小麦E3泛素连接酶基因TaAIRP2-1B克隆及功能分析

干旱等非生物胁迫严重影响农作物生产。本研究克隆了小麦(Triticum aestivum L.)TaAIRP2-1B基因,探讨其对非生物胁迫的响应机制,为促进小麦抗旱性的遗传改良提供基因资源。组织特异性表达模式分析显示,TaAIRP2-1B基因在小麦抽穗期的各个组织中均有表达,在茎组织中的表达水平较高,而根系中的表达水平较低。非生物胁迫表达模式分析显示,Ta AIRP2-1B受ABA、PEG及冷胁迫诱导表达。过表达TaAIRP2-1B拟南芥在0.4μmol/L的ABA处理条件下,种子发芽率显著低于野生型,表明TaAIRP2-1B提高了拟南芥种子萌发期对ABA的敏感性。ABA处理抑制转基因和野生型拟南芥幼苗的根系生长,但转基因拟南芥受抑制程度显著高于野生型,表明TaAIRP2-1B提高了拟南芥幼苗对ABA的敏感性。转基因结果表明超表达TaAIRP2-1B增强了拟南芥的抗旱性,并且转基因株系的保水率显著高于野生型。总之,本研究发现小麦基因Ta AIRP2-1B参与了植物对非生物胁迫的应答,可能是通过ABA途径正向调控植物的抗旱性。     

水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应

锌指蛋白在调控植物生长发育和应对逆境过程中发挥着重要作用.为进一步研究锌指类蛋白参与植物非生物胁迫响应的分子机制,对水稻(Oryza sativa)中一个编码含有B-box锌指结构域蛋白的OsBBX25基因进行了功能分析.OsBBX25受盐、干旱和ABA诱导表达.异源表达OsBBX25的转基因拟南芥(Arabidopsis thaliana)与野生型相比对盐和干旱的耐受性增强,且盐胁迫条件下转基因植物中KIN1、RD29A和COR15的表达上调,干旱胁迫下KIN1、RD29A和RD22的表达上调.外源施加ABA时,转基因植物的萌发率与野生型之间没有明显差异.OsBBX25可能作为转录调控的辅助因子调节胁迫应答相关基因的表达,进而参与植物对非生物胁迫的响应.     

拟南芥抵抗丁香假单胞杆菌过程中AZI1基因功能研究

AZI1属于脂转移蛋白家族,它在拟南芥抵抗病原菌侵染过程中可能起着传递信号物质的作用。该实验以过表达和T-DNA插入突变体及野生型拟南芥植株为材料,通过RNA印迹、蛋白质免疫印迹和原位免疫组织化学方法,研究了拟南芥壬二酸诱导基因AZI1对丁香假单胞杆菌的抗性功能。结果表明:(1)AZI1基因可以被丁香假单胞杆菌、H2O2和乙烯利诱导,它可能参与水杨酸和乙烯介导的抗菌途径。(2)蛋白质免疫印迹实验结果显示,丁香假单胞杆菌侵染叶片的叶柄渗出液中存在AZI1蛋白及其同源物EARLI1,并能够与其他蛋白质形成复合体,说明AZI1有可能通过维管组织移动到个体的其他部位,与信号分子的转移有关。(3)AZI1及其同源物EARLI1主要在花序茎的木质化部位表达,过表达AZI1基因能够促进木质素的合成,提高拟南芥对丁香假单胞杆菌的抗性。     

陆地棉GhCDPK1基因响应干旱胁迫的功能初探

钙依赖性蛋白激酶(CDPKs)是一类重要的钙信号感受蛋白和响应蛋白,在植物干旱、低温、盐碱等非生物胁迫应答中起着重要的调控作用。为探讨陆地棉GhCDPK1基因在干旱胁迫下所起的作用,该研究利用实时荧光定量PCR技术分析了PEG模拟干旱胁迫下该基因的表达量,发现GhCDPK1基因受干旱胁迫诱导。通过构建植物表达载体pCAMBIA2300-GhCDPK1,采用农杆菌介导的叶盘法转化模式植物烟草,发现干旱胁迫下转基因植株保水能力明显高于野生型植株,叶绿素、脯氨酸、可溶性蛋白含量及POD、SOD活性也高于野生型植株,而丙二醛含量低于野生型植株。研究结果表明,GhCDPK1基因作为正向调控因子响应干旱胁迫诱导,过表达GhCDPK1基因可以使植株积累更多的渗透调节物质、增强抗氧化系统酶的活性和维持细胞膜的稳定性来提高植物抵御外界干旱胁迫的能力。     

