RNAi沉默CsHB1降低茶树叶片愈伤组织咖啡碱积累  

RNAi Silencing CsHB1 Reduces the Accumulation of Caffeine in Tea Callus

作　　者：康馨 刘平 马雯慧 张媛媛[1] 林晓蓉[1] 李斌[1,2] 陈忠正 KANG Xin;LIU Ping;MA Wenhui;ZHANG Yuanyuan;LIN Xiaorong;LI Bin;CHEN Zhongzhen(College of Food Science,South China Agricultural University,Guangzhou 510642,China;Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods,Guangzhou 510642,China)

机构地区：[1]华南农业大学食品学院,广州510642 [2]广东省功能食品活性物重点实验室,广州510642

出　　处：《园艺学报》2020年第12期2373-2384,共12页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31770727,31270726);广州市科技计划项目(201607010139);国家现代农业产业技术体系建设专项资金项目(CARS-19);广东省科技计划项目(2015A030302065)。

摘　　要：以‘英红9号’茶树叶片为材料,克隆HD-Zip转录因子基因CsHB1,原核表达CsHB1重组蛋白,利用本氏烟草进行CsHB1表达蛋白的亚细胞定位;构建CsHB1的RNAi转基因载体,经农杆菌介导转化‘英红9号’茶树叶片愈伤组织,并对沉默CsHB1愈伤组织系进行基因表达分析和咖啡碱含量测定。结果表明,克隆基因与NCBI登记的‘龙井43’茶树Cs HB1(MF033534)有6个碱基差异,但编码相同的氨基酸序列;原核诱导表达获得CsHB1的49 kD不可溶蛋白;将CsHB1蛋白亚细胞定位于细胞核和细胞质。在沉默表达CsHB1愈伤组织中,CsHB1表达显著下降,催化茶叶咖啡碱合成的N–甲基转移酶基因yhNMT1表达下调,愈伤组织中咖啡碱含量显著降低。推测转录因子基因CsHB1的沉默抑制了靶基因yhNMT1的表达,进而降低‘英红9号’愈伤组织中咖啡碱的合成积累。In this study,the HD-Zip transcription factor CsHB1 gene was cloned from‘Yinghong 9’(Camellia sinensis)tea plant,and the recombinant CsHB1 protein was expressed in Escherichia coli.The subcellular localization of CsHB1 was carried out using tobacco as an expression host.The RNAi transgenic vector of CsHB1 was constructed and transformed into‘Yinghong 9’tea callus mediated by Agrobacterium,followed by the analysis of gene expression and caffeine content on the silence CsHB1 callus.Our results showed that the cloned CsHB1 gene shared the same amino acid sequence exception 6 nucleotides difference with NCBI registered CsHB1 gene(accession No.MF033534)isolated from‘Longjing 43’tea plant.A 49 kD insoluble recombinant protein corresponding to the CsHB1 gene was obtained by prokaryotic induction.The expression protein infused with GFP as GFP-CsHB1 located in the cell nucleus and cytoplasm of tobacco.In the silence expression of CsHB1 callus,the expression of the CsHB1 gene decreased significantly,and the expression of yhNMT1,an N-methyltransferase,which catalyzes the synthesis of caffeine in tea,was also found decreased by real-time fluorescence quantitative PCR analysis.HPLC analysis showed that the content of caffeine in the transgenic callus decreased markedly.Based on these results,we suggest that the silencing of CsHB1 suppresses the expression of yhNMT1,and then reduces the accumulation of caffeine in the callus of‘Yinghong 9’.

关 键 词：茶树 咖啡碱 HD-Zip转录因子 RNA干涉 

分 类 号：S571.1[农业科学—茶叶生产加工]