超量表达响应β-氨基丁酸诱导的StWRKY8基因提高马铃薯晚疫病抗性  

Overexpression of β-aminobutyric Acid Inducible Gene StWRKY8 Enhances Late Blight Resistance in Potato(Solanum tuberosum)

作　　者：王海霞 余小玲[1] 戚烨通 田振东[1,2] WANG Hai-Xia;YU Xiao-Ling;QI Ye-Tong;TIAN Zhen-Dong(Key Laboratory of Potato Biology and Biotechnology, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China;Key Laboratory of Horticultural Plant Biology （HZAU）, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China)

机构地区：[1]华中农业大学农业部马铃薯生物学与生物技术重点实验室,武汉430070 [2]华中农业大学园艺植物生物学教育部重点实验室,武汉430070

出　　处：《农业生物技术学报》2018年第7期1107-1115,共9页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2013AA102603);国家自然科学基金(No.31171603和No.31471550)

摘　　要：晚疫病是马铃薯(Solanum tuberosum)生产中最为严重的病害,抗晚疫病是马铃薯育种的主要目标之一。前期研究中,在具有田间抗性的马铃薯材料中筛选到一个受广谱诱抗剂β-氨基丁酸(β-aminobutyric acid,BABA)诱导表达的转录因子StWRKY8基因片段。为了进一步研究该基因在马铃薯晚疫病抗性中的功能,本研究利用qRT-PCR技术进一步分析了该基因受β-氨基丁酸和晚疫病菌—卵菌致病疫霉(Phytophthora infestans)诱导表达的模式;从马铃薯中克隆了该基因cDNA,构建了35S启动子驱动的植物表达载体,通过农杆菌(Agrobacterium tumefaciens)介导的方法进行了马铃薯的遗传转化;对获得的超量表达转基因株系进行了离体叶片晚疫病接种抗性鉴定,同时利用qRT-PCR技术检测了StWRKY8下游植保素合成相关基因—3-羟-3-甲戊二酰辅酶A还原酶基因2(3-hydroxy-3-methylglutaryl Co A reductase 2,HMGR2)和NADP-苹果酸酶基因(NADP-malic enzyme,NADP-ME)的表达。研究结果表明,BABA和P.infestans都能诱导StWRKY8基因上调表达,BABA诱导24 h达到表达高峰,而P.infestans诱导36 h达到表达高峰。本研究克隆的StWRKY8基因编码区长1 605 bp,编码534个氨基酸,与已报道马铃薯StWRKY8(NP_001274836.1)蛋白序列相似性达到98%,含有两个WRKYGQK保守结构域。获得了StWRKY8超量表达转基因株系,晚疫病接种抗性鉴定表明,4个表达量高的转基因株系晚疫病抗性显著提高。另外,本研究结果表明,受StWRKY8调控的HMGR2和NADP-ME基因的表达量在超量表达转基因株系中并未明显提高。但是,当转基因株系受到晚疫病菌液体培养物(culture filtrate,CF)诱导时,这两个基因在转基因株系中显著上调表达。本研究结果为进一步探讨StWRKY8抗性调控机理以及利用该基因提高马铃薯晚疫病抗性提供了重要信息。Late blight resistance is a major aim of potato （Solanum tuberosum） breeding. In order to investigate the molecular mechanisms of inducible late blight resistance in potato, previously, we had identified a transcription factor gene StWRKY8 fragment which was induced by a broad spectrum chemical inducer- v-aminobutyric acid （BABA） in a potato material with filed late blight resistance, For further looking into its function, the expression patterns of StWRKY8 responded to BABA and Phytophthora infestans was investigated by qRT-PCR. Full length cDNA of StWRKY8 was cloned from potato and over-expression vector （controlled by CaMV 35S promoter） was constructed and transformed into potato. Detached leave assay was performed to evaluate late blight resistance on transgenic plants. In addition, the expression levels of two of StWRKY8 downstream defense-related genes, 3-hydroxy-3-methylglutaryl CoA reductase 2 （HMGR2） （involving phytoalexin synthesis） and NADP-malic enzyme （NADP-ME） were analyzed by qRT-PCR. The results showed that StWRKY8 was induced by both BABA and P. infestans. The expression of StWRKY8 reached a peak at 24 h after BABA treatment and at 36 h after P. infestans inoculation. Sequence analysis showed that the open reading frame （ORF） of cloned StWRKY8 was 1 605 bp. It codes 534 aa with two conserved WRKYGQK domains and shares 98% identity with a StWRKY8 （NP_001274836.1） deposited in GenBank. Detached leaf assay of resistance showed that late blight resistance enhanced significantly in 4 transgenic potato lines compared to control. HMGR2 and NADP-ME were significantly upregulated in transgenic potato lines after treatment with liquid P. infestans culture filtrate （CF）. This study provides valuable information for further investigation of defense regulating mechanism of StWRKY8 and utilization of this gene to improve late blight resistance in potato breeding.

关 键 词：马铃薯 StWRKY8 超量表达 晚疫病抗性 功能研究 

分 类 号：S532[农业科学—作物学]