中间锦鸡儿CiMYB15基因正调控拟南芥黄酮代谢

目的:R2R3-MYB类转录因子参与调控植物初生和次生代谢。方法:从中间锦鸡儿(Caragana intermedia)干旱转录组数据库中搜索并克隆了一个R2R3-MYB基因,命名为CiMYB15(GenBank登录号MH678649);将CiMYB15基因编码区转入野生型拟南芥中,利用分光光度法测定了野生型和转基因拟南芥中总黄酮含量,并用qRT-PCR检测了转基因植物中At CHS基因的表达情况。同时采用染色体步移法克隆了CiMYB15基因的启动子序列。结果表明:(1) CiMYB15基因g DNA长度为1 960 bp,包含三个外显子(134、131和521 bp)和两个内含子(281和893 bp);开放阅读框长度为786 bp,编码262个氨基酸。(2)克隆得到1 580 bp的启动子序列,序列中主要包含损伤诱导元件G-box和P-box、盐诱导作用元件GT1-motif、参与干旱诱导的反应元件MBS,以及真菌侵害应答元件BOX-W1、植物-病原菌互作元件EIER;此外,还包含调节黄酮合成基因的MYB转录因子的结合位点。(3) CiMYB15基因的表达受到紫外胁迫的诱导。(4) CiMYB15基因过表达株系的总黄酮含量高于野生型。(5)过表达植物中At CHS基因的表达量亦高于野生型。以上结果说明,CiMYB15基因正调控拟南芥黄酮代谢。     

盐穗木水通道蛋白基因的克隆与功能鉴定

非生物胁迫(如盐渍和干旱)会引起植物水分代谢的紊乱,导致植物细胞水分丧失,直接抑制植物的生长发育。研究水通道蛋白(aquaporins,AQPs)在非生物胁迫下对植物生理功能的影响十分重要。本研究利用RACE技术克隆获得一个盐穗木(Halostachys caspica)水通道蛋白家族亚类质膜内嵌蛋白(plasma intrinsic protein,PIPs)基因,命名为Hc PIP1。Hc PIP1基因全长序列为1 244 bp,包含858 bp的开放阅读框(open reading frame,ORF)序列,编码285个氨基酸,分子量大小约为30.6 k D。q RT-PCR检测表明Hc PIP1在盐穗木根部的表达量显著高于同化枝中,盐胁迫诱导其上调表达。在酿酒酵母INVSc1中Hc PIP1的异源过表达显著提高了重组菌的耐盐能力;和野生型植株相比过表达Hc PIP1的拟南芥能够显著缓解渗透胁迫和离子胁迫对生长的抑制,并且根长明显比野生型的长。结果表明Hc PIP1在胁迫时能够通过增加根的生长以抵御胁迫的影响。     

中间锦鸡儿CiNAC1基因促进转基因拟南芥叶片的衰老

NAC转录因子家族是植物特有的、最大的转录因子家族之一,参与植物生物胁迫和非生物胁迫应答、激素信号转导、植物次生生长、细胞分裂和植物衰老等多种过程,在植物生长发育过程中起着重要的作用。以中间锦鸡儿Ci NAC1基因的过表达拟南芥纯合体株系为材料,以野生型为对照,对Ci NAC1基因功能进行分析。结果发现,乙烯处理后,Ci NAC1基因过表达株系与野生型拟南芥相比,叶片衰老提前、叶绿素含量降低、离子渗透率升高。实时荧光定量PCR检测发现,乙烯处理后Ci NAC1基因过表达株系中与叶绿素降解相关的基因SGR1、SGR2、PPH,以及与衰老相关的基因SAG13、SAG29、ORE1、SINA1、VNI2和乙烯信号途径中的重要转录因子EIN3的表达量均明显高于野生型拟南芥。表明Ci NAC1基因在乙烯诱导的叶片衰老过程中发挥重要作用。     

大豆转录因子GmC2H2基因转化拟南芥效果分析

GmC2H2转录因子基因是本实验室获得的一个编码172个氨基酸携带516bp核苷酸的转录因子,属于经典C2H2型锌指蛋白.通过构建植物表达载体GmC2H2-pCAMBIA1304,借助优化的Floral-dip法转化模式植物拟南芥,经潮霉素Hygromycine( 45-50 mg/L)抗性筛选获得转基因拟南芥植株.GUS组织染色分析表明,GmC2H2基因在生长12d的转基因拟南芥幼苗中,表达部位主要集中在根部.对转基因拟南芥进行了低温(1℃)和脱落酸(200 μmol/L)胁迫处理,测定其生理生化指标,通过real-time qPCR确定目的基因在转基因拟南芥中的表达情况.结果表明,携带GmC2H2目的基因的转基因拟南芥中脯氨酸和可溶性糖水平要高于野生型植株,而丙二醛水平要低于野生型,在抗逆性方面明显优于野生型拟南芥植株;并且胁迫处理下的转基因拟南芥中GmC2H2基因的表达量要高于未胁迫处理的转基因植株,说明GmC2H2基因的表达受低温和ABA的诱导,初步明确了该转录因子基因的功能.     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.     

