花生抗青枯病分子标记研究

利用抗、感青枯病的花生品种为素本配制杂交组合中花5号×远杂9102,构建重组近交系,以其F6为研究材料,分析青枯痛抗性遗传规律,结果表明,花生青枯病抗性是由两时主效基因控制的遗传,并且主效基因的遗传力较高,为84%;同时采用AFLP技术和BSA分析方法,获得两个与花生青枯病抗性连锁的分子标记,标记与抗性间的遗传距离分别为8.12cM和11.46cM.利用获得的分子标记对抗、感青枯病的花生种质进行了分子鉴定,证实了标记P3M59与膏枯病抗性的符合率为70%,标记PIM58的符合率为50%,从而为花生青枯病抗性辅助选择育种提供理论基础.     

茄科植物抗青枯病特性研究及其应用展望

对 8个属 2 1种野生或栽培茄科植物的抗青枯病特性进行观测研究 ,筛选出水茄、乳茄、曼陀罗等 3种抗青枯病能力很强的物种。将番茄、烟草嫁接在水茄、乳茄上 ,嫁接苗也获得较强的抗病能力。将此结果结合现代抗病分子生物学技术及其研究成果 ,探讨这些抗性材料的抗病机理及其在抗病基因工程中的应用前景。     

抗稻瘟病水稻资源抗性基因Pita、Pib、Pi9以及Pikm的分布研究

利用抗稻瘟病水稻资源品种杂交,聚合多个抗性基因是培育持久抗稻瘟病水稻新品种的主要育种途径.利用分子标记技术对水稻抗性资源进行基因型鉴定是分子辅助聚合育种的基础.通过以亚华种业科学院稻瘟病病圃抗病水稻资源为材料,利用特异性分子标记对Pi9、Pita、Pib以及Pikm基因在水稻抗稻瘟病资源的分布进行了鉴定,初步建立了抗性基因数据库.同时对抗性基因及与抗性反应的相关性进行了探讨,结果表明以Pi9为主效基因,同时聚合Pita和Pib抗性基因能提高持久抗稻瘟病能力.     

小麦抗赤霉病研究现状与展望

小麦是我国最重要的口粮作物之一。在小麦生产所面临的各种病害中,赤霉病的发生具有愈来愈严重的趋势,引起小麦产业界的高度关注。近几十年来,科研人员在小麦抗赤霉病遗传育种以及防控技术领域进行了持续不懈的努力,在赤霉病病原菌致病基因、小麦赤霉病抗性基因定位、克隆及功能研究以及抗赤霉病分子育种等方面取得了重大进展。本文主要从赤霉病抗性基因资源的发掘和鉴定、不同抗源遗传基础解析、小麦赤霉病抗性基因、抗赤霉病分子标记辅助选择育种与基因聚合以及小麦抗赤霉病基因的克隆和功能研究等方面进行了综述,分析了目前小麦抗赤霉病研究中存在的问题,并提出应加强基因克隆、功能分子标记开发以及应用单体型辅助选择(HAS)和标记组辅助选择(MSAS)等小麦抗赤霉病研究的相关建议。     

大豆品种早熟18抗疫霉根腐病基因的SSR分子标记

大豆品种早熟18是抗疫霉根腐病的有效抗源。本研究鉴定和分子标记早熟18的抗疫霉根腐病基因,以期为该品种的有效利用及分子辅助育种奠定基础。以感病大豆品种Williams与早熟18杂交建立分离群体。抗性遗传分析表明,早熟18对大豆疫霉菌抗性由1个显性单基因控制,该基因被定名为RpsZS18。SSR标记连锁分析表明,RpsZS18位于大豆分子遗传连锁群D1b上的SSR标记Sat_069和Sat_183之间,与这两个标记的遗传距离分别为10．0cM和8．3cM。RpsZS18是D1b连锁群上鉴定的第一个抗疫霉根腐病基因。     

