番茄Pti基因瞬时表达与互作检测

丁香假单胞菌所导致的细菌性斑点病是影响番茄产量和品质的重要病害,而番茄色氨酸苏氨酸激酶(Pto)是植物识别、防御这一病原菌的重要抗性蛋白。文章通过克隆番茄Pti(Pto interaction protein)基因Pti4、Pti5和Pti6,分别构建植物表达载体,利用农杆菌介导方法在烟草叶片中进行瞬时表达和western blotting检测,并进一步利用免疫共沉淀技术,在植物体系中检测了它们与番茄Pto蛋白的相互作用。实验结果表明,利用所构建Pti4、Pti5和Pti6基因植物表达载体,在烟草中获得了预期大小Pti4、Pti5和Pti6蛋白,并在植物体系中验证了它们与Pto蛋白的相互作用。该工作为深入研究Pti基因在番茄分子免疫途径的功能奠定了基础。     

水杨酸对烟草GRPs基因表达的影响

通过RT-PCR得到富含甘氨酸RNA结合蛋白（GRPs）基因片段,利用northern杂交技术对水杨酸诱导不同时间不同烟草GRPs基因的表达模式进行研究.结果表明：水杨酸能诱导烟草GRPs基因上调表达,3 h达到最高,然后开始下降;但3个TMV抗性不同的供试烟草增加幅度不同,基因表达幅度与抗性有一定关系,抗性强的红花大金元表达增幅大于抗性较弱的云烟85.本研究明确了水杨酸诱导下烟草该基因的表达谱,揭示了SA诱导烟草不同抗性的品种的幅度不同,并且发现诱导后3 h为关键点,表明GRPs基因可作为烟草水杨酸代谢途径中的一个标记基因研究其途径对逆境的应答.     

核黄素诱导番茄幼苗抗白粉菌机理研究

研究了2mmol/L核黄素诱导感病番茄品种Moneymaker对白粉菌(Oidium neolycopersici)的抗性及相关生理反应:叶片H2O2积累、丙二醛(malonaldehyde,MDA)含量、超氧阴离子(O2-)产生速率和苯丙氨酸解氨酶(phenylalanine ammonialyase,PAL)、过氧化物酶(peroxidase,POD)活性的变化.核黄素诱导后番茄MM对O.ne-olycopersici的抗性增强,诱发了过敏反应(hypersensitive response,HR),番茄叶片内MDA的含量、O2-产生速率、PAL,POD活性均高于核黄素未处理,其变化趋势与抗病番茄品种G1.1560与白粉菌组成的非亲和组合相似.结果表明,H2O2的积累、膜脂过氧化、O2-,PAL和POD在核黄素诱导感病番茄对白粉菌的抗性反应中发挥重要作用.     

番茄转录因子SlWRKY53的分离及生物学功能鉴定

构建番茄SlWRKY53的过量表达载体,通过农杆菌介导转入番茄（Solanum lycopersicum）品种Ailsa Craig（AC+）,获得转基因阳性植株.定量分析显示,这些转基因株系中SlWRKY53基因表达量显著高于野生型.表型分析显示,转基因植株对盐胁迫抗性强于野生型.对抗病性进行检测,转基因植株抗性稍强,但与野生型相比无明显差异.由此推测番茄SlWRKY53基因的过量表达能够一定程度增强植株抵抗盐胁迫的能力.     

番茄青枯病拮抗菌的筛选及防治效果

为了筛选出具有抑制青枯雷尔氏菌作用的拮抗菌,采用形态指标、生理生化特征以及16S rRNA序列分析等方法对菌株进行了研究,并且通过阴性对照（CK）、番茄青枯病菌阳性胁迫处理（RB）和拮抗菌K+番茄青枯病菌处理（KRB）3个盆栽番茄土壤样品进行了防卫反应基因的表达研究.结果表明,拮抗菌K5菌落呈乳白色且表面光滑,经革兰氏染色为阳性,初步确定为芽孢杆菌,通过分子鉴定后确定为贝莱斯芽孢杆菌;番茄青枯病的盆栽防治效果达到23.20%,K5菌株在接种48 h后能诱导番茄根部抗病基因表达,CTR、ETR基因的表达量提高,表明K5通过植物抗病相关基因的表达,能诱导植物产生抗病性,并能有效地抑制青枯病菌的繁殖,从而达到防治青枯病的作用.     

