Screening and Bioinformatics Analysis of Proteins Interacting with StMAPKK1 in Potato by Yeast Two-Hybrid System

Liao Yuqiu¹　Wang Fangfang¹　Zhu Xi²　Zhang Ning^1*　Si Huaijun^1,3

1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070; 2 College of Agronomy, Gansu Agricultural University, Lanzhou 730070; 3 Gansu Provincial Key Laboratory of Aridland Crop Science, Lanzhou 730070

*Corresponding author, ningzh@gsau.edu.cn

Abstract　Mitogen-activated protein kinase (MAPK) cascade reaction is one of important and complex signal networks involved in plant growth and development, hormone and stress response. As one of its main members, mitogen-activated protein kinase kinase (MAPKK) is located in the middle of the cascade reaction and plays a key role in signal collection and divergence. There were shown that potato (Solanum tuberosum L.) StMAPKK1 (PGSC0003DMT400000744) gene responded to drought stress. Therefore, StMAPKK1 gene was firstly selected to screen its interacting protein. In this study, the bait vector pGBKT7-StMAPKK1 was constructed by homologous recombination and used to screen potato cDNA library by yeast (Saccharomyces cerevisiae) two-hybrid system. Five StMAPKK1-interacting proteins, hydrolase (hydrolyzing O-glycosyl compound), RING-H2 subgroup RHE protein, cyanate hydratase, ARF GTPase activator, and a C2 domain-containing protein, were obtained through this screening and were identificated by bioinformatics analysis, and the interaction was verified by small-scale hybridization verification. The results provided theoretical basis for further study on the signal pathway and biological function of potato StMAPKK1.

Keywords　Potato, StMAPKK1, Yeast two-hybrid, Interacting protein

促丝裂原活化蛋白激酶(Mitogen-activated protein kinase, MAPK)级联反应是一种广泛存在于真核生物中、在进化过程中高度保守的生物信号转导模块(MAPK Group, 2002)。植物中，MAPK信号途径几乎参与各种生长发育过程以及多种生物与非生物胁迫的应激反应，使细胞内形成庞大复杂、相互沟通交叉的生物信号转导网络，为植物生长发育及应对生长发育过程中的胁迫提供了足够的信息快速集散的基本条件。

MAPK级联反应通过其主要的三种激酶，促分裂原激活蛋白激酶激酶激酶(MAPKKK)、促分裂原激活蛋白激酶激酶(MAPKK)和促分裂原活化蛋白激酶(MAPK)，在特定的基序位点上磷酸化作用于某些成员的上游和下游从形成级联信号通路(Iftikhar et al., 2017)。其中，MAPKKs成员数量最少，可分为A、B、C、D四个亚族。MAPKKs是一种双重激酶，既可被上游MAPKKK磷酸化激活，也可通过磷酸化反应激活下游MAPK。植物中MAPKK响应干旱胁迫，AtMAPKKK18-AtMKK3-AtMPK1/2参与ABA诱导依赖的抗旱性调控(Li et al., 2017)，拟南芥中过表达ZmMKK1或ZmMKK4均增强了抗旱性 (Cai et al., 2014; Kong et al., 2011)。GhMKK3可调节气孔大小及根毛生长的方式增强棉花的抗旱性(Wang et al., 2016)。另外，有研究发现，OsMKK1参与水稻盐胁迫信号传导(Wang et al., 2014)，OsMKK6在低温和盐胁迫中起作用(Xie et al., 2012)。

已有研究对马铃薯MAPKKs家族基因进行了鉴定(刘雪, 2017)，获得5个StMAPKK基因，并根据番茄同源性分析命名为StMAPKK1 (PGSC0003DMT400000744)、StMAPKK2 (PGSC0003DMT400023739)、StMAPKK3 (PGSC0003DMT400014637)、StMAPKK4 (PGSC0003DMT400083995)和StMAPKK5 (PGSC0003DMT400039329)。不同处理下(包括4℃、45℃、20% PEG、200 mmmol/L NaCl、10 mmol/L H₂O₂、100 µmol/L MeJA、100 µmol/L SA、100 µmol/L ABA)马铃薯StMAPKK基因定量结果发现StMAPKK1基因在干旱胁迫下表达量显著升高。故选择StMAPKK1基因为实验研究对象，通过酵母双杂交技术筛选获得与StMAPKK1互作的蛋白质，并采用生物信息学方法进行鉴定分析，从而探寻马铃薯中StMAPKK1基因在干旱胁迫下作用的信号通路。

