Gene Cloning and Expression Characteristics of Transcription Factor IiMYB from Isatis indigotica

Ma Xueqi ¹　Li Shunuo ¹　Zhang Guoning ¹　Li Yuanyu ¹　Chen Wansheng ^1,2　Xiao Ying ^1*　Sun Lianna ^1*

1 Teaching and Research Department of Chinese Medicine Processing, Research and Development Center of Chinese Medicine Resources and Biotechnology, Institute of traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203; 2 Medical Guarantee Center, Changzheng Hospital, Naval Medical University, Shanghai, 200003

* Co-corresponding authors, xiaoyingtcm@shutcm.edu.cn; sssnmr@163.com

Abstract　MYB transcription factors play an important role in plant growth and development, biosynthesis of secondary metabolites, as well as resistance to stresses. To explore the expression characteristics of a specific MYB transcription factor from Isatis indigotica, the full-length cDNA of the target MYB transcription factor encoding gene IiMYB is cloned by rapid amplification of cDNA ends (RACE), and its genomic DNA sequence is further obtained. qRT-PCR is used to detect the expression levels of IiMYB in different organs of tetraploid and diploid I. indigotica and under various stress conditions. The full-length cDNA of IiMYB gene is 926 bp (GenBank accession number: DQ468346) long with an open reading frame (ORF) of 600 bp encoding a polypeptide of 199 amino acid residues. The corresponding genomic DNA sequence contains 3 exons and 2 introns. Bioinformatics analysis shows that IiMYB has the highest homology and closest genetic distance with AtMYBL2 from Arabidopsis, and also contains only one myb domain (R3). IiMYB is expressed in the roots, stems and leaves of both diploid and tetraploid I. indigotica, with the highest expression level in leaves, and its expression in tetraploid one is higher than that in diploid. Stress treatments including salt (NaCl), methyl jasmonate (MeJA), gibberellin (GA3), abscisic acid (ABA), and salicylic acid (SA) can all increase IiMYB expression level, while low temperature (4 °C) treatment has no significant effect on IiMYB expression. In this study, a MYB transcription factor is cloned from I. indigotica for the first time and its expression characteristics are analyzed, which provides a possibility to further elucidate the MYB relevant molecular mechanism of polyploidy vigor of I. indigotica, and also prompts to cultivate high-quality I. indigotica through molecular breeding.

Keywords　Isatis indigotica Fortune; MYB transcription factor; Molecular cloning; Bioinformatics; Expression analysis

菘蓝(Isatis indigotica Fortune)为十字花科菘蓝属(Cruciferae)草本植物。其根入药为板蓝根，其叶入药为大青叶，有清热解毒、凉血消斑、利咽止痛的功效，广泛应用于临床(胡永胜等, 2015)。本课题组前期经过多倍体育种培育了四倍体菘蓝优良品系，与原二倍体亲本相比，四倍体菘蓝的根、叶增产显著，且表现出稳定的高水平抗病毒活性(乔传卓等, 1989)。为了探究两种倍性菘蓝品质差异的分子机制，课题组前期通过拟南芥(Arabidopsis thaliana)基因芯片对二倍体和四倍体菘蓝的全基因组基因表达水平进行比较(Lu et al., 2006a)，并对筛选获得的一系列差异基因进行表达特征及功能研究，包括信号转导基因IiCPK1 (Pan et al., 2008)和IiCPK2 (Lu et al., 2006b)、气孔密度调节基因IiSDD1 (Xiao et al., 2010)、抗病毒活性成分木脂素生物合成相关基因IiPAL (Lu et al., 2006c; Ma et al., 2016)及转录因子IiWRKY34 (Xiao et al., 2020)等，以期获得影响四倍体菘蓝优良性状形成的关键遗传靶点。例如：基因芯片的结果显示转录因子IiWRKY34在四倍体菘蓝中的表达显著高于二倍体亲本，以此为依据，分离获得了IiWRKY34编码基因并进行靶向调控，发现IiWRKY34的高表达确实能促进菘蓝根系发育、激活木脂素类成分的生物合成，并能提升抗寒抗旱能力(Xiao et al., 2020)。

