全基因组DNA甲基化和转录组联合分析鉴定杜梨耐盐相关转录因子  

Identification of Salt-Tolerant Transcription Factors in the Roots of Pyrus betulaefolia by the Association Analysis of Genome-Wide DNA Methylation and Transcriptome

作　　者：李慧[1] 张雨峰 李晓刚[1] 王中华[1] 蔺经[1] 常有宏[1] LI Hui;ZHANG YuFeng;LI XiaoGang;WANG ZhongHua;LIN Jing;CHANG YouHong(Institute of Pomology,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,Nanjing 210014;College of Biology and the Environment,Nanjing Forestry University,Nanjing 210037)

机构地区：[1]江苏省农业科学院果树研究所/江苏省高效园艺作物遗传改良重点实验室,南京210014 [2]南京林业大学生物与环境学院,南京210037

出　　处：《中国农业科学》2023年第7期1377-1390,I0004-I0007,共18页Scientia Agricultura Sinica

基　　金：江苏省自然科学基金(BK20191238);江苏现代农业(梨)产业技术体系项目[ATS(2021)436];国家自然科学基金面上项目(31772287)。

摘　　要：【目的】鉴定不同杜梨株系根中响应盐胁迫信号的相关转录因子,分析盐胁迫下基因序列DNA甲基化变化与基因表达改变之间的关系,探讨参与调控不同杜梨株系耐盐能力的转录因子成员。【方法】以杜梨耐盐株系和普通株系为试材,在苗期使用200 mmol·L^(-1) NaCl对90日龄组培生根苗进行水培处理,以Hoagland营养液为对照。利用火焰石墨炉原子吸收光谱仪测定钠离子含量;利用全基因组DNA甲基化和转录组测序技术从表观遗传修饰和转录调控水平对盐胁迫下转录因子进行生物信息学分析;最后用McrBC-PCR和qPCR对差异转录因子进行验证。【结果】外源NaCl处理24 h后,杜梨植株中钠离子含量显著增加,其中耐盐株系的增加幅度比普通株系小,为普通株系钠含量的73.1%,但根中积累的钠是普通株系含量的1.1倍;杜梨根中检测到69类共2682个转录因子的表达,盐胁迫后243个转录因子在两个株系中都发生了差异表达,包括AP2/ERF(37个)、bHLH(19个)、bZIP(7个)、HD-Zip(10个)、MYB(30个)、NAC(18个)、WRKY(8个)和ZFP(23个)等家族成员;盐胁迫后,耐盐株系基因组中转录因子甲基化水平下降,而普通株系转录因子甲基化水平上升,其发生DNA差异甲基化区域主要在基因启动子位置,差异甲基化类型主要为mCHH,占mCG、mCHG、mCHH三种类型总和的93%以上。AP2/ERF、bHLH、DREB、GRAS、GT因子、HB Zip、MYB、NAC、Trihelix和Zinc finger ZFP家族的23个转录因子响应盐胁迫表达量上调而甲基化水平降低,可能参与调节钠在根中的吸收和积累。对部分候选基因的表达模式和启动子区域分别进行实时荧光定量(qPCR)和甲基化依赖型限制性内切酶PCR(McrBC-PCR),验证了生物信息学分析结果。【结论】盐胁迫后在两个杜梨株系根中均差异表达的转录因子数目为243个,其中8个转录因子(PbERF2、PbGT3、PbZAT10.1、PbSCL33、PbDREB1、PbZAT10.2、PbERF53、PbNAC72)DNA序【Objective】Here,two ecotypes of P.betulaefolia from Huaguo Mountain,Lianyungang(the salt-tolerant ecotype,D)and Purple Mountain,Nanjing(the common ecotype,U)were collected for this research.The purpose of this study was to analyze the role of transcription factor genes in the roots of two ecotypes of P. betulaefolia differing in terms of salt stress. Transcription factors involving in the regulation of the salt tolerance of different P. betulaefolia ecotypes were identified on the grounds of differential expression under salt stress and the relationship between the methylation status and the relative expression level of relevant tolerance genes after exposure to salt stress was investigated. 【Method】The 90-day-old P. betulaefolia seedlings were grown hydroponically in Hoagland’s nutrient solution supplemented with 200 mmol·L^(-1) NaCl, with seedlings grown in Hoagland’s nutrient solution as the control. The sodium ion content in the tissues was determined by flame graphite furnace atomic absorption spectrometry. Whole-genome DNA methylation analysis and transcriptome sequencing were performed on three replicates for the following four root samples: ecotype D and ecotype U, each grown in the presence or absence of salt stress. Bioinformatics analysis of transcription factor gene expression under salt stress at the levels of transcriptional regulation and epigenetic methylation were carried out using transcriptome sequencing data and whole-genome DNA methylation results, respectively. Then, McrBC-PCR and real-time fluorescence quantitative PCR (qPCR) were used to confirm the levels of methylation and transcription of differential transcription factor genes. 【Result】After exogenous NaCl treatment for 24 h, the concentration of sodium ions in P. betulaefolia roots increased significantly, with the increase in sodium ion concentration in the salt-tolerant ecotype being significantly less than that in the common ecotype. In the whole seedling, the final salt concentration of tolerant ecotype was only 7

关 键 词：杜梨 盐胁迫 转录因子 DNA甲基化 转录组 

分 类 号：S661.2[农业科学—果树学]