海岛棉GbRar1和GbSgt1基因的过量表达提高烟草离体叶片对赤星病的抗性

RAR1 和 SGT1 是两个植物抗病蛋白 (R) 防卫反应的信号元件,在 R 蛋白下游和活性氧产生之间发挥作用. 利用简并引物 PCR 及3′RACE 等技术,克隆了海岛棉 GbRAR1 和 GbSGT1的编码基因. Northern blot 表明,海岛棉GbRar1和GbSgt1基因在转录水平上受棉花黄萎病菌的诱导,诱导后 mRNA 表达量显著增加. 构建组成型植物表达载体 pRar 和 pSgt,分别转化烟草品种 NC89. 离体叶片抗病性鉴定表明,两类转基因烟草对赤星病的抗性明显提高,为植物防卫反应信号元件RAR1 和 SGT1在广谱抗病基因工程中的应用提供了实验证据.     

拟南芥中的诱导抗病性的研究新进展

植物信号分子水杨酸和茉莉酸在诱导抗病性中有重要作用,它们介导的通道间的对话可以抑制JA介导的防卫反应。在拟南芥中,SAR和ISR可有效对付广谱的病原物,包括叶部病原物丁香假单孢菌番茄致病变种。SAR和ISR都需要关键调节蛋白NPR1通过平行的激活NPR-1介导的防卫反应防治丁香假单孢菌。     

番茄黄化曲叶病毒病抗性材料的筛选鉴定

 为评估新培育番茄栽培品种对番茄黄化曲叶病毒（tomato yellow leaf curl virus,TYLCV）的抗性水平,更全面地了解这些品种的抗病性差异,以金棚1 号和金曼为感病对照,采用带毒烟粉虱自然传毒方式,对各品种的发病时间、发病率及病情指数等参数进行比较,结合PCR 及ELISA 对TYLCV 的检测结果,综合分析了18 个番茄品种对番茄黄化曲叶病毒的抗病性。结果表明,不同品种对TYLCV 的抗性差异较大。金棚1 号和金曼两份材料发病率都达到100%,病情指数在65.2 以上,属于典型的感病品种;秋光15-6、PC-88 和秋光69 号3 份材料发病率和病情指数均为0,属于抗性较高的品种;其他13 份材料的发病率和病情指数分别在5.6％～45.2%和2.6～18.0 之间,分别表现了不同程度的抗、耐病水平。     

番茄诱导抗病性的研究进展

本文介绍了植物诱导抗病性特点、诱导抗性机理和激发子,综述了诱导番茄抗细菌、真菌病害的研究进展和发展前景。     

核黄素,烟酸对烟草抗赤星病诱导的交互作用

用核黄素、烟酸单独或二者组合分别诱导烟草植株，１６ｈ提取总ＲＮＡ，用５种防卫基因的ｃＤＮＡ探针作印迹杂交。结果表明，病程相关蛋白ＰＲ－ｌａ在４种处理中均表现出转录活性增强，其中以核黄素诱导效应最好。几丁质酶（ＣＨＴ）、查尔酮合成酶酶（ＣＨＳ）及脂氧合酶（ＬＯＸ）基因的表现为：受核黄素、烟酸／核黄素诱导后活性略有增强，而受烟酸、核黄素／烟酸诱导后活性下降。苯丙氨酸氨裂解酶（ＰＡＬ）基因在４种处理中都     

组成型表达线粒体小分子热激蛋白提高番茄抗冷性

线粒体小分子热激蛋白不但是线粒体的主要热诱导产物,而且也被低温诱导,线粒体小分子热激蛋白可以提高植物的耐热性,但目前还不清楚它是否与植物的耐冷性有关．本实验利用基因工程方法,将番茄线粒体小分子热激蛋白基因（LeHsp23．8）导入番茄,Northern—blotting及Western—blotting分析结果表明,在35sCaMV启动子的驱动下,导入的线粒体小分子热激蛋白基因呈现组成型表达．转基因番茄呈现耐低温性更强的表型,说明组成型表达线粒体小分子热激蛋白基因能提高番茄的抗冷性．     

Systemic defense signaling in tomato

The wound-inducible expression of proteinase inhibitors （PIs） genes in tomato provides a powerful model system to elucidate the signal transduction pathway of systemic defense response. An increasing body of evidence indicates that systemin and jasmonic acid （JA） work in the same signaling pathway to activate the expression of Pls and other defense-related genes. However, little is known about how systemin and JA interact to regulate cell to cell communication over long distances. Genetic analysis of the systemin/JA signaling pathway in tomato plants provides a unique opportunity to dissect the mechanism by which peptide and oxylipin signals interact to coordinate systemic expression of defense-related genes. Previously, it has been proposed that systemin is the long-distance mobile signal for systemic expression of defense related genes. However, recent genetic approach provided new evidence that jasmonic acid, rather than systemin, functions as the systemic wound signal, and that the peptide systemin works to regulate the biosynthesis of JA.     