转录因子(transcription factor, TF)作为转录调控最重要的成员，参与植物生长发育、信号转导、代谢调控及抗逆响应等各个关键环节，发挥中枢核心调控作用(肖莹等, 2019)。通过比较两种倍性菘蓝差异的转录因子，发现与WRKY类似，四倍体菘蓝中MYB转录因子的表达显著高于二倍体亲本(Lu et al., 2006a)，提示四倍体菘蓝中MYB转录因子的高表达很可能也是影响其优势性状特征形成的重要遗传因素。MYB转录因子家族是一类含有myb结构域的转录因子，作为最大的转录因子家族之一，对调控植物生长发育、次生代谢产物生物合成及胁迫响应等发挥重要作用。例如：青蒿Artemisia annua中MYB转录因子AaTAR2对青蒿腺毛发育、青蒿素和类黄酮的生物合成具有积极的调控作用(Zhou et al., 2020)；在拟南芥中异源表达二色补血草(Limonium bicolor)的MYB转录因子LbTRY能显著促进根毛生长(Leng et al., 2021)；转录因子MdMYB73通过水杨酸途径提高苹果(Malus sieversii)对真菌(Botryosphaeria dothidea)的抗性(Gu et al., 2020)。综上，本研究聚焦前期实验提示的四倍体菘蓝中高表达的目标MYB转录因子(Lu et al., 2006a)，对其进行基因克隆和表达特征研究，以期为通过分子育种培育高产高抗、抗病毒活性成分高积累的高品质菘蓝品系提供基础。

1结果与分析

1.1 IiMYB基因克隆

通过RACE-PCR技术克隆获得IiMYB全长cDNA序列为926 bp (GenBank登录号: DQ468346) (图1A)，包含一个600 bp的开放阅读框(ORF)，编码199个氨基酸。IiMYB的cDNA含有长度为117 bp的5’UTR，209 bp的3’UTR(图 2)，四倍体菘蓝中IiMYB基因的cDNA序列与二倍体中完全一致。扩增IiMYB基因全长cDNA对应的基因组序列的PCR得到了一条清晰的条带(图1B)。测序结果表明IiMYB基因组DNA序列为979 bp，含有2个内含子，它们的剪接遵守GT/AG法则(图2)。

1.2 IiMYB的生物信息学特征

IiMYB的等电点(pI)为9.71，分子量大小约为22.70 kD。IiMYB只含有一个myb域(R3)，位于氨基端的A31~A81间(图2)。IiMYB的氨基酸序列与其他植物来源的MYB氨基酸序列的比对结果显示，在A31～A81这个区域，IiMYB与其他植物的MYBs之间有较高的同源性，均高于90.0%；且都含有Phe39、Trp58，Trp77这三个保守的氨基酸残基(图3)，参与空间结构疏水核心的形成。在IiMYB的C端存在两个丝氨酸富集区，分别位于A123~A132和A181~A191；并预测了IiMYB的多聚腺嘌呤信号位点(图2)，是mRNA前体多聚腺苷化所必需的Poly(A)加尾信号(陆颖等, 2006)，因此推测IiMYB可能具有转录激活功能。IiMYB与AtMYBs的系统进化树结果显示IiMYB与AtMYBL2有较高的相似度，遗传距离最近(图4)。

1.3 IiMYB基因的表达特征

使用qRT-PCR比较IiMYB基因在二倍体和四倍体菘蓝不同器官中的表达情况。结果表明，IiMYB在两种倍性菘蓝的根、茎和叶中均有表达；在两种倍性菘蓝中IiMYB基因均在叶中的表达量最高，在根中的表达量最低；且IiMYB在四倍体菘蓝根、茎、叶中的表达水平均高于二倍体，分别约为二倍体的1.2、1.5和1.5倍(图5)，与基因芯片MYB基因差异表达的结果相符(Lu et al., 2006a)。

