转录因子基因CsSHN1调控脐橙蜡质基因表达的分子机理研究

‘Ganqi 3’ is a novel cultivar originated from ‘Newhall’ navel orange (Citrus sinensis [L.] Osbeck cv. Newhall) with bright glossy fruit peels. The decrease expression of various wax related genes resulted in the decline of aliphatic wax amounts and the wax crystal numbers, and finally leading to the glossy surface of ‘Ganqi 3’ fruits. These down-regulated wax genes contain several downstream genes of WIN1/ SHN1, which is the first and most studied wax-related transcription factor identified from plants. In our previous study, the full length cDNA squences of CsSHN1 were cloned from ‘Newhall’ navel orange and ‘Ganqi 3’, which were completely identical. We also obtained the F2 transgenetic Arabidopsis overexpressed CsSHN1 for further study.This project will further study the CsSHN1 transcription factor activities including transactivation activity and DNA binding trait. The up- and down-stream regulated genes of CsSHN1 will be isolated using yeast one-hybrid and digital gene expression profiling analysis. Moreover, the expression difference between ‘Newhall’ navel orange and ‘Ganqi 3’of these genes will be studied using real-time PCR. The results of this project will help us to construct the theoretic model of CsSHN1 regulating the expression of citrus wax-related genes, and find out more wax-related genes that can be used to improve the exterior quality of citrus.

“赣脐3号”是申请者培育的脐橙果皮光泽型新品种，其果皮中大量蜡质基因下调表达，导致果皮蜡质含量下降，蜡质晶体数目减少，最终造成果皮光亮。这些下调表达蜡质基因，包含了WIN1/ SHN1转录因子下游目标基因。WIN1/ SHN1是植物中第一个被鉴定的参与蜡质合成途径的转录因子。前期研究中，申请者从母本纽荷尔脐橙和“赣脐3号”中克隆出CsSHN1基因全长序列完全一致，并获得了超量表达CsSHN1的F2代转基因拟南芥。本项目拟继续深入研究CsSHN1的转录激活和DNA结合特性等转录因子相关活性，通过酵母单杂交、数字表达谱分析等技术分离CsSHN1上下游调控基因，通过实时定量PCR分析这些基因在纽荷尔脐橙和“赣脐3号”果皮中表达差异，建立CsSHN1调控柑橘蜡质基因表达分子机理的理论模型，顺着CsSHN1调控的上下游基因，找到更多蜡质基因，为使用基因工程改良柑橘外观品质提供新的基因资源和理论基础。

表皮蜡质是覆盖在柑橘表皮的疏水屏障，具有抵御生物和非生物胁迫等重要生理功能。SHN1是参与调控植物表皮蜡质合成的关键转录因子。本项目在前期研究的基础上，鉴定了脐橙CsSHN1基因功能，并研究了CsSHN1调控下游蜡质和角质基因表达的分子机理。研究结果表明，与野生型拟南芥相比，超量表达CsSHN1的转基因拟南芥中多个蜡质和角质相关基因表达量显著增加，导致其表皮蜡质和角质含量增加，表皮蜡质数目增多，抗旱性增强。进一步研究表明，CsSHN1转录因子具有转录激活活性，可以结合在CsLACS2基因启动子上，调控其表达。另外，CsSHN1启动子上含有多个激素响应和非生物胁迫响应顺式作用元件。通过酵母单杂交，我们筛选出了CsMYB16、CsMYB106等CsSHN1的上游调控基因。在果实发育过程中，相对于“纽荷尔脐橙”， CsSHN1的上下游调控基因在纽荷尔脐橙果皮蜡质缺失突变品种“赣脐3号”果皮中显著下调表达。综上所述，CsSHN1具有调控下游蜡质和角质基因表达，提高植物抗旱性的功能。