柑橘转录因子CsbHLH3调控柠檬酸代谢的功能解析

Functional Analysis of Transcription Factor CsbHLH3 in Regulating Citric Acid Metabolism of Citrus Fruit

转录因子CsbHLH3调控柑橘果实柠檬酸代谢过程中的具体机制尚不清楚。以冰糖橙果实cDNA为材料,克隆了CsbHLH3。瞬时转化烟草亚细胞定位显示CsbHLH3蛋白定位在细胞核。异位表达CsbHLH3的阳性番茄果实中CsbHLH3的表达显著上升,同时柠檬酸含量显著降低。定量分析表明,CsbHLH3在番茄中异位表达引起了柠檬酸降解相关基因SlACO3b、SlGDH及液泡膜质子泵基因SlPH8的上调。相反,在冰糖橙叶片中瞬时抑制CsbHLH3会引起柠檬酸降解相关基因CsGABP、CsACO2、CsACLα1、Cs ACLα2及液泡膜质子泵基因CsPH8、CsVHP2、CsVHP3的下调表达,其柠檬酸含量显著高于空载对照。通过酵母单杂交和EMSA试验证实CsbHLH3均能够靶定PH8和GABP启动子调控其表达。上述结果表明转录因子CsbHLH3是通过调控柠檬酸代谢途径多个相关基因包括PH8和降解途径基因GABP的表达综合负调控柠檬酸的水平。

Transcription factor CsbHLH3 plays an important role in regulating citrate metabolism in citrus fruits,but the specific mechanism remains unclear.In this study,the CsbHLH3 gene was cloned,and transient transformation in tobacco leaves demonstrated the subcellular localization of CsbHLH3 protein in the nucleus.CsbHLH3 overexpression tomato fruit showed a significant increase in the expression of CsbHLH3 while a significant decrease in the content of citrate relative to the wide type.Quantitative analysis revealed that the overexpression of CsbHLH3 induced the up-regulation of SlACO3b and SlPH8.On the contrary,transient suppression of the CsbHLH3 gene in Bingtangcheng leaves resulted in a significantly higher content of citrate than that of the control.In addition,the suppression of CsbHLH3 expression down-regulated the expression of the Cs ACO2,CsACLα1,CsACLα2and CsGABP genes in the citrate degradation pathway and proton pump genes CsPH8,CsVHP2 and CsVHP3.Moreover,CsbHLH3 was further confirmed to target the promoter of PH8 and GABP genes to regulate their expression through Y1H and EMSA.These results suggested that the transcription factor CsbHLH3 negatively regulates the citrate level by regulating the expression of several genes related to citrate metabolism,including PH8 and the degradation gene GABP.