通过对四倍体菘蓝幼苗进行多种胁迫处理以进一步考察IiMYB的表达是否受到一些胁迫因素及信号分子的影响。结果表明，分别用高盐(NaCl, 250 mmol/L)、水杨酸(SA, 1 mmol/L)、茉莉酸甲酯(MeJA, 500 μmol/L)、赤霉素(GA3, 100 μmol/L)和脱落酸(ABA, 100 μmol/L)处理8 h后的样品中，IiMYB的表达与对照相比均提高了大约2倍(P<0.01)，但冷处理对IiMYB的表达几乎没有影响(图6)。

2讨论

MYB是植物中最大的转录因子家族之一，参与植物生长发育、生理代谢、细胞的形态和模式建成等生理过程(牛义岭等, 2016; Li et al., 2019)，且在次生代谢调控(Cao et al., 2020)及干旱、盐渍、冷害等胁迫反应中均发挥重要作用(Tiwari et al., 2020)。前期利用拟南芥基因芯片筛选发现四倍体菘蓝中特异MYB基因的表达水平显著高于二倍体亲本(Lu et al., 2006a)，提示四倍体菘蓝的优良性状(高产高抗, 抗病毒活性成分高积累)可能与MYB基因的高表达密切相关。由于拟南芥基因芯片获得的四倍体菘蓝比二倍体高表达的MYB探针At1g71030对应的基因为AtMYBL2 (Kirik and Bäumlein, 1996)，本研究利用AtMYBL2序列进行特异引物设计，以保证扩增获得的IiMYB是菘蓝MYB基因大家族中与AtMYBL2同源的基因成员。基因表达水平检测发现IiMYB在四倍体菘蓝根、茎和叶中的表达显著高于二倍体亲本，进一步明确了IiMYB在两种倍性菘蓝中的表达差异，为研究与之相关的四倍体菘蓝优良性状形成的分子机制提供了科学依据。

本研究获得的IiMYB是菘蓝中首个报道的MYB基因，与拟南芥中AtMYBL2的同源性较高，且具有相似的结构特征。该结果一方面进一步证明了同源克隆获取目标基因的准确性，另一方面提示IiMYB可能具有与AtMYBL2相似的功能。文献报道拟南芥中AtMYBL2作为转录抑制因子(Ye et al., 2012)，负调控拟南芥花青素的生物合成(Xie et al., 2016)，而花青素是保护植物组织免受许多环境胁迫的类黄酮化合物(Kim et al., 2017)，说明AtMYBL2参与了拟南芥对外界环境的响应。IiMYB转录因子在菘蓝中是否发挥类似功能值得进一步研究。

环境改变和植物激素能诱导胁迫相关基因的表达从而使植物产生抗逆活性(黎家和李传友, 2019)。本研究发现NaCl，SA，MeJA，GA3和ABA处理均能上调IiMYB的表达水平，提示IiMYB可能参与以上胁迫响应，这与其它植物中MYB参与抗逆反应的功能相似。例如，MeJA处理可以显著提高茶树(Camellia sinensis)中CsMYB8和CsMYB20的表达水平(Wang et al., 2018)；低温、干旱、高盐和激素处理能提高菠萝(Ananas comosus)中AcoMYB4的表达水平，且通过ABA信号转导途径调控渗透胁迫(Chen et al., 2020)；多种胁迫因素(包括NaCl, GA3, MeJA, ABA, IAA和低温)能诱导TaMYB86B表达提升，且过表达TaMYB86B会增加小麦(Triticum aestivum)中胁迫相关基因的表达量(Song et al., 2020)。综上，四倍体菘蓝抗逆活性的增强是否与IiMYB表达的提升相关值得深入研究。本研究推测在四倍体菘蓝中转录因子IiMYB的高表达可能会影响到一批受之调控的基因的表达，从而对四倍体菘蓝的抗逆活性等多种性状产生影响。接下来本研究将通过遗传转化、蛋白互作实验等筛选IiMYB调控的下游基因并深入研究其在调控四倍体菘蓝优良性状形成中的作用。

