林烟草蛋白激酶NsylCIPK3基因的克隆与功能分析

CIPK蛋白激酶家族(CBL-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶家族,与类钙调磷酸酶B亚基CBL(calcineurin B-like protein)蛋白共同形成CBL-CIPK网络,在植物的生长发育和逆境胁迫响应过程中发挥重要作用。烟草中该家族的研究还比较少,本研究从林烟草(Nicotiana sylvestris)中获得一个CIPK家族基因,该基因与拟南芥和杨树中的CIPK3同源性分别为68.4%和87.5%,将其命名为Nsyl CIPK3。氨基酸序列分析表明,Nsyl CIPK3具有CIPK蛋白家族的典型结构特征,在N端和C端分别具有典型的激活环结构域和NAF结构域。进化树分析显示,Nsyl CIPK3属于CIPK蛋白亚家族Ⅱ。表达模式研究表明,该基因在林烟草的叶和腋芽中的表达量相对较高,在主根中的表达量次之,在侧根、茎、花瓣和萼片中的表达量相对较低,并且在烟草成熟期的叶中表达量明显升高。在高盐、紫外光和低钾胁迫下该基因的表达发生不同程度的上调。酵母双杂交结果显示,Nsyl CIPK3可与Nsyl CBL9互作。推测Nsyl CIPK3可能通过与Nsyl CBL9互作形成信号通路,激活下游靶蛋白,参与烟草响应非生物逆境胁迫的信号转导过程。     

条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

小麦TaCIPK8基因的表达分析及其与TaCBLs的互作

CIPK是植物钙感受器钙调磷酸酶B类似蛋白特定靶向的一类丝氨酸/苏氨酸蛋白激酶。根据拟南芥At CIPK8基因序列,利用同源克隆的方法从抗逆性较强的小麦品种石4185中克隆了一个编码序列全长为1350 bp的蛋白激酶基因Ta CIPK8(Gen Bank号:KJ561804.1)。序列分析表明该基因编码的蛋白含有449个氨基酸,分子量为52.24 k D,理论等电点为7.16,具有CIPKs家族蛋白所特有的N端激酶域和C端NAF/FISL结构域;与拟南芥At CIPK8蛋白序列相似度达83.5%。为进一步研究其功能,采用Real-time PCR方法检测该基因在胁迫条件下的响应情况,发现Ta CIPK8基因受高盐、外源ABA和低温(4℃)胁迫诱导表达。利用植物启动子数据库Plant CARE和PLACE对Ta CIPK8基因启动子序列进行分析,结果显示Ta CIPK8基因启动子存在大量应答脱水胁迫、干旱、低温和ABA的顺式作用元件,也存在一些响应激素GA和茉莉酸甲酯的元件。利用酵母双杂交方法研究Ta CIPK8和Ta CBL家族蛋白的互作,结果显示,共转化含有Ta CIPK8和Ta CBL3基因载体的酵母菌能够在SD/-Trp/-Leu、SD/-Trp/-Leu/X-α-Gal/Ab A和SD/-Trp/-Leu/-Ade/-His/X-α-Gal/Ab A三种培养基上生长,且在后两种培养基上长出蓝色菌落。结果说明Ta CIPK8与Ta CBL3发生了互作,进而引起了报告基因MEL1、AUR1-C、HIS3和ADE2的表达。该研究结果对于研究Ta CIPK8基因的功能以及与CBL蛋白的互作调控网络具有一定的参考作用。     

CBL-CIPKs信号系统的研究进展

Ca2+是植物体中普遍存在的第二信使,参与了植物众多的生长发育和逆境胁迫调控过程。钙调磷酸酶B类蛋白(calcineurin B-like protein,CBL)能够与一类蛋白激酶(CBL-interacting protein kinase,CIPK)互作来解码特异"钙信号"。该文总结了近几年在植物CBL-CIPKs信号系统研究领域的最新进展,包括CBL与CIPK互作特点及生理功能等,并对未来的研究方向作了展望。     

