Characterization and Expression Analysis of the Target Genes Regulated by ERF in Cassava

SONG Na; ZHANG Liuningying; Cao Min; GUO Wenya; HONG Yuhui; WU Jinshan; CHEN Yinhua; YU Xiaohui

doi:10.15886/j.cnki.rdswxb.2020.02.008

The defense response of plants to stress is largely regulated by the level of gene transcription, and the recognition and combination of the transcription factor and cis-acting elements on the target gene promoter plays a key regulatory role. ERF is a type of transcription factor that plays an important role in the growth and development of plants. There is a conserved GCC-box in the promoter region of downstream target genes regulated by it. In order to analyze the regulatory pathway of ERF in the growth and development of cassava, 204 genes containing GCC-box cis-acting elements in the promoter region were screened. Chromosomal position distribution, analysis of expression patterns under pathogen infection, and promoter structure prediction of these genes were analyzed using bioinformatics methods. The qRT-PCR results showed that Manes.02G189600, Manes.03G039700, Manes.06G002000, Manes.09G063700, Manes.14G141600, Manes.15G181900 might be involved in regulating plant disease resistance pathways, and play an important way in ethylene-mediated signal transduction pathways.

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在自然环境中，植物总会遭受各种生物或非生物的胁迫，在长期的进化过程中，自然也会形成一定的防御机制。转录因子在植物响应逆境胁迫过程中具有重要功能，它们通过与多个基因启动子区域顺式作用元件的特异性结合参与植物的生长、发育和压力信号传递等过程^[1-2]。乙烯响应因子（ERF，Ethylene Response Factor）是植物对生物和非生物胁迫响应反应中的主要调节因子^[3-5]。转录因子识别特定的基序，并作为特定基因的激活或抑制因子发挥作用^[6]。ERF转录因子含有57～70个氨基酸组成的ERF域^[7-9]，可以特异地与存在于植物细胞内的病原菌响应基因启动子中的GCC-box（GCC-box：AGCCGCC）结合，从而激活或者抑制ET，JA偶联的防御途径^[8，10]。在许多病程相关基因启动子区都有GCC-box，如β−1,3−葡聚糖酶、几丁质酶和渗透蛋白，转录因子通过与GCC-box的结合直接或间接调节PR基因的表达，从而介导这些关键基因在植物响应生物胁迫中发挥作用^[11]。木薯（Manihot esculenta）不仅是主要的粮食和经济作物，也是一种重要的再生能源作物^[12-13]。广西、广东、海南、云南和福建是我国木薯的主要种植省份。由于种植省份的特殊地理位置，使木薯经常受到台风、海水倒灌使土地海盐量升高等的影响，极大地影响了木薯的产量。如时涛，李超萍等^[14-15]的研究表明，低温则使得木薯低产甚至绝收，湿热则容易让木薯受到病害的威胁。目前，已发现ERF转录因子在木薯应对生物或非生物胁迫时能够发挥一定的作用^[16]。因此，笔者利用生物信息学手段，挖掘木薯ERF转录因子的下游靶基因，旨在为木薯响应生物及非生物胁迫分子机理的解析提供基因资源和理论基础。

1. 材料与方法

1.1. 材料和试剂

供试材料为华南8号（SC8, South China 8）品种木薯，由本实验室种植和保存。菌株：由本实验室在广西分离的Xam11菌株。主要试剂：多糖多酚植物总RNA提取试剂盒（TAINGEN, DP441），反转录试剂盒（Thermo，K1622）, TB GREENII（TaKaRa，RR820A）, DNA Marker（TaKaRa），引物由上海生工生物工程有限公司合成。

1.2. 木薯ERF转录因子靶基因的筛选及生物信息学分析

木薯ERF转录因子靶基因的生物信息学分析通过相应的在线数据库和软件完成。在Phytozome网站上下载木薯全基因组数据。利用在线网站Softberry对木薯全部基因的启动子区进行含有GCC-box顺式作用元件的基因的筛选；利用MapInspect软件分析MeBFs基因的染色体定位情况；在NCBI网站的SRA（Sequence Read Archive）数据库中下载病原菌侵染木薯的转录组数据。转录组数据来源于用Xam弱致病菌株ORST4和强致病菌株ORST4+TALE1侵染苗龄8周的木薯（品种为MCOL1522）组培苗的实验^[17]。计算木薯ERF转录因子靶基因在这些转录组中的FPKM值，并将相互之间表达差异大于2倍的76个基因用Heatmap进行可视化；在Phytozome数据库中下载ERF转录因子靶基因ATG上游2 000 bp序列，利用PlantCARE在线网站对这2 000 bp序列的顺式作用元件进行预测分析，筛选与生物胁迫或非生物胁迫相关的顺式作用元件，再利用TBtools软件进行可视化分析。