3材料与方法

3.1实验材料的处理

二倍体和四倍体菘蓝种子采集自海军军医大学药学院植物园。培养基质为泥炭土和珍珠岩按1:1的体积比混合而成。培养温度25/18 ℃ (白天/夜晚)，光照强度120 µmol/m²/s，光周期16/8 h (白天/夜晚)。生长两个月后，分别采集两种倍性菘蓝的根、茎和叶，用于提取DNA和RNA，每种实验材料采集三个生物学重复。

3.2菘蓝核酸的提取以及RACE cDNA文库的构建

分别按照CTAB法和TRIzol法提取四倍体菘蓝的DNA和RNA，并检测浓度和纯度。按SMART^TM RACE cDNA Amplification Kit操作程序以总RNA为模板构建RACE cDNA文库。5’-ready和3’-ready RACE cDNA文库于-20 ℃保存。

3.3菘蓝IiMYB基因全长cDNA及对应的基因组序列的获得

根据SMART^TM RACE cDNA Amplification Kit实验手册进行RACE-PCR实验，扩增获得IiMYB基因全长cDNA及其对应的基因组序列(表1)。PCR扩增反应体系为50 μL，并通过琼脂糖凝胶电泳对目的片段进行回收纯化，纯化后的PCR产物连接pMD-18T载体，转入大肠杆菌菌株DH5α，菌液送至上海生工生物工程有限公司进行测序。

3.4 IiMYB的生物信息学研究

基于ProtParam (http://us.expasy.org/tools/protparam.html)的预测和分析获得IiMYB的蛋白质基本性质。通过Vector NTI Advance 11.5.1软件预测基因ORF并完成核苷酸的翻译。

使用https://www.ncbi.nlm.nih.gov/网站以IiMYB的核苷酸序列进行BLAST筛选相似序列(表2)，并使用http://tcoffee.crg.cat/网站、Jalview和Vector NTI Advance 11.5.1对这些基因编码的蛋白序列进行多重序列比对和分析。

通过文献调研筛选到同为十字花科植物拟南芥中38条功能明确的MYB基因，并在NCBI找到其氨基酸序列。使用MEGA 7.0软件以neighbor-joining (NJ)法，在重复1 000次计算下构建这38条AtMYBs与IiMYB蛋白的系统进化树。

3.5 IiMYB的器官及胁迫表达特征

胁迫处理方法：将四倍体菘蓝幼苗置于4 °C冰箱8 h进行冷处理；分别使用250 mmol/L NaCl、1 mmol/L SA、500 μmol/L MeJA、100 μmol/L GA3和100 μmol/L ABA五种溶液喷洒于四倍体菘蓝幼苗的叶表面，收集处理8 h后的叶片样品。分别将未作冷处理或喷洒蒸馏水的四倍体菘蓝叶片作为对照样品。每种实验材料采集三个生物学重复。

采用TRIzol法提取生长两个月的两种倍性菘蓝的根、茎和叶，以及不同胁迫处理后的四倍体菘蓝叶片的RNA。用NovoScript® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge)试剂盒将不同器官及不同胁迫处理后的菘蓝总RNA反转录成cDNA，作为qRT-PCR反应的模板。qRT-PCR按照TaKaRa TB Green^® Premix Ex Taq^TMⅡ试剂盒的操作程序进行，IiMYB基因特异引物为MYB-qRT-F、MYB-qRT-R，内参基因引物为actin-F、actin-R (表1)。基因相对表达量用2^−ΔΔCt方法计算，每个样品独立重复三次，通过Student’s t-test分析处理样品与对照中基因相对表达量的差异显著性 (差异极显著：**P<0.01；差异显著：*P<0.05)。

作者贡献

马雪祺完成了本文的基因克隆、表达分析实验和论文初稿的撰写；李姝诺完成了生物信息学分析；张国宁协助数据分析及论文修改；李元玉完成了相关图表的制作；陈万生是本实验的指导者；肖莹提出实验想法，设计实验及指导研究、完成文章的修改和定稿；孙连娜指导并推进整个项目的进展工作。全体作者都阅读并同意最终的文本。

致谢

本研究由国家自然科学基金资助项目(81874335和31872665)、上海市青年科技启明星计划(18QB1402700)和上海中医药大学项目(A1-GY20-306-02-08)共同资助。

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