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。     

大豆GmGolS2基因启动子的克隆及功能分析

本研究利用PCR技术克隆了大豆Gm Gol S2基因的启动子序列。系统进化树分析表明,Gm Gol S2与沙冬青的Gols亲缘关系最近。利用Plant CARE分析启动子元件发现,在Gm Gol S2基因启动子序列中含有多个与逆境胁迫、激素等反应相关的元件。在The Bio-Analytic Resource for Plant Biology数据库中分析了Gm Gol S2的同源基因At Gol S2在不同逆境胁迫处理时的基因表达情况,结果表明该基因具有组织表达特异性,并参与盐胁迫、渗透胁迫及干旱胁迫等反应。RT-PCR结果表明Gm Gol S2基因受干旱诱导上调表达。     

大豆GmNFYB1基因功能预测及表达分析

本研究对大豆Gm NFYB1(Glyma02g17310)进行基因功能和表达分析,利用Plant CARE分析启动子元件发现,在基因启动子序列中含有多个与ABA、抗旱、光反应相关的元件。在The Bio-Analytic Resource for Plant Biology数据库中分析了Gm NFYB1的同源基因At NFYB5在不同逆境胁迫和不同激素处理时的基因表达情况,表明该基因表达具有组织表达特异性,参与盐胁迫、渗透胁迫等反应。Gm NFYB1在大豆的q RT-PCR结果表明,该基因受ABA、Na Cl、PEG诱导上调表达,参与大豆抗逆反应。     

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。     

大豆GmDAO1基因的功能预测及表达分析

本研究通过在大豆基因组数据库中检索拟南芥AtDAO1在大豆中的同源基因,获得了GmDAO1基因序列。通过对GmDAO1基因编码的氨基酸序列及启动子序列进行生物信息学分析,我们发现GmDAO1基因CDS序列全长951bp,编码316个氨基酸。GmDAO1编码的蛋白为亲水性蛋白,具有1个N-糖基化位点、3个激酶磷酸化位点与1个豆蔻酰化位点。结构域分析表明GmDAO1含有双加氧酶与2OG-Fe(II)加氧酶结构域,是2-酮戊二酸依赖性双加氧酶基因(2-ODD)家族的成员。GmDAO1预测的启动子区域含有与激素、胁迫、光应答、生物钟调控和转录因子结合相关的顺式作用元件。系统进化分析结果表明DAO1在豆科植物进化过程中比较保守。组织特异性表达分析结果显示GmDAO1在叶片中表达量最低,在根中表达量最高。因此我们推测其可能参与生长素的代谢途径。     

大豆VAS1基因家族的鉴定及参与侧根发育的研究

拟南芥VAS1基因编码一个磷酸吡哆醛依赖性氨基转移酶,可将吲哚丙酮酸转化为吲哚乙酸的生物合成前体色氨酸,是生长素代谢调控中的一个关键酶。对大豆VAS1基因家族进行全基因组鉴定与表达模式分析,并探究其在根系发育中的作用,为深入挖掘大豆VAS1基因的功能奠定基础。运用生物信息学方法对该基因家族成员进行鉴定和分析,采用实时荧光定量分析该家族在不同逆境处理下的表达模式,克隆GmVAS1-1,进一步研究其功能,并通过酵母双杂交试验筛选大豆GmVAS1-1蛋白的互作因子。结果表明,大豆VAS1基因家族共有2个成员,命名为GmVAS1-1和GmVAS1-2。系统进化树、基因结构和保守基序分析表明,大豆VAS1与豆科植物亲缘关系更近。启动子序列分析发现多个逆境和光响应元件。表达模式分析表明,大豆VAS1家族基因在不同种子发育时期的胚乳中表达量较高。荧光定量PCR分析表明,在干旱和盐胁迫下,大豆根系VAS1基因表达量显著上调。拟南芥过量表达GmVAS1-1植株侧根数较野生型显著减少。酵母互作试验及预测共筛选到17个与GmVAS1-1互作的蛋白。大豆VAS1基因家族成员在胚乳中表达量较高,且响应多种逆境及...     

玉米MCM2基因的克隆与功能验证北大核心CSCD

内复制是影响玉米胚乳发育的关键因素。通过降低CDK(周期蛋白依赖性激酶)活性,胚乳细胞可实现有丝分裂向内复制转变,进而推动籽粒快速灌浆。该研究以玉米微染色体维持蛋白ZmMCM基因家族为对象,对其基本生物信息学特征和非生物胁迫条件下的表达特征进行系统分析,并对其中低温胁迫响应明显的ZmMCM2通过转基因和酵母双杂的方法进行功能验证和分子互作分析。结果表明:(1)在玉米基因组中共鉴定到17个MCM家族成员,分布于6条染色体,而且部分基因间存在串联重复基因和片段复制基因;不同物种MCM蛋白的系统进化树可分为6个亚组,玉米、水稻和拟南芥的MCM2蛋白同属于第Ⅳ亚组;启动子序列分析显示,MCM家族基因启动子序列含有许多与激素响应、胁迫应答以及生长发育调节相关的顺式作用元件。(2)逆境胁迫响应分析表明,MCM基因表达受到NaCl和ABA抑制,对PEG、高温以及低温表现出不同程度的应答,尤其对低温具有明显响应。(3)ZmMCM2基因的过表达会对内复制产生一定的抑制作用,进而导致拟南芥植株矮小、莲座叶数减少以及种子体积缩小。(4)亚细胞定位结果显示,ZmMCM2基因定位于细胞核内;cDNA文库筛选和回转验证发现,MCM2与CDC73相互作用。该研究结果为进一步深入了解MCM2蛋白的分子作用机理奠定了基础。     

