CclSAUR49基因的表达特征及对类柠檬苦素生物合成的影响  

Characteristics of CclSAUR49 expression and its effect on limonoids biosynthesis in citrus

作　　者：余洪 易倩[1] 张曼曼[1] 朱世平[1] 王福生 赵晓春[1] YU Hong;YI Qian;ZHANG Manman;ZHU Shiping;WANG Fusheng;ZHAO Xiaochun(Citrus Research Institute of Southwest University/Chinese Academy of Agricultural Sciences,National Citrus Engineering Research Center,Chongqing 400712,China)

机构地区：[1]西南大学·中国农业科学院柑桔研究所·国家柑桔工程技术研究中心,重庆400712

出　　处：《果树学报》2021年第8期1240-1251,共12页Journal of Fruit Science

基　　金：国家自然科学基金(31901995);财政部和农业农村部:国家现代农业产业技术体系建设专项资金(CARS-26)。

摘　　要：【目的】探究CclSAUR49基因对柑橘类柠檬苦素生物合成的影响。【方法】通过高效液相色谱(HPLC)检测叶片中的诺米林和柠檬苦素含量;利用实时荧光定量PCR分析基因的表达量。分别从柚和湖北早实枳中克隆CclSAUR49基因编码序列(coding sequence,CDS)及启动子,利用瞬时转化烟草对CclSAUR49蛋白进行表达定位;通过遗传转化湖北早实枳分析该基因的组织表达特性及其对类柠檬苦素生物合成的影响。【结果】类柠檬苦素含量和CclSAUR49表达水平在2个沙田柚品种间以及叶片的生长发育中均呈显著负相关。CclSUAR49蛋白定位于质膜和细胞核。从湖北早实枳中克隆得到的CclSAUR49基因的启动子序列中不含吲哚乙酸(IAA)响应元件。启动子活性分析发现,Ccl-SAUR49基因主要在茎、根和老叶中表达,在嫩叶中没有表达。超量表达CclSAUR49导致类柠檬苦素含量显著降低,植株的叶片和节间显著伸长,茎增粗。【结论】柑橘CclSAUR49基因的表达有明显的组织特异性,对类柠檬苦素的合成或积累有抑制作用。【Objective】Limonoids are secondary metabolites widely distributed in the Rutaceae and Meliaceae,with biological and pharmacological activities such as anti-oxidation,anti-allergy,anti-obesity,anti-osteoporosis,anti-cancer,anti-bacteria,anti-virus.The genetic modulation of limonoids biosynthesis is still poorly understood.So the investigation of genes regulating the limonoids biosynthesis and the study of their functions will help to understand the regulatory mechanism of limonoids biosynthesis and provide a theoretical basis for further cultivation of high-quality citrus.It has been shown that the SAUR significantly affects the biological yield and production of secondary metabolites by regulating the hormone metabolism in the plant,but whether it is involved in the biosynthesis of limonoids has not been reported.To study the role of CclSAUR49 gene in biosynthesis of limonoids and characteristics of its promoter in citrus,the open reading frame(ORF)and promoter of CclSAUR49 gene were cloned from citrus.Limonoids contents,gene expression levels,subcellular location and promoter activity were analyzed.【Methods】Limonoids contents and expression of the CclSAUR49 in the leaves of different developmental stages from two genotypes of Shatianyou pomelos[Citrus grandis(L.)Osbeck]varied in limonoids content were analyzed with HPLC and Real-time qRT-PCR.Specific primers were designed to amplify the target fragment using the sequence in the Clementine genome(http://phytozome.jgi.doe.gov/pz/portal.)as a reference.The coding region and promoter of the CclSAUR49 were isolated from Shatianyou pomelo and precocious trifoliate orange(Poncirus trifoliata L.Raf.)via PCR amplification,respectively.Subcellular localization of the CclSAUR49 protein was accomplished with transient expression of 35S::SAUR49-GFP fusion protein in the leaves of Nicotiana benthamiana and observed using confocal microscope.cis-acting element of the promoter was predicted through the PlantCARE website.In order to analyze the characteristics of the pro

关 键 词：柚 枳 CclSAUR49基因 类柠檬苦素 启动子 顺式作用元件 超量表达 

分 类 号：S666[农业科学—果树学]