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca²⁺. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca²⁺ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

Progesterone moderates damage in Arabidopsis thaliana caused by infection with Pseudomonas syringae or P. fluorescens

Brassinosteroids are known to protect plants against various abiotic and biotic stresses, however, very limited information is available about the role of progesterone. Therefore the effects of Pseudomonas syringae pv. syringae (P.s.) wild type strain 61, its hrcC mutant, and the saprophytic P. fluorescens (P.f.) strain 55 were investigated in wild type Arabidopsis thaliana cv. Columbia and its rbohF knock-out mutant, with and without progesterone pre-treatment. The reactions of wild type and rbohF mutant Arabidopsis to bacterial inoculations were similar, although 2 h after injection of P.s. a larger increase of electrolyte leakage was measured in wild type than in rbohF knockout mutant leaves. The hrcC mutant caused weak necrotic symptoms and increased leakage in both types of Arabidopsis, although to a much lesser extent than P.s. The P.f. did not induce any visible symptom, but slightly increased the electrolyte leakage in both types of Arabidopsis. Inoculation by all Pseudomonas bacteria led to significant alterations in photosystem 2 efficiency as compared to control plants. Pre-treatment of leaves with progesterone diminished the necrotic symptoms, the electrolyte leakage and improve the efficiency of photosystem 2 caused by Pseudomonas bacteria.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling

Arabidopsis thaliana calmodulin binding protein 60g (CBP60g) contributes to production of salicylic acid (SA) in response to recognition of microbe‐associated molecular patterns (MAMPs) such as flg22, a fragment of bacterial flagellin. Calmodulin binding is required for the function of CBP60g in limiting growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) ES4326 and activation of SA synthesis. Here, we describe a closely related protein, SARD1. Unlike CBP60g, SARD1 does not bind calmodulin. Growth of Pma ES4326 is enhanced in sard1 mutants. In cbp60g sard1 double mutants, growth of Pma ES4326 is greatly enhanced, and SA levels and expression of PR‐1 and SID2 are dramatically reduced. Expression profiling placed the CBP60g/SARD1 node between the PAD4/EDS1 and SA nodes in the defense signaling network, and indicated that CBP60g and SARD1 affect defense responses in addition to SA production. A DNA motif bound by CBP60g and SARD1, GAAATTT, was significantly over‐represented in promoters of CBP60g/SARD1‐dependent genes, suggesting that expression of these genes is modulated by CBP60g/SARD1 binding. Gene expression patterns showed a stronger effect of cbp60g mutations soon after activation of a defense response, and a stronger effect of sard1 mutations at later times. The results are consistent with a model in which CBP60g and SARD1 comprise a partially redundant protein pair that is required for activation of SA production as well as other defense responses, with CBP60g playing a more important role early during the defense response, and SARD1 to playing a more important role later.     

A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

Arabidopsis C3HC4‐RING finger E3 ubiquitin ligase AtAIRP4 positively regulates stress‐responsive abscisic acid signaling

Degradation of proteins via the ubiquitin system is an important step in many stress signaling pathways in plants. E3 ligases recognize ligand proteins and dictate the high specificity of protein degradation, and thus, play a pivotal role in ubiquitination. Here, we identified a gene, named Arabidopsis thaliana abscisic acid (ABA)‐insensitive RING protein 4 (AtAIRP4), which is induced by ABA and other stress treatments. AtAIRP4 encodes a cellular protein with a C3HC4‐RING finger domain in its C‐terminal side, which has in vitro E3 ligase activity. Loss of AtAIRP4 leads to a decrease in sensitivity of root elongation and stomatal closure to ABA, whereas overexpression of this gene in the T‐DNA insertion mutant atairp4 effectively recovered the ABA‐associated phenotypes. AtAIRP4 overexpression plants were hypersensitive to salt and osmotic stresses during seed germination, and showed drought avoidance compared with the wild‐type and atairp4 mutant plants. In addition, the expression levels of ABA‐ and drought‐induced marker genes in AtAIRP4 overexpression plants were markedly higher than those in the wild‐type and atairp4 mutant plants. Hence, these results indicate that AtAIRP4 may act as a positive regulator of ABA‐mediated drought avoidance and a negative regulator of salt tolerance in Arabidopsis.