小麦锌指蛋白基因TaLOL2的克隆及特征分析

通过电子克隆与RT-PCR相结合的方法,在条锈菌诱导的小麦叶片中克隆获得1个新的LSD1型锌指蛋白基因TaLOL2,并用qRT-PCR技术分析了其转录表达特征。结果显示:(1)小麦锌指蛋白基因TaLOL2的cDNA全长1 095bp,编码179个氨基酸。(2)TaLOL2含有3个典型的zf-LSD1型(CxxCxRxxLMYxxGASxVxCxxC)保守结构域,与水稻、拟南芥、大麦等植物LSD1型锌指蛋白序列具有高度相似性,其中与水稻OsLOL2相似度达86.0%。(3)进化树分析表明,TaLOL2与水稻、拟南芥和大麦中部分含有3个保守zf-LSD1锌指结构的基因亲缘关系较近,而与其它包含不同数目的zf-LSD1锌指结构的基因亲缘关系较远。(4)qRT-PCR定量分析表明,TaLOL2在条锈菌侵染前期呈上调表达,在亲和及非亲和反应中差异表达。研究表明,TaLOL2参与了条锈菌诱导的小麦抗病防卫反应,很可能作为正调控因子参与了小麦-条锈菌非亲和互作中对条锈菌的抗性信号途径。     

小麦翻译控制肿瘤蛋白TCTP与蔗糖非酵解型蛋白激酶SnRK1的相互作用

翻译控制肿瘤蛋白(Translationally controlled tumor protein, TCTP)广泛存在于真核细胞中,参与调节细胞分裂、植物生长发育,并介导植物抵御病原物侵染。蔗糖非酵解型蛋白激酶(SNF1-related protein kinase, SnRK1)在酵母、动物和植物中非常保守,并参与包括糖代谢和抵抗非生物和生物胁迫在内的一系列生理过程。本实验室前期工作证明TaTCTP响应叶锈菌侵染并参与诱发寄主产生防卫反应。为了深入探讨TaTCTP在叶锈菌侵染小麦诱发的防卫反应中发挥的作用,采用串联亲和纯化(TAP)与质谱(MS)联用技术,鉴定出SnRK1可能为TaTCTP潜在互作蛋白。文中对TCTP和SnRK1的相互作用进行了研究。酵母双杂交结果表明,同时携带TCTP和SnRK1的酵母可以在SD/-Leu/-Trp/-His/-Ade(SD/-LWHA,四缺)培养基上生长,说明TCTP与SnRK1在酵母双杂交系统中可以发生相互作用;通过双分子荧光互补实验,发现TCTP与SnRK1发生相互作用的荧光信号分布在细胞质中;进一步用Co-IP实验证明TCTP和SnRK1可以发生相互作用。本研究为深入研究TaTCTP在小麦与叶锈菌互作过程中的作用机制奠定了重要基础,对进一步完善小麦抵御叶锈菌侵染的分子机理具有重要意义。     

菜用大豆CIPK基因对逆境胁迫及激素的响应特征

CIPK(calcineurin B-like-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶,在植物响应逆境胁迫和激素信号转导中发挥重要作用。本研究利用大豆基因组数据库,在全基因组水平鉴定获得52个CIPK蛋白激酶。蛋白比对分析发现所有Gm CIPK含有高度保守特征性的N端激酶区、连接区和C端调控区。系统进化树分析发现大豆Gm CIPK与拟南芥、水稻CIPK分类一致,分为4个亚家族,且每个亚家族含有3个不同物种的成员,表明Gm CIPK基因的分化早于物种的分化。启动子分析表明,多数Gm CIPK基因的启动子区含有逆境和激素应答元件。组织表达分析发现,Gm CIPK基因呈现多样化的组织表达特性。进一步选取组织表达量相对较高的14个Gm CIPK进行荧光定量PCR分析,结果表明这些菜用大豆CIPK基因在不同程度上均受高温、干旱、高盐胁迫以及ABA、ACC、SA、Me JA激素的诱导表达。采用蛋白同源比对和蛋白互作在线数据库对拟南芥及大豆同源CIPK蛋白激酶与其他蛋白的互作关系进行了预测分析,发现17对同源CIPK与其他蛋白(激酶、磷酸酶、转录因子等)存在互作。本研究为菜用大豆CIPK基因的功能研究与利用奠定了基础。     

斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

【目的】蜕皮激素在昆虫变态发育中起着关键的调控作用。蜕皮激素活化为20-羟基蜕皮酮(20-hydroxyecdysone,20E)后与蜕皮激素受体二聚体〔蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle protein,USP)〕结合,启动20E诱导的级联反应。本研究旨在从互作蛋白角度探究EcR/USP自身调控的分子机理。【方法】以重要农业害虫斜纹夜蛾Spodoptera litura为研究对象,通过构建酵母双杂交筛选系统分别筛选了与EcR和USP相互作用的蛋白。【结果】文库转化效率达到3.0×106cfu/μg,文库滴度达到1.3×108cfu/m L,文库插入片段大小在500~2 000 bp之间。4种诱饵质粒(EcRA/p GBKT7,EcRB1/p GBKT7,USP1/p GBKT7和USP2/p GBKT7)对酵母细胞无毒性,并且无自激活性,说明构建的酵母双杂交文库质量可靠。用以上4种诱饵质粒筛选酵母双杂交文库,共得到110个互作蛋白,其中与EcRB1,USP1和USP2互作的蛋白分别有26,52和32个,未筛选到与EcRA互作的蛋白。随后,从中挑选了Dna J-5(Hsp40 homolog 5,一种热激蛋白分子伴侣),MBF2(mediator of Bm FTZ-F1 type 2,一种转录共激活子),polyubiquitin(多聚泛素类蛋白),esr16(ecdysteroid regulated 16k Da protein,一种蜕皮激素调控蛋白)和NEDD8-like(neural precursor cell expressed,developmentally down-regulated protein 8,一种泛素调节相关蛋白)5种蛋白,利用酵母双杂交和Far-Western印迹法进一步验证了蛋白间的互作关系。【结论】分子伴侣和泛素化修饰等在蜕皮激素受体调控中可能起着重要作用。本研究对深入理解昆虫变态发育的分子机理具有重要意义。     

玉米ZmCIPK31基因原核表达及启动子序列分析

与钙传感器类钙调磷酸酶B蛋白(calcineurin B-like protein,CBL)互作的蛋白CIPK(CBL-interacting protein kinase)在植物特定的生长发育和应答胁迫过程中起重要作用。对前期研究得到的玉米ZmCIPK31基因构建原核表达重组载体,进行原核表达分析,转化重组质粒的大肠杆菌BL21菌株能够诱导出期望的目的蛋白。蛋白可溶性分析表明,它是可溶性的蛋白,通过淀粉树脂柱对其进行纯化,为下一步激酶活性分析提供了有活性的目的蛋白。同时,克隆了ZmCIPK31基因起始密码子ATG上游包括2189bp的启动子区段,顺式作用元件预测分析表明此区段不仅具有TATA-box和CAAT-box等启动子共有序列,还具有光应答、胁迫应答、发育相关、激素相关及其他功能未知的调控结构域。聚乙二醇(PEG)胁迫下,ZmCIPK31基因的诱导表达进一步表明其能够应答胁迫,且在地上部和根中的表达模式不同。     

采用酵母双杂交系统筛选GmDREB5的互作蛋白

以无自激活活性的GmDREB5蛋白73～226位氨基酸区段为诱饵,采用酵母双杂交系统筛选干旱处理5 h大豆cDNA文库.结果发现:一个互作蛋白含有保守的TPR(Tetratricopeptide repeat)结构域,与拟南芥的TPR蛋白仅有14%的相似性,说明其可能是一类新的大豆TPR蛋白,将其定名为GmTPR1;表达特性分析表明,GmTPR1基因受干旱、低温、高盐、ABA的诱导;证明GmTPR1不仅参与植物对非生物胁迫的响应,同时参与对GmDREB5蛋白水平的调控.     

Simultaneous editing of three homoeologues of TaCIPK14 confers broad-spectrum resistance to stripe rust in wheat

Wheat stripe rust caused by the fungus Puccinia striiformis f. sp. tritici (Pst) is one of the most destructive wheat diseases resulting in significant losses to wheat production worldwide. The development of disease-resistant varieties is the most economical and effective measure to control diseases. Altering the susceptibility genes that promote pathogen compatibility via CRISPR/Cas9-mediated gene editing technology has become a new strategy for developing disease-resistant wheat varieties. Calcineurin B-like protein (CBL)-interacting protein kinases (CIPKs) has been demonstrated to be involved in defence responses during plant-pathogen interactions. However, whether wheat CIPK functions as susceptibility factor is still unclear. Here, we isolated a CIPK homoeologue gene TaCIPK14 from wheat. Knockdown of TaCIPK14 significantly increased wheat resistance to Pst, whereas overexpression of TaCIPK14 resulted in enhanced wheat susceptibility to Pst by decreasing different aspects of the defence response, including accumulation of ROS and expression of pathogenesis-relative genes. We generated wheat Tacipk14 mutant plants by simultaneous modification of the three homoeologues of wheat TaCIPK14 via CRISPR/Cas9 technology. The Tacipk14 mutant lines expressed race-nonspecific (RNS) broad-spectrum resistance (BSR) to Pst. Moreover, no significant difference was found in agronomic yield traits between Tacipk14 mutant plants and Fielder control plants under greenhouse and field conditions. These results demonstrate that TaCIPK14 acts as an important susceptibility factor in wheat response to Pst, and knockout of TaCIPK14 represents a powerful strategy for generating new disease-resistant wheat varieties with BSR to Pst.     

TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust

Calcineurin B‐like interacting protein kinase (CIPKs) has been shown to be required for biotic stress tolerance of plants in plant‐pathogen interactions. However, the roles of CIPKs in immune signalling of cereal crops and an in‐depth knowledge of substrates of CIPKs in response to biotic stress are under debate. In this study, we identified and cloned a CIPK homologue gene TaCIPK10 from wheat. TaCIPK10 was rapidly induced by Puccinia striiformis f. sp. tritici (Pst) inoculation and salicylic acid (SA) treatment. In vitro phosphorylation assay demonstrated that the kinase activity of TaCIPK10 is regulated by Ca²⁺ and TaCBL4. Knockdown TaCIPK10 significantly reduced wheat resistance to Pst, whereas TaCIPK10 overexpression resulted in enhanced wheat resistance to Pst by the induction of defense response in different aspects, including hypersensitive cell death, ROS accumulation and pathogenesis‐relative genes expression. Moreover, TaCIPK10 physically interacted with and phosphorylated TaNH2, which was homologous to AtNPR3/4. Silencing of TaNH2 in wheat resulted in enhanced susceptibility to the avirulent Pst race, CYR23, indicating its positive role in wheat resistance. Our results demonstrate that TaCIPK10 positively regulate wheat resistance to Pst as molecular links between of Ca²⁺ and downstream components of defense response and TaCIPK10 interacts with and phosphorylates TaNH2 to regulate wheat resistance to Pst.     

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca²⁺ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL-CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL-CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.     

Isolation and characterisation of cDNA encoding a wheat heavy metal‐associated isoprenylated protein involved in stress responses

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal‐associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy‐terminal end. To investigate the roles of wheat heavy metal‐associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal‐associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real‐time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up‐regulated by ABA exposure or wounding. Additionally, TaHIPP1 over‐expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu²⁺ and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1‐GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus‐induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.     

甘蓝型油菜BnaCIPK15基因的表达分析与功能鉴定

CBL-CIPK是高等植物中广泛存在的一类解析Ca~(2+)信号的蛋白。该研究在前期工作基础上,对甘蓝型油菜(Brassica napus L.)的BnaCIPK15基因进行了亚细胞定位、双分子荧光互补(BiFC)、酵母双杂交和qRT-PCR检测等一系列分析,以探究BnaCIPK15蛋白在ABA激素响应中的作用。结果显示:(1)亚细胞定位发现,BnaCIPK15蛋白定位于细胞质和细胞核中; BiFC分析发现,BnaCIPK15蛋白与BnaCBL1/3/4/9蛋白之间的互作较强,与BnaCBL10仅有微弱互作。(2)qRT-PCR检测发现,BnaCIPK15基因受ABA和冷胁迫的诱导极显著上调表达,而对百草枯(Paraquat)、活性氧(H_2O_2)和热胁迫的诱导较弱,表明BnaCIPK15基因很可能参与ABA和冷胁迫的调控过程。(3)酵母滴定实验结果显示,BnaCIPK15蛋白与脱落酸(ABA)信号通路中的BnaHAB1蛋白(属于蛋白磷酸酶PP2C家族)存在明显的互作,而与BnaABFs/AREB3/ABI5转录因子无明显互作;BiFC验证显示,BnaCIPK15与BnaHAB1蛋白之间存在互作信号,而BnaCIPK15与BnaHAB2组合没有观察到信号,证明BnaCIPK15与BnaHAB1磷酸酶具有特异互作特征,推测BnaCIPK15可能参与调控ABA信号转导。研究认为,甘蓝型油菜中可能存在基于BnaCIPK15-BnaHAB1的互作模块,并参与ABA的信号转导和网络调控。     

CBL‐mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores

During adaptation and developmental processes cells respond through nonlinear calcium‐decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B‐like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL‐interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N‐terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII‐independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra‐ and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.