1.3. 激素与病原菌处理及qPCR表达分析

用100 mg·L⁻¹ ACC（1−氨基环丙烷−1−羧酸）喷施处理木薯（SC8）组培幼苗，处理后分别在0，0.5，1，2，4，6 h取样。用刀片刮伤法给苗龄30 d的木薯田间苗接种Xam11病原菌，分别在接种后0，5，8，24，48 h取样。取样时设置3个生物学重复，其中以10 μmol·L⁻¹ MgCl₂溶液作为病原菌处理的负对照，ddH₂O作为激素处理的负对照。采取的叶片鲜样用液氮速冻，置于−80 ℃冰箱备用。

用多糖多酚植物总RNA提取试剂盒（TAINGEN，DP441）提取样品的RNA，用1.5%的琼脂糖凝胶电泳检测提取的总RNA的质量。以获得的总RNA为模板，利用反转录试剂盒（Thermo，K1622）进行cDNA反转录。反转录完成后，用木薯内参基因EF1a进行RT-PCR检测cDNA，以cDNA为模板进行后续实验。使用Oligo7设计qPCR引物，以未处理、激素处理和病原菌处理的不同时间点的cDNA为模板，以EF1a为内参基因，在Rotor-Gene Q（QIAGEN）仪器上进行qPCR分析，实时荧光定量PCR体系为：TB GREENII酶10 μL，上下游引物各0.5 μL，木薯cDNA模板2 μL，ddH₂O 7 μL，每个时间点设3个技术性重复。

3. 讨　论

在对各种外界胁迫的适应过程中，植物体内发生了一系列的信号传递，并激发其防御体系，使植物产生防卫反应，提高其自身抵抗生物和非生物胁迫的能力。这些防卫反应涉及大量相关基因在转录水平上的调整，其中，各种转录因子与其目标基因启动子上顺式作用元件的识别和结合起关键作用^[18-19]。GCC-box通常存在于植物PR基因的启动子中，如在拟南芥的PDF2.1、烟草的PR3等基因的启动子中均含有GCC-box，在一些非生物胁迫应答基因的启动子中也含有GCC-box^{[11, 20-21]}。番茄ERF2能分别结合乙烯合成相关基因ACS3和ACO3启动子中的GCC盒和DRE顺式作用元件，调控乙烯的合成^[22]。在过表达ERF104的拟南芥转基因植株的基因表达谱分析中，发现534个表达水平上调3倍以上的基因，包括PDF1.2，PR5，MKK4，RBOHD，ERF4，WRKY33和TGA1.3等一些已知的防卫基因或抗病信号调控基因，其中，有2个PDF1.2基因的表达水平升高近1 000倍，而表达水平上调10倍以上的基因启动子中含有GCC-box^[23]。GCC-box是ERF转录因子的主要顺式作用元件，一些ERF转录因子能通过结合启动子中GCC-box来调控靶标基因的表达。GCC-box存在于大量的PR基因启动子中，ERF转录因子可以通过结合GCC-box直接调节PR基因的表达，如拟南芥ERF1和ORA59能直接与PR基因PDF1.2启动子中的GCC顺式作用元件结合，并激活其表达^[24]。

本研究从木薯全基因组中筛选出了204个启动子区含有GCC-box顺式作用元件的基因，它们在木薯的18条染色体上均有分布。通过分析这些基因在病原菌侵染下的表达模式，从中选取表达差异比较显著的基因，并对它们的启动子区顺式作用元件进行预测分析，发现这些基因的启动子区都含有多种胁迫响应元件，多种顺式作用元件的存在说明这些基因可能受到复杂的调控，在木薯遭遇胁迫时发挥一定的作用。本研究利用qPCR验证的结果进一步表明，这些基因可以响应病原菌对木薯的侵染，初步推断这些基因可能参与了木薯的抗病途径。本实验结果为木薯抗病机制及抗病育种研究奠定了良好的基础。

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运行ID Run ID	样品名称 Sampling
SRR1050891	ORST4, collected at 0 d
SRR1050892	ORST4, collected at 5 d
SRR1050893	ORST4, collected at 7 d
SRR1050894	ORST4+TALE1Xam, collected at 0 d
SRR1050895	ORST4+TALE1Xam, collected at 5 d
SRR1050896	ORST4+TALE1Xam, collected at 7 d
SRR1050897	ORST4, collected at 0 d
SRR1050898	ORST4, collected at 5 d
SRR1050899	ORST4, collected at 7 d
SRR1050900	ORST4+TALE1Xam, collected at 0 d
SRR1050901	ORST4+TALE1Xam, collected at 5 d
SRR1050902	ORST4+TALE1Xam, collected at 7 d

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Characterization and Expression Analysis of the Target Genes Regulated by ERF in Cassava

doi: 10.15886/j.cnki.rdswxb.2020.02.008

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通讯作者: 陈斌, bchen63@163.com

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Characterization and Expression Analysis of the Target Genes Regulated by ERF in Cassava

doi: 10.15886/j.cnki.rdswxb.2020.02.008

1. College of Tropical Ccrops, Hainan University, Haikou, Hainan 570228

2. School of Life and Pharmaceutical Sciences, Hainan University, Haikou, Hainan 570228, China