1结果与分析

1.1 酵母双杂交诱饵载体pGBKT7-StMAPKK1的构建

马铃薯StMAPKK1基因(PGSC0003DMT400000744)转录序列全长为1 666 bp，根据同源重组引物设计原则设计引物扩增的插入片段会在插入片段5’和3’末端分别带有和线性化载体两端对应的同源序列21 bp，故产物大小约为1 708 bp，与电泳结果相符(图1A)。pGBKT7质粒大小为7 303 bp，经限制性内切酶Pst Ⅰ和NdeⅠ双酶切获得线性质粒，大小约为7 270 bp，电泳检测符合预期大小(图1 B)。

重组产物转化至感受态大肠杆菌，涂布于LB固体平板(50 mg/L Kan)，挑取6个单菌落进行菌液PCR鉴定，均在1 708 bp左右有明显条带(图1 C)。StMAPKK1基因中在572 bp和582 bp处有NdeⅠ酶切位点，NdeⅠ和PstⅠ双酶切重组质粒pGBKT7-StMAPKK1应会获得三个片段大小分别约为7 270 bp、1 126 bp和572 bp，电泳结果(图1D)与预期结果相符。同时经测序结果比对，表明StMAPKK1基因已成功插入pGBKT7载体(图2)。

图1  酵母双杂交诱饵载体pGBKT7-STMAPKK1的构建 注: A: 马铃薯StMAPKK1基因扩增 M: Trans2K DNA分子标记, 1~3: 马铃薯StMAPKK1基因PCR产物; B: pGBKT7质粒双酶切线性化 M: DL15000 DNA分子标记, 1: pGBKT7载体质粒, 2: pGBKT7经Pst I和Nde Ⅰ限制性内切酶双酶切后所得线性化载体; C: 诱饵载体pGBKT7重组质粒的菌液PCR鉴定 M: Trans2K DNA分子标记; 1~6: 单菌落菌液PCR产物; D: 诱饵载体pGBKT7重组质粒的双酶切鉴定M1: DL15000 DNA分子标记, 1: 载体酶切片段及StMAPKK1基因克隆片段混合物电泳条带,  2, 3: Nde I和Pst I双酶切产物, M2: Trans2K DNA分子标记 Figure 1 Construction of yeast two-hybrid bait vector pGBKT7-StMAPKK1 Note: A: Amplification of StMAPKK1 gene M: Trans2K DNA molecular marker, 1-3: PCR product of StMAPKK1 gene; B: The double enzyme digestion linearization of pGBKT7 plasmid  M: DL15000 DNA molecular marker, 1: pGBKT7 vector plasmid, 2: Pst Ⅰ and Nde Ⅰ restriction endonuclease digestion to obtain the linear vector of pGBKT7; C: Identification of recombinant plasmid pBGKT7-StMAPKK1 by PCR M: Trans2K DNA molecular marker, 1-6: PCR product of single colony solution; D: Identification of the recombinant plasmid pGBKT7-StMAPKK1 digestion by Nde Ⅰ and Pst Ⅰ M1: DL15000 DNA molecular markers, 1 and 2: Products of pGBKT7-StMAPKK1 digestion by Nde Ⅰ and Pst Ⅰ, M2: Trans2K DNA molecular markers

