids, sugars, and fatty acids and ethylene releasing were observed, which emphasizes the significance of these metabolic changes and ethylene emancipation during fruit ripening. These results shed a new light into the relation between CgDREB and ripening process.
引文
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