枣WRKY转录因子的鉴定及其对枣疯病植原体和激素处理的应答

doi:10.11707/j.1001-7488.20180808

摘要/Abstract

摘要： [目的]分析枣WRKY转录因子对枣疯病植原体以及不同激素的响应，探讨WRKY基因在枣疯病发病过程中的作用，为进一步研究枣WRKY转录因子的生物学功能及枣与植原体相互作用机制奠定基础。[方法]利用同源比对从NCBI数据库以及转录组数据库鉴定出枣假定WRKY转录因子，并利用SMART等对其进行生物信息学分析；从转录组分析中挑选出差异表达的6个基因，设计定量引物，验证其在灰枣枣疯病发病过程中的表达量变化情况；以‘蜂蜜罐’枣胚培苗为材料，外源施加0.1 mmol·L^-1水杨酸（SA）和茉莉酸甲酯（MeJA）后，利用实时荧光定量PCR （qPCR）技术分析枣WRKY基因的表达量变化情况。[结果]确定了69个枣假定WRKY转录因子，重新命名为ZjWRKY1-69，可分为3个组GroupⅠ、GroupⅡ和GroupⅢ，GroupⅠ含有17个成员，GroupⅡ又可细分为Ⅱa、b、c、d、e 5个亚组，分别包含3、11、15、3、9个成员，GroupⅢ有11个成员；69个枣WRKY转录因子基因中，有43个ZjWRKY基因可以定位到11条染色体上，但在染色体上呈不均匀分布，7号染色体上没有ZjWRKY基因存在；大部分ZjWRKY蛋白都包含有WRKYGQK保守序列，有2个ZjWRKY蛋白（ZjWRKY23和ZjWRKY46，GroupⅡc）变异为WRKYGKK；GroupⅠ中的WRKY转录因子含有较多的保守基序（10个），且同一个组内WRKY蛋白的保守基序类型及个数相似。从枣疯病发生过程中枣叶片的转录组数据中检测到28个ZjWRKY转录因子基因的差异表达主要集中在嫁接后38~52周。枣疯病发病过程中ZjWRKY8、ZjWRKY52、ZjWRKY61、ZjWRKY69基因表达量发生明显变化（上调）。SA处理后，ZjWRKY42和ZjWRKY52表达量显著升高；MeJA的积累诱导ZjWRKY42和ZjWRKY61的表达量发生较明显变化。[结论]初步确定了枣69个WRKY转录因子，分为3组；43个枣WRKY基因可以定位到11条染色体上，呈不均匀分布，7号染色体上尚没有发现WRKY基因的存在。枣疯病植原体侵染枣树后，诱导了ZjWRKY5、ZjWRKY8、ZjWRKY42、ZjWRKY52、ZjWRKY61基因的表达，SA和MeJA处理后分别使ZjWRKY42、ZjWRKY52和ZjWRKY42、ZjWRKY61表达量显著升高。

关键词: 枣, WRKY, 枣疯病, 水杨酸, 茉莉酸

Abstract: [Objective] To study the biological function of jujube WRKY transcription factor (TF) and interaction mechanisms between jujube and ‘Candidatus Phytoplasma ziziphi’, we analyzed the response of jujube WRKY to ‘Candidatus Phytoplasma ziziphi’ and salicylic acid or methyl jasmonate treatments.[Method] The putative jujube WRKY TFs were explored and identified from NCBI and transcriptomics database by homologous alignment, and their structure and bioinformation were analyzed using SMART software. Six differentially expressed jujube WRKY were screened from the transcriptomics analyses, and the expression changes of them were verified by real time PCR during the development of jujube witches' broom (JWB) of Ziziphus jujuba ‘Huizao’. The embryo cultured seedling of jujube (Z. jujuba ‘Fengmiguan’) were sprayed with 0.1 mmol·L^-1 salicylic acid (SA) and methyl jasmonate (MeJA), and relative expression levels of the selected six WRKY genes were analyzed by qPCR.[Result] 69 putative jujube WRKY TFs were confirmed and named ZjWRKY1-69, which could be divided into three groups:GroupⅠ, GroupⅡ, GroupⅢ. GroupⅠ contained 17 members, whereas groupⅡ can be further divided into GroupⅡa, b, c, d, e subgroups, contained 3, 11, 15, 3, and 9 members, respectively. GroupⅢ included 11 members. 43 of 69 putative jujube WRKY genes were mapped to the 11 jujube chromosome, and were unevenly located on different chromosomes such as no WRKY on chromosome 7. Most of the jujube WRKY proteins contained conserved WRKYGQK sequences, while two WRKY proteins (ZjWRKY23 and ZjWRKY46, GroupⅡc) varied to WRKYGKK. The WRKY TFs in GroupⅠ contained 10 conserved motifs, the number of motifs in GroupⅠ was the largest among the three groups. The conserved motif type and number of WRKY protein are similar in the same group. The expression pattern of 28 jujube WRKY genes were detected to be different during the JWB developing in jujube leaves, and differentially expressed ZjWRKY genes were mainly detected from 39 to 52 weeks after infection. The expression levels of ZjWRKY8, ZjWRKY52, ZjWRKY61, and ZjWRKY69 were significantly up-regulated during JWB developing. After SA treatment, the expression levels of ZjWRKY42 and ZjWRKY52 were significantly increased. The expression levels of ZjWRKY42 and ZjWRKY61 were significantly up-regulated in response to MeJA treatment.[Conclusion] 69 jujube WRKY TFs were preliminarily confirmed, which can be divided into three groups. 43 WRKY genes were mapped to 11 chromosomes,no WRKY genes on Chr 7. After the infection of ‘Candidatus Phytoplasma ziziphi’, the expression of ZjWRKY5, ZjWRKY8, ZjWRKY42, ZjWRKY52, and ZjWRKY61 were induced. The expression of ZjWRKY42 and ZjWRKY52, ZjWRKY42 and ZjWRKY61 were significantly up-regulated by SA and JA treatments, respectively.

Key words: Ziziphus jujuba, WRKY, ‘Candidatus Phytoplasma ziziphi’, salicylic acid, methyl jasmonate

中图分类号:

S718.46

付冰, 叶霞, 王会鱼, 陈鹏, 李继东, 郑先波, 谭彬, 冯建灿. 枣WRKY转录因子的鉴定及其对枣疯病植原体和激素处理的应答[J]. 林业科学, 2018, 54(8): 65-78.

Fu Bing, Ye Xia, Wang Huiyu, Chen Peng, Li Jidong, Zheng Xianbo, Tan Bin, Feng Jiancan. Identification of WRKY Transcription Factors in Jujube and Their Responses to ‘Candidatus Phytoplasma ziziphi’ and Salicylic Acid or Methyl Jasmonate Treatments[J]. Scientia Silvae Sinicae, 2018, 54(8): 65-78.

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