图2  pGBKT7-StMAPKK1测序结果比对 Figure 2 Sequencing result of pGBKT7-StMAPKK1

1.2 酵母双杂交诱饵载体的毒性和自激活活性检测

将含有pGBKT7空载体与pGBKT7-StMAPKK1诱饵载体的Y187酵母涂布于单缺培养基SD/-Trp/X-α-gal上培养，观察可得到两种菌株生长数量大致相同(图3)，说明该诱饵载体对酵母细胞没有毒性。同时两种菌株在二缺平板SD/-Trp/-His/X-α-gal和SD/-Trp/-Ade/X-α-gal上均未生长，说明该菌株没有自激活活性，无法单独激活下游报告基因。

图3 酵母双杂交诱饵载体pGBKT7-StMAPKK1的毒性检测与自激活活性检测 Figure 3 Detection of toxicity and self-activating activity of yeast two-hybrid bait vector pGBKT7-StMAPKK1

1.3 阳性克隆小规模杂交验证

将诱饵蛋白与文库的杂交液涂布于50个QDO培养基上培养，共挑选直径较大的单菌落406个进行初步X-α-Gal染色鉴定筛选，其中210个菌落在48 h内生长并显蓝色，视为阳性克隆，阳性率为51.72%。经电泳检测及测序，共鉴定5个不同的StMAPKK1互作蛋白，分别命名为C1~C5，分别占比为87%、2.9%、4.8%、2.9%和2.9%。

随机挑选5个StMAPKK1互作蛋白基因质粒，重新转入AH109酵母中，与含有诱饵载体的Y187分别进行杂交，涂布于QDO/X-α-Gal培养基进行培养。培养两天后，5个点种位置均长出蓝色深浅不一的菌落(图4)。说明5个阳性克隆基因均能通过小规模杂交的回转验证，其互作关系真实可信。

图4 阳性克隆的小规模杂交验证 Figure 4 Small-scale hybridization verification of positive clones 注: +: 阳性对照AH109 (pGADT7-RecT)×Y187(pGBKT7-53); -: 阴性对照AH109 (pGADT7-RecT)×Y187(pGBKT7-Lam); C1~C5: 阳性克隆编号 Note: +: Positive control AH109 (pGADT7-RecT)×Y187 (pGBKT7-53); -: Negative control AH109 (pGADT7-RecT) ×Y187 (pGBKT7-Lam); C1~C5: Numbers of positive clones

1.4 阳性克隆基因鉴定

通过对阳性克隆测序比对分析，得到5个马铃薯StMAPKK1互作蛋白C1~C5：水解酶(水解 O-糖基化合物)、RING-H2亚家族RHE蛋白、氰酸酯酶、ARF (ADP-ribosylation factor) GTPase活化因子以及含有C2结构域的蛋白。采用生物信息学方法获取其基因序列基本信息和注释(表1)。

表 1 马铃薯StMAPKK1互作蛋白信息 Table 1 Details information of proteins interacting with StMAPKK1

2 讨论

经对阳性克隆测序结果比对，共获得5种马铃薯StMAPKK1互作蛋白：水解酶(水解 O-糖基化合物)、RING-H2亚家族RHE蛋白、氰酸酯酶、ARF GTPase活化因子以及含有C2结构域的蛋白。

