丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）是一种高度保守的蛋白激酶，广泛存在于动物、植物和微生物等各类真核生物中[1]。植物中的MAPK级联途径由3种激酶组成，即MAPKK激酶（MAPKKK）、MAPK激酶（MAPKK）和MAP激酶（MAPK），它是真核生物中感受并且传导胁迫信号的一种重要的生理途径，在调节植物对环境的适应方面起着主导作用[2]。当植物感受外界刺激信号时，通过这3种激酶的依次磷酸化（MAPKKK→MAPKK→MAPK）进行信号传递，最终由MAPK磷酸化下游底物（包括细胞骨架蛋白、蛋白激酶和细胞凋亡因子等）调控响应基因的表达，促使植物对逆境做出反应[3-4]。MAPKKK位于MAPK级联途径的最上游，分为3种亚型，分别为Raf、MEKK和ZIK[5]。植物MAPKKK是MAPK家族中成员最多的一类，拟南芥中有80个MAPKKK，其中48个属于Raf亚型，21个属于MEKK亚型，11个属于ZIK亚型[6]。水稻中存在75个MAPKKK，这3种亚型分别含有43、22和10个成员[7]。本课题组前期对玉米中的MAPKKK家族基因进行鉴定发现玉米中共含有71个MAPKKK，其中37个为Raf亚型，26个为MEKK亚型，8个为ZIK亚型[8]。研究表明，MAPKKK基因在植物的抗逆机制中发挥着重要作用[9]，苦荞中鉴定出的MAPKKK家族中FtMAPKKK8、FtMAPKKK10和FtMAPKKK24可积极响应干旱、高温和盐碱等非生物胁迫[10]。此外，研究[11]表明，苹果对苹果轮纹病菌的抗性受MAPKKK1和MdBSK1的互作调控。在拟南芥中过表达MAPKKK18可通过激活下游MAPKKK3途径显著增强其耐旱性[12]。通过对高粱MAPK级联基因进行分析[13]，发现SbMPK14基因表现出对干旱的超敏反应，故将该基因作为一个干旱胁迫的负调控因子。上述研究表明可通过获得关键抗逆MAPKKK基因并结合转基因等手段导入作物的方法创制抗旱新种质。
本实验室前期鉴定了玉米MAPKKK家族成员，并对玉米品种ZD619进行干旱胁迫下转录组测序并分析表达差异基因，发现包括ZmMAPKKK21基因在内的8个基因在干旱胁迫下出现上调表达，在玉米自交系J24、J853、X178、E28、C8605-2、200B、Q319和B73中进行干旱胁迫后发现除了干旱敏感自交系B73之外，ZmMAPKKK21基因在其余7个品种中干旱胁迫下均表达上调，一致性较高，充分说明该基因作用的广泛性；此外对于转录组测序筛选出的其他基因，例如将ZmMAPKKK26基因在多品种中进行表达分析，发现部分品种干旱处理后其表达下调，对干旱的响应程度不高，因而选择ZmMAPKKK21基因进行深入研究[16]。本试验以抗旱玉米自交系J24为材料，克隆ZmMAPKKK21基因并对其进行结构分析、启动子分析、蛋白理化性质分析、系统进化树分析、亚细胞定位预测以及互作蛋白分析，对该基因的组织表达情况进行了实时荧光定量PCR分析以及干旱处理后的基因表达分析，进一步确定了该基因是一个潜在的重要抗旱基因，为后续阐明该基因在水分胁迫下的调控机制提供理论依据。
1 材料与方法
1.1 试验材料及处理
玉米自交系J24由北京市农林科学院玉米研究所提供，试验于2022年在北京市农林科学院玉米研究所温室内进行。收集玉米自交系J24不同生长时期[出苗（VE）、三叶期（V3）、七叶期（V7）、吐丝期（R1）]的的不同组织材料（根、茎、叶、雌穗和雄穗），样品采集经液氮速冻存于-80 ℃，用于RNA提取。
对玉米自交系J24进行干旱盆栽试验。玉米幼苗正常生长至三叶一心期进行如下处理，对照组CK和处理组D用水分测量仪SM150T（Delta-T Devices，英国）将土壤含水量分别控制在16%~ 18%和8%~10%，处理14 d后进行取样，用液氮冻存于-80 ℃。
1.2 试验方法
1.2.1 引物设计与合成
根据NCBI中获得ZmMAPKKK21的全长序列以及启动子序列，设计引物用于PCR扩增，引物由北京擎科生物科技有限公司合成（表1）。
表1 引物序列及用途
Table 1
| 引物名称 Primer name | 引物序列（5'-3'） Primer sequence (5'-3') | 用途 Usage |
|---|---|---|
| ZmMAPKKK21-F | AACGATGGAAACGGACCGAA | 基因克隆 |
| ZmMAPKKK21-R | TGGGAATCTTGGCGTTGACA | |