菜用大豆液泡膜内在蛋白(TIPs)基因鉴定及表达分析

TIPs(tonoplast intrinsic proteins)是一类定位于液泡膜或液泡形成体上的水通道蛋白,在植物响应逆境胁迫中发挥重要作用。本研究利用大豆基因组数据库,在全基因组水平鉴定到23个GmTIPs基因。染色体定位分析发现GmTIPs基因分布在大豆17条染色体上。蛋白多序列比对分析发现所有GmTIPs含有典型的6个保守的跨膜螺旋(TM1 to TM6)和2个保守的氨基酸元件NPA盒(Asp-Pro-Ala box)。蛋白特性分析发现GmTIPs的氨基酸数目在237~255之间,分子量在24.08~27.11k D之间,等电点在5.08~10.01之间。进化关系分析显示,大豆GmTIPs可划分为5个分支(TIP1,TIP2,TIP3,TIP4和TIP5),与拟南芥分类一致,且每个分支均含有这两个物种中的TIPs成员,暗示同一分支TIPs基因成员可能具有相似的基因功能。进一步利用q RT-PCR技术分析了5个GmTIPs候选基因对逆境胁迫(干旱和高盐)及激素信号(脱落酸ABA、乙烯前体ACC)的响应情况。结果显示,这些环境胁迫及外源激素均可以诱导GmTIPs基因在根和叶这两种组织中的上调或下调表达。其中,干旱和高盐两种胁迫诱导GmTIP2;6在根中上调表达最为明显。然而,干旱胁迫分别诱导GmTIP2;1、GmTIP2;2和GmTIP4;1在叶中显著上调表达。激素ACC处理诱导GmTIP2;2在根中明显上调表达。以上结果为进一步探讨GmTIPs基因的抗逆功能、分子机制及其在菜用大豆分子育种中的应用提供重要依据。     

拟南芥GHMP基因家族成员的组织表达及生物信息学分析

利用生物信息学方法获得拟南芥全基因组中12个GHMP基因家族成员。通过实时定量PCR技术研究这12个基因在不同组织中的表达,结果显示它们具有组织表达特异性。构建了拟南芥中GHMP基因家族成员的系统进化树。启动子区调控元件分析表明,大多数GHMP成员包含有光响应、生物钟及其它逆境胁迫响应的相关元件,预测这些GHMP基因家族成员可能参与了植物的光信号、生物钟及相关的逆境胁迫信号转导途径。     

大豆Glyma03g24460基因功能预测及表达分析

大豆Glyma03g24460基因,与拟南芥ECERIFERUM1(CER1)基因具有高度的同源性,是一种植物角质层蜡质基因,参与植物角质层蜡质的合成。本研究对其进行基因功能预测和表达分析,利用Plant CARE分析启动子元件发现在基因启动子序列中含有黄酮合成、激素响应、生物和非生物胁迫相关的元件。氨基酸序列比对发现Glyma03g24460与其他物种的脂肪醛脱羧酶基因家族成员有很高的相似度。Glyma03g24460在大豆的q RT-PCR结果表明,它主要在植物的地上器官表达,并且可以受到ABA及干旱等非生物胁迫的诱导表达。     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of expressed receptor-like kinases （RLKs） in soybean

Receptor-like kinases （RLKs） play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags （ESTs） survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.     

Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.)

Background

Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca²⁺ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola.

Results

In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop.

Conclusions

Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.     

Analysis of expressed receptor-like kinases (RLKs) in soybean

Receptor-like kinases (RLKs) play crucial roles in cellular signal perception and propagation. To study the evolutionary relationships among RLKs in soybean, a large-scale expressed sequence tags (ESTs) survey for RLKs-related sequences was conducted. By doing BLAST analysis using our database and The Gene Index Database, 605 putative RLK genes were identified. Based on the phylogeny of the kinase domain, these soybean RLKs were classified into 58 different small subfamilies. The phylogenetic analysis of RLKs in soybean, rice and Arabidopsis showed that different subfamilies of RLKs had different functions and could have experienced different selective pressures.     

Comprehensive structural,interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice

Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine–threonine protein kinases designated as CBL-interacting protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL–CIPK mediated signaling pathways, expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly, expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes. Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolved by many tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4.     

The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high-salt conditions. Interestingly, the Na(+) content of the cbl10 mutant, unlike other salt-sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca(2+)-signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and the CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance pathway that regulates the sequestration/compartmentalization of Na(+) in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi-functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.