其中，互作蛋白C1为水解酶(水解 O-糖基化合物)，另一注解为半乳糖醇-蔗糖半乳糖基转移酶，属于糖苷水解酶家族36 (Glycoside hydrolase family 36)中11个亚家族(GH36A到GH36K)的GH36C亚族，该家族所含成员又可称为棉子糖合成酶(Raffinose synthase, RS)或种子自吸蛋白(Seed imbibition protein I, Sip I)。棉子糖合成酶(EC 2.4.1.82)是将蔗糖导入棉子糖低聚糖途径的关键酶，在植物种子获得抗旱能力与种子寿命延长方面起着重要作用(Peterbauer et al., 2002)。有研究表明，棉子糖合成酶在玉米种子中是棉子糖系列寡糖(Raffinose family oligosaccharides, RFO)分解的唯一原因(Andreas et al., 2008)。而黄瓜叶片和果实中低温和外源激素脱落酸(ABA)可诱导CsRS表达，RS活性和棉子糖含量逐渐增加(Sui et al., 2012)。同样在甜菜(Beta vulgaris L.)中被鉴定出来的两种棉子糖合成酶被证明参与冷胁迫和盐胁迫的响应(Kito et al., 2018)。互作蛋白C2为RING-H2亚家族RHE蛋白，为具有E3泛素连接酶活性的含有RING结构的蛋白，属于ATL (Arabidopsis toxicos para levadura)基因家族(MartínezGarcía et al., 1996)。在杨树杂交品种中证明PtaRHE1参与其次生韧皮部纤维发育(Baldacci-Cresp et al., 2015)。有研究表明ATL基因家族可能参与植物对病原体攻击的早期防御反应(Salinas-Mondragón et al., 1999)。实验还筛选得到的互作蛋白C3氰酸酯酶，又称氰酸酯裂解酶(EC:4.2.1.104)，负责氰酸酯的水解，存在于细菌及植物中，使生物体克服环境氰酸盐的毒性(Sung and Fuchs, 1988)。互作蛋白C4 ARF (ADP-ribosylation factor) GTPase活化因子常参与囊泡运输运输，尤其是高尔基潴泡间的外被体包裹的囊泡运输(Rothman and Wieland, 1996)。同时，还筛选得到互作蛋白C5，一个含有C2结构域蛋白，该结构域参与调节膜转运(Thomas and Rizo, 1996)。C2结构域显示出与多种不同的配体和底物结合的显著特性，包括Ca²⁺、磷脂、磷脂肌醇以及胞内蛋白(Cho and Stahelin, 2006)。水稻中一个含有C2结构域的蛋白质OsPBP1被证明可能通过Ca²⁺和磷脂信号通路从而调控花粉育性(Yang et al., 2008)。

本实验中，通过酵母双杂交技术筛选获得的马铃薯StMAPKK1互作蛋白与预期目标结果有一定差距，未筛选到MAPKs级联反应中其它类型激酶，如MAPKKK和MAPK。有研究表明，除典型的MAPK途径MAPKKK-MAPKK-MAPK外，还存在有其它级联途径，可能和其它信号通路途径有交叉传递。

3材料与方法

3.1 实验材料

马铃薯四倍体栽培品种'紫花白'的酵母双杂交cDNA文库（梁丽娜，2017）、酵母(Saccharomy-cescerevisiae)菌株Y187、AH109，大肠杆菌(Esche⁃richia coli)菌株 DH5α及诱饵载体pGBKT7均由甘肃农业大学生命科学技术学院实验室保存。

3.2 酵母双杂交诱饵载体pGBKT7-StMAPKK1的构建

使用Pst Ⅰ和Nde Ⅰ限制性内切酶将载体pGBKT7线性化并进行凝胶回收。PCR反应扩增马铃薯StMAPKK1基因目的片段反应体系为：2×Power Taq PCR Master MIX 10 μL，马铃薯‘大西洋’叶片cDNA模板1 μL，10 μmol/L StMAPKK1-F (5’-tcagaggaggacctgcatat gGTTGCCTGGAAGCAATTTCACA-3’)、StMAPKK1-R(5’-ctagttatgcggccgctgcagTTCATTGAAAGGTCTCTAAATTGATCTC-3’)（下划线部分为载体同源序列）各1 μL，ddH₂O 7 μL。反应程序为：94℃ 5 min；94℃ 30 sec，62℃ 30 s，72℃ 67 s，共35个循环；72℃ 10 min；4 ℃保存。电泳检测并进行凝胶回收，片段大小为1 708 bp。将回收所得线性化载体与StMAPKK1基因PCR产物根据Vazyme公司的ClonExpress® II One Step Cloning Kit说明书计算构建同源重组反应体系，反应程序为37℃ 30 min后4 ℃保存。将重组产物通过热激法转化大肠杆菌DH5α，涂布于含有50 mg/L Kan的LB培养基37 ℃培养过夜。挑取单菌落进行菌液PCR检测及质粒双酶切检测并测序。