| PZmMAPKKK21-F | GTCCAACTCTGACCCTAAGCG | 启动子克隆 |
| PZmMAPKKK21-R | CTACCAGTTCCAGTGTCTGC | |
| QZmMAPKKK21-F | GATGCAGAGGTGGAGCAACT | 实时荧光 定量PCR |
| QZmMAPKKK21-R | GTGGACGCCTGAATGCATAG | |
| ZmGPN1-F | TGACCAAGGTGAAGAGCACTGT | |
| ZmGPN1-R | CAAATCTCACGTGGCTATGAAAC |
1.2.2 供试主要试剂
高效植物基因组DNA提取试剂盒（离心柱型）和RNAprep Pure植物总RNA提取试剂盒（离心柱型）均购自天根生化科技（北京）有限公司；PrimeScript RT reagent kit with gDNA Eraser（Perfect Real Time）反转录试剂盒以及TB Green Premix Ex TaqTM II（TIi RNaseH Pius）荧光定量PCR试剂盒购自宝日医生物技术（北京）有限公司。
1.2.3 总DNA提取以及目标基因克隆
用植物基因组DNA提取试剂盒提取玉米叶片总DNA，具体提取方法参照试剂盒说明书（高效植物基因组DNA提取试剂盒）。利用表1中设计的引物扩增目的基因，1%琼脂糖凝胶电泳检测所提取DNA的完整性。以2 µL基因组DNA为模板，采用20 µL的PCR反应体系进行PCR扩增，体系包括2× KOD one mix 10 µL，上游引物（10 µmol/L）1 µL、下游引物（10 µmol/L）1 µL，2 µL DNA模板和6 µL ddH2O。PCR反应扩增程序为94 ℃预变性2 min；98 ℃变性10 s，68 ℃退火5 s，68 ℃延伸12 s，循环34次；68 ℃修复延伸10 min。PCR扩增产物经1%琼脂糖凝胶电泳检测，并送北京擎科生物科技有限公司进行测序。
1.2.4 生物信息学分析
利用在线软件对ZmMAPKKK21基因编码蛋白进行理化性质、跨膜区域和亲疏水性分析。进行二级结构和三级结构预测，并对磷酸化位点、亚细胞定位以及互作蛋白进行预测（表2）。设计引物扩增ZmMAPKKK21基因的启动子序列，利用在线网站根据测序结果分析启动子区域的顺式作用元件和CpG岛，并利用TBtools软件对顺式作用元件在基因上的位置进行可视化处理。利用MEGA 7.0构建进化树以分析不同物种中ZmMAPKKK21基因间的同源性。
表2 生物信息学分析网址
Table 2
| 功能Function | 网址Website |
|---|---|
| 启动子分析Promoter analysis | |
| 保守结构域预测Prediction of conserved domain | |
| 磷酸化位点预测Phosphorylation site prediction | |
| 亲疏水性预测Hydrophilicity prediction | |
| 信号肽预测Signal peptide prediction | |
| 跨膜结构预测Transmembrane structure prediction | |
| 理化性质预测Physical and chemical properties prediction | |
| 二级结构预测Secondary structure prediction | |
| 三级结构预测Three-level structure prediction | |
| 亚细胞定位Subcellular localization | |
| 蛋白互作预测Protein interaction prediction |
1.2.5 总RNA提取与cDNA第一链合成
根据植物总RNA提取试剂盒，依据操作说明分别对玉米自交系J24组织材料进行总RNA提取。使用1%琼脂糖凝胶电泳检验提取结果，选择完整性较好、条带清晰且无降解的总RNA进行反转录。
在0.2 mL的PCR管中加入5× gDNA Eraser Buffer 2 μL，gDNA Eraser 1 μL，RNA定量1 µg，补足RNase Free dH2O至10 μL，42 ℃反应2 min。之后进行反转录反应，在去除基因组DNA反应液中加入PrimeScript RT Enzyme Mix I和RT Primer Mix各1 μL，5× PrimeScript Buffer 2（for Real Time）和RNase Free dH2O各4 μL，37 ℃反应15 min，85 ℃反应5 s，反应结束后于-20 ℃保存。