The structural basis for activation of plant immunity by bacterial effector protein AvrPto

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.     

Enhanced late blight resistance of transgenic potato expressing glucose oxidase under the control of pathogen-inducible promoter

To engineer crop disease resistance by utilizing natural defense mechanism that was expressed in the incompatible host-pathogen interactions is expected to result in a durable and broad-spectrum resistance. In order to prove this viewpoint, we amplified the coding region of the glucose oxidase (GO) gene from Aspergillus niger via PCR and fused it to the pathogen-inducible promoter, Prp1-1. The chimeric gene was cloned into a plant expression vector and conjugated into Agrobacterium. Twenty-three transgenic potato plants were obtained by Agrobacterium-mediated transformation. The integration of GO gene was confirmed by Southern hybridization and the GO gene expression was identified with KI-starch color reaction. Phytophthora infestans inoculation revealed that the expression of the chimeric transgene was induced by pathogen infection. Most of the transgenic plants exhibited various degrees of enhanced disease resistance. Four of them had lesion sizes reduced to less than half of the non-transgenic controls. One plant showed disease resistance of the hypersensitive response. These results testified the feasibility of our strategy of expressing GO transgene under the control of the disease-inducible promoter in engineering crop disease resistance.     

短日照处理诱导油松容器苗针叶差异表达蛋白质的功能分析

【目的】基于蛋白质水平探讨短日照处理调控油松(Pinus tabulaeformis)容器苗基础代谢和抗逆性机理的研究,完善油松容器苗的育苗和造林技术,为短日照处理育苗技术在我国北方地区困难立地造林中推广应用提供理论依据。【方法】以经日照长度为10 h、持续3 周短日照处理的油松容器苗针叶为研究对象,采用改良的酚法提取针叶蛋白。应用双向电泳结合二级质谱分析的蛋白质组学技术,研究短日照处理诱导油松容器苗针叶蛋白质表达的变化,分析差异表达蛋白质的鉴定和功能。【结果】成功获取5个短日照处理诱导表达差异显著的蛋白质,上调表达的是叶绿体放氧增强蛋白1(oxygen-evolving enhancer protein 1, OEE1)(蛋白点404)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)(蛋白点539)、延伸因子-Tu(elongation factor-Tu, EF-Tu)(蛋白点654)、核酮糖-1,5-二磷酸羧化酶/加氧酶活化酶(ribulose bisphosphate carboxylase/oxygenase activase, RCA)(蛋白点681); 下调表达的是磷酸甘油酸激酶1(phosphoglycerate kinase 1, PGK1)(蛋白点641)。这些蛋白质的功能主要涉及光合代谢、糖代谢和胁迫防御。【结论】短日照处理诱导油松容器苗蛋白质差异表达,对油松容器苗的基础代谢和抗逆性产生重要影响。     

Bacterial disease resistance in Arabidopsis through flagellin perception

Plants and animals recognize microbial invaders by detecting pathogen-associated molecular patterns (PAMPs) such as flagellin. However, the importance of flagellin perception for disease resistance has, until now, not been demonstrated. Here we show that treatment of plants with flg22, a peptide representing the elicitor-active epitope of flagellin, induces the expression of numerous defence-related genes and triggers resistance to pathogenic bacteria in wild-type plants, but not in plants carrying mutations in the flagellin receptor gene FLS2. This induced resistance seems to be independent of salicylic acid, jasmonic acid and ethylene signalling. Wild-type and fls2 mutants both display enhanced resistance when treated with crude bacterial extracts, even devoid of elicitor-active flagellin, indicating the existence of functional perception systems for PAMPs other than flagellin. Although fls2 mutant plants are as susceptible as the wild type when bacteria are infiltrated into leaves, they are more susceptible to the pathogen Pseudomonas syringae pv. tomato DC3000 when it is sprayed on the leaf surface. Thus, flagellin perception restricts bacterial invasion, probably at an early step, and contributes to the plant's disease resistance.     

农杆菌介导的双价抗虫基因对番茄遗传转化的研究

采用农杆菌介导法将含有苏云金芽孢杆菌毒蛋白基因(CryIAc)与半夏凝集素抗虫基因(Pta)的高效植物表达载体pCAMBIA3300转入番茄品系Micro Tom的子叶外植体中。经过共培养、除草剂筛选和分化再生,获得了24个具有除草剂抗性的株系。再将转化后的番茄植株经过PCR检测和Southern Blot检测,确定检测后呈阳性反应的株系为8个。通过小菜蛾幼虫初步抗性试验证明,转基因株系表现出较强的抗虫性。实验结果为进一步研究番茄抗虫性和培育抗虫番茄新品种奠定了重要基础。     

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.     

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.