3.3 酵母双杂交诱饵载体的毒性和自激活活性检测

将pGBKT7-StMAPKK1诱饵载体质粒和pGBKT7空载体质粒通过聚乙二醇/醋酸锂法分别转化酵母菌Y187。转化产物分别涂SD/-Trp平板，30℃培养1~2天。挑取2~3 mm大小、从30 ℃生长2天后长出的pGBKT7-StMAPKK1诱饵载体和pGBKT7空载体单菌落各一个，于装有500 μL SD/-Trp的液体培养基中混匀，各取80 μL涂布在SD/-Trp/X-α-Gal，SD/-His/-Trp/X-α-Gal和SD/-Ade/-Trp/X-α-Gal缺陷培养基上，剩余的菌液4℃保存。将涂好的平板放在30℃培养3~4天。根据菌落生长情况判断诱饵载体是否具有毒性和自激活活性。

3.4 诱饵蛋白与酵母文库的杂交

挑取诱饵载体单菌落至50 mL SDO/-Trp/Kan (20 µg/mL)培养基中，30℃ 230 r/min过夜培养(16~24 h)，离心后用5 mL SDO/-Trp液体培养基重悬沉淀。取诱饵载体Y187菌液5 mL和文库AH109菌液1 mL于2 L三角瓶中。加入50 mL 2×YPDA/Kan (50 μg/mL)，30℃摇床低速(30~50 r/min)孵育20~24 h。把杂交液5 000 rpm离心10 min，弃上清，沉淀用10 mL 0.5×YPDA/Kan重悬。将重悬液涂布于50个QDO培养基 (SD/-Ade/-His/-Leu/-Trp)，每个板80 μL，30℃培养5~7天，进行高严谨互作筛选。挑选较大(直径>2 mm)的单菌落点种到涂布有100 μL X-α-Gal的QDO培养基进行初步染色鉴定筛选。在选择培养基QDO/X-α-Gal上可以水解底物X-α-Gal并呈现明显的蓝色，视为阳性克隆。将阳性克隆进行PCR鉴定，引物为根据pGADT7载体多克隆位点两端序列设计的pGADT7-F (5’-TAATACGACTCACTATAG GGCGAGCG-3’)、pGADT7-R (5’-GTGAACTTGCGGGGTTTTTCAGTAT-3’)。所得PCR产物经电泳检测并送苏州金唯智生物科技有限公司测序。

3.5 阳性克隆小规模杂交验证

参照徐文琳等(2003)的方法提取阳性单克隆酵母质粒。挑选含正确ORF的阳性质粒转化酵母AH109感受态细胞，再与含有诱饵载体的酵母Y187重新杂交。AH109 (pGADT7-RecT)×Y187 (pGBKT7-53)和AH109 (pGADT7-RecT)×Y187 (pGBKT7-Lam)分别用作阳性和阴性对照。上述杂交液各取20 μL滴在新鲜的QDO/X-α-Gal平板上使其形成圆形菌斑，于30℃黑暗培养，观察生长及变色情况，验证互作的真实性。

3.6 阳性克隆基因的生物信息学分析鉴定

阳性克隆测序结果于NCBI (https://www.ncbi.nlm.nih.gov/)、马铃薯PGSC (http://solanaceae.plantbiology.msu.edu/ pgsc_download.shtml)以及Phytozome v12.1 (https://phytozome.jgi. doe.gov/pz/portal.html)中的马铃薯(Solanum tuberosum L.)数据库进行Blast比对分析，得到StMAPKK1互作蛋白的蛋白名称，染色体位置及其全长基因和蛋白质序列。

作者贡献

廖钰秋是本研究的实验设计者和实验研究的执行人，进行数据整理及论文初稿的写作；王芳芳和朱熙参与部分实验；张宁和司怀军是项目的负责人，指导实验设计、数据统计、论文写作与修改。全体作者都同意最终的文本。

致谢

本研究由国家自然科学基金(No.31960444)和甘肃农业大学干旱生境作物学重点实验室开放基金课题(No.GSCS-2019-Z03)共同资助。

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