1.2.6 ZmMAPKKK21基因荧光定量PCR（qRT- PCR）分析
用Primer Premier 5.0软件设计目的基因及内参基因引物序列（表1）。qRT-PCR扩增程序如下，95 ℃预变性30 s，随后进行40个循环：95 ℃变性5 s，60 ℃退火34 s。以玉米甘油醛-3磷酸脱氢酶1（glyceraldehyde-3-phosphate deHaseN1，GPN1）为内参基因，利用2-ΔΔCt法计算出基因的相对表达量，使用软件IBM SPSS v20.0进行数据统计分析并用GraphPad Prism 8绘制图形。
2 结果与分析
2.1 玉米自交系J24 DNA提取以及ZmMAPKKK21基因克隆
提取玉米叶片总DNA凝胶电泳结果显示，提取的DNA条带清晰完整（图1a），Nanodrop 2000微量紫外分光光度计检测到DNA浓度为182.5 ng/L，OD260/280值为1.68。结果表明所提取基因组DNA浓度和纯度满足后续试验要求。
图1
图1
PCR扩增电泳图
(a) 基因组DNA电泳图，(b) ZmMAPKKK21基因全长扩增电泳图，(c) ZmMAPKKK21基因启动子扩增电泳图，M：1 kb DNA分子量标准。
Fig.1
Electrophoresis of PCR amplification
(a) Electrophoresis of genomic DNA, (b) Electrophoresis for amplification of full-length ZmMAPKKK21, (c) Electrophoresis for amplification of ZmMAPKKK21 promoter, M: 1 kb DNA marker.
PCR扩增产物电泳结果如图1b所示，在2 kb处有一条明显的扩增条带。PCR产物测序后将序列进行拼接组装，得到最终序列。测序结果表明该基因含有1个外显子，无内含子，开放阅读框长度为1419 bp，共编码472个氨基酸。
利用启动子扩增引物扩增基因ZmMAPKKK21上游2 kb的启动子区域，如图1c所示，在2~3 kb间有目的产物条带，将该产物送去测序，并拼接组装得到ZmMAPKKK21基因的启动子序列用于后续分析。
2.2 ZmMAPKKK21基因生物信息学分析
2.2.1 ZmMAPKKK21基因启动子分析
通过在线网站Plantcare（
表3 ZmMAPKKK21基因启动子顺式作用元件分析
Table 3
| 顺式作用元件Cis-acting element | 序列Sequence | 功能Function | 数量Number |
|---|---|---|---|
| ABRE，ABRE3a | ACGTG, CACGTGTACGTG | 脱落酸响应元件 | 5 |
| CGTCA基序，TGACG基序CGTCA-motif, TGACG-motif | CGTCA, TGACG | 茉莉酸响应元件 | 5 |
| G盒G-box | TACGTG | 光响应元件 | 4 |
| GARE基序GARE-motif | TCTGTTG | 赤霉素响应元件 | 1 |
| 富含TC重复序列TC-rich repeats | GTTTTCTTAC | 参与防御和应激反应元件 | 1 |
| TGA元件TGA-element | AACGAC | 生长素响应元件 | 1 |
| MRE | AACCTAA | 参与光响应的MYB结合位点 | 1 |
| AAGAA基序AAGAA-motif | gGTAAAGAAA | 发育相关基序 | 1 |
| CCAAT盒CCAAT-box | CAACGG | MYBHv1结合位点 | 1 |
| CCGTCC盒CCGTCC-box | CCGTCC | 分生组织特异性激活响应元件 | 1 |
| STRE | AGGGG | 热诱导响应元件 | 4 |
| MYB | CAACCA | 干旱响应元件 | 1 |
| MYC | CATGTG, CATTTG | 干旱响应元件 | 2 |
图2
图2
ZmMAPKKK21启动子顺式作用元件分析（a）和CpG岛分析（b）
(b) 蓝色区域为CpG岛。
Fig.2
Analysis of ZmMAPKKK21 gene promoter cis-acting elements (a) and CpG island (b)
(b) The blue region represents CpG island.
2.2.2 ZmMAPKKK21蛋白理化性质分析
利用ProtParam在线软件对ZmMAPKKK21蛋白理化性质进行分析。结果（表4）表明，ZmMAPKKK21基因编码蛋白质的氨基酸数为472，分子量49 516.58 kD，分子式C2149H3399N663O657S15，理论等电点为6.41，为酸性蛋白。带正电荷氨基酸残基总数（Lys+Arg）为53，带负电荷氨基酸残基总数（Asp+Glu）为57。脂肪系数为76.12，总平均疏水系数为-0.23。半衰期为30 h，不稳定系数42.45，根据不稳定参数值在40以下才是稳定蛋白的标准，可推测该基因编码不稳定蛋白。
表4 ZmMAPKKK21蛋白一级结构预测结果
Table 4
| 一级结构特征 Characteristic of primary structure | 预测结果 Prediction result |
|---|---|
| 氨基酸数量Number of amino acids | 472 |
| 等电点Isoelectric point (pI) | 6.41 |
| 分子量Molecular weight (kD) | 49 516.58 |
| 分子式Molecular formula | C2149H3399N663O657S15 |
| 正电荷残基Arg+Lys | 53 |
| 负电荷残基Asp+Glu | 57 |
| 平均疏水性Average hydrophobicity | -0.23 |
| 脂肪系数Aliphatic index (AI) | 76.12 |
| 不稳定系数(Ⅱ) Instability coefficient (Ⅱ) | 42.45 |
| 半衰期Estimated half-life (h) | 30.00 |
运用Prot Scale工具分析ZmMAPKKK21基因编码蛋白产物的亲水性和疏水性，亲水性分析中最大峰值位于多肽链第417位的赖氨酸，对应峰值为-3.289，而疏水性分析中最大峰值位于第391位的缬氨酸，对应峰值为2.411（图3a）。由于亲水性氨基酸多于疏水性氨基酸，推测ZmMAPKKK21基因编码的蛋白质为亲水性蛋白。
图3
图3
ZmMAPKKK21蛋白理化性质分析
(a) 亲疏水性分析，(b) 信号肽预测，(c) 跨膜结构预测，(d) 磷酸化位点预测。
Fig.3
Analysis of physical and chemical properties of ZmMAPKKK21 protein
(a) Analysis of hydrophilicity and hydrophobicity, (b) Signal peptide prediction, (c) Prediction of transmembrane structure, (d) Prediction of phosphorylation sites.
利用Signal P在线软件和TMHMM在线软件对ZmMAPKKK21蛋白进行信号肽和跨膜结构预测，由图3b~c所示发现无信号肽和跨膜结构，说明ZmMAPKKK21基因编码的蛋白不属于分泌型蛋白，并且不在细胞中发生迁移。
运用NetPhos-3.1在线网站分析ZmMAPKKK21蛋白磷酸化位点，由图3d可知该蛋白可知共含有33个磷酸化位点，其中18个位于丝氨酸（Ser）残基上，14个位于苏氨酸（Thr）残基上，1个位于酪氨酸（Tyr）残基上。该蛋白的磷酸化位点主要集中在丝氨酸和苏氨酸残基上，这些残基使蛋白结构发生变化，进一步引起蛋白质活性变化，这些磷酸化氨基酸对蛋白质功能发挥有重要意义。
2.2.3 ZmMAPKKK21基因进化树构建和保守结构域分析
为了分析玉米ZmMAPKKK21蛋白的亲缘进化关系，采用MEGA7.0对该基因编码的氨基酸序列同其他18种植物蛋白氨基酸序列进行系统进化树的构建并进行保守基序分析。结果如图4所示，这些蛋白序列均有MEKK家族拥有的保守性结构域G（T/S）Px（W/F）MAPEV，以及磷酸化激活位点S/TxxxxxS/T基序。ZmMAPKKK21蛋白与谷子（Setaria italica）（SETIT_004745mg）和高粱（Sorghum bicolor）（SORBI_3003G268900）聚为一簇，具有较近的同缘关系并且均含有motif-9，由图4可以看到另一簇基序与该簇基序相比均少motif-9，猜测可能是造成同源性差异的原因。依据进化树分析结果，选取与ZmMAPKKK21蛋白同源性高的5个蛋白（水稻OSNPB_010699600、高粱SORBI_3003G268900、谷子SETIT_004745mg、拟南芥AT2G32510和AT1G05100），使用NCBI的Conserved Domain Database数据库对上述6种蛋白的氨基酸序列进行保守结构域分析，并通过TBtools处理得到可视化图形（图5）。由图5可知，这6种同源性较高的蛋白结构域中均有STKc_ MAPKKK结构域。
图4
图4
ZmMAPKKK21蛋白与其他植物MAPKKK基因编码的氨基酸序列的系统进化树
Fig.4
Phylogenetic tree of amino acid sequences encoded by ZmMAPKKK21 and MAPKKK from other plants
图5
图5
ZmMAPKKK21蛋白及其同源蛋白的保守结构域分析
Fig.5
Conserved domains analysis of ZmMAPKKK21 protein and homologous proteins
2.2.4 MAPKKK家族蛋白结构分析
利用SOPMA在线网址对上述MAPKKK家族蛋白进行二级结构预测，结果表明这些蛋白二级结构由α螺旋、不规则卷曲结构和β转角组成。其中，ZmMAPKKK21蛋白与水稻、高粱和谷子的同源蛋白中α螺旋各有193、179、167和197个，不规则卷曲结构各有186、193、204和195个，β转角各有28、30、26和21个。而拟南芥（AT2G32510和AT1G05100）蛋白中α螺旋各有143和138个，不规则卷曲各有154和130个，β转角各有18和23个。
图6
图6
ZmMAPKKK21蛋白及其同源蛋白三级结构预测
Fig.6
The prediction of tertiary structural model of ZmMAPKKK21 protein and homologous proteins
2.2.5 亚细胞定位分析和蛋白互作分析
使用在线网址Cell-PLoc-2（
图7
图7
亚细胞定位预测（a）和蛋白互作网络预测（b）
Fig.7
Subcellular localization prediction (a) and protein interaction prediction (b)
蛋白质互作网络（protein-protein interaction networks，PPI）是由蛋白通过彼此之间的相互作用构成，参与生物信号传递、基因表达调节、能量和物质代谢及细胞周期调控等生命过程的各个环节，因而了解和分析蛋白质的互作网络具有很重要的意义。
通过STRING （
2.3 实时荧光定量PCR（qRT-PCR）表达分析
2.3.1 ZmMAPKKK21基因表达模式分析
应用qRT-PCR技术分析ZmMAPKKK21基因在不同时期中不同组织的表达量（记VE根的表达量为1），结果如图8所示，在玉米自交系J24不同组织（根、茎、叶、雌穗和雄穗）以及不同时期（VE、V3、V7和R1）中均检测到ZmMAPKKK21基因的表达，但在各组织中的表达量不同。其中，在R1的根中表达量最高，其次是茎，在VE、V3和V7根中表达量明显高于其他组织，在雌穗和雄穗中表达量较低。
图8
图8
ZmMAPKKK21基因在各器官的相对表达量
R：根，L：叶，S：茎。
Fig.8
Relative expression of ZmMAPKKK21 gene in various organs
R: root, L: leaf, S: stem.
2.3.2 干旱胁迫对ZmMAPKKK21基因表达的影响
干旱胁迫后ZmMAPKKK21基因在根和叶中的表达结果如图9所示，记正常水分处理的叶片中ZmMAPKKK21基因表达量为1，与CK相比，干旱处理后，ZmMAPKKK21基因在叶片和根中表达量均显著升高。
图9
图9
干旱胁迫下ZmMAPKKK21基因在根、叶组织中的的表达
“**”代表P < 0.01水平上的显著性差异。
Fig.9
Expression of ZmMAPKKK21 gene in roots and leaves under drought stress
“**”represents significant differences at the level of P < 0.01.
3 讨论
ZmMAPKKK21基因在不同组织中的表达量检测结果显示，该基因在R1期的根、茎以及V3期根中较其他组织中表达量较高，与小麦[24]、番茄[25]和拟南芥[26]中的组织特异性表达结果一致，说明ZmMAPKKK21基因主要在根组织中表达。对ZmMAPKKK21基因在干旱胁迫下进行表达分析，结果表明该基因可上调表达以响应干旱胁迫。此外，我们进行差异表达分析的品种本身在抗旱性上存在差异，研究[27-28]发现在这些农艺性状各异的材料中，在水分胁迫下该基因表达更高的品种，抗旱性更为突出。植物根系是植物从环境中吸收水分和养分的主要器官，在轻度干旱胁迫下，玉米植株的根系变长以求获得更多的水分吸收，而在严重干旱下根长变短以致活力丧失[29]。在试验证明ZmMAPKKK21基因在根系中高表达并积极响应干旱胁迫，但具体功能和作用模式还需要进一步验证。本文为进一步研究玉米抗旱相关基因ZmMAPKKK21的分子机理，并利用生物技术手段创制抗旱玉米新材料奠定基础。
4 结论
本研究基于前期数据挖掘筛选得到干旱胁迫下差异表达基因ZmMAPKKK21，从抗旱性强玉米自交系J24中克隆出该基因并对其进行了生物信息学分析，发现该基因属于MAPKKK家族MEKK亚系，且在启动子序列区含有多种与逆境胁迫相关的顺式作用元件，亲缘关系与高粱和谷子较近，且该基因在根中特异性高表达，并积极响应干旱胁迫，进一步验证了该基因在抗旱中潜在的重要作用。
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