水稻细菌性条斑病和抗性育种研究进展

水稻细菌性条斑病(简称细条病)是由Xanthom onas oryzae pv. oryzicola侵染引起的全球性病害,对水稻生产构成严重威胁。发掘和利用新抗源、定位克隆抗性基因及深入了解病原菌—水稻之间的相互作用机理等对于水稻抗细条病研究有重要意义。本文主要介绍了细条病的抗性鉴定与抗源筛选、抗性基因遗传分析与分子标记定位、抗性基因的克隆、抗性育种的研究现状,提出了加快抗细条病育种研究进程的建议。     

茄子种质资源抗青枯病的鉴定与评价

对304份茄子种质资源进行抗青枯病苗期人工接种鉴定,筛选出免疫材料10份,高抗材料51份,抗病材料35份,中抗材料32份,感病或高感材料176份,分别占鉴定材料的3.3%、16.8%、11.5%、10.5%和57.9%.茄子野生近缘种Solanum sisymbriifolium和S.torvum对青枯病有较强的抗病性,可作为茄子青枯病的抗源材料.获得4份抗青枯病的种间体细胞杂种.茄子对青枯病的抗性遗传较为复杂,主要由多基因控制.     

青枯病病菌对茄子相关防卫信号基因表达及活性氧含量的影响

本文以茄子抗青枯病自交系和感病自交系为试材,在接种青枯病菌后,对调控抗性反应的不同信号传导基因进行表达特性分析。结果表明,在9个基因中,EDS1、PAD4、NPR1、SGTI和WRYK70等5个基因的表达均随着接种诱导时间延长而增加,而且在抗病自交系‘E-31’中的表达量要高于感病自交系‘E．32’;而JAR1、NDR1、EIN2YeaRARl等基因的表达随着接种诱导时间的增加,表达水平变化不大。初步推断EDS1、PAD4、NPRI、SGTI和WRYK70可能与调节茄子抗青枯病抗性反应有关。同时茄子在接种后活性氧的含量均增加,但是感病植株的含量高于抗病植株。     

抗褐飞虱水稻品种的培育及其抗性表现

褐飞虱Nilaparvata lugens(Stl)是危害水稻的主要虫害之一,发掘和利用新的抗褐飞虱基因培育抗性品种是目前防治褐飞虱最经济有效的方法之一。抗褐飞虱基因来自药用野生稻的抗虫品种B5,对褐飞虱生物型1和2具有高度抗性,B5携带的抗性基因Bph14被定位在第3染色体上。本研究以B5-10为抗源,以优良杂交稻亲本扬稻6号为受体亲本,通过复交和回交,利用与Bph14紧密连锁的分子标记MRG2329在后代中进行分子标记辅助选择,通过苗期分子标记检测和成株期农艺性状选择,最后育成恢复系R476和杂交组合广两优476。采用苗期群体鉴定技术对R476和广两优476的褐飞虱抗性进行了鉴定,R476和广两优476的抗性水平分别为中抗和中感。广两优476在稻飞虱发生较重的稻田进行试种示范,与对照品种扬两优6号和两优培九相比,广两优476对稻飞虱表现出明显的抗性。研究结果表明在育种过程中利用分子标记辅助选择Bph14基因是培育抗褐飞虱水稻品种的有效途径之一。     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum . This locus named RCH2 (for recognition of C. higginsianum ) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 ( resistance to Ralstonia solanacearum 1 ) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2 , were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum . The screening identified an additional susceptible mutant ( rps4-21 ) that has a 5-bp deletion in the neighboring gene, RPS4-Ws , which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 ( Pst - avrRps4 ). The rps4-21 / rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4 . Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.     

ERECTA, an LRR receptor-like kinase protein controlling development pleiotropically affects resistance to bacterial wilt

Bacterial wilt, one of the most devastating bacterial diseases of plants worldwide, is caused by Ralstonia solanacearum and affects many important crop species. We show that several strains isolated from solanaceous crops in Europe are pathogenic in different accessions of Arabidopsis thaliana. One of these strains, 14.25, causes wilting symptoms in A. thaliana accession Landsberg erecta (Ler) and no apparent symptoms in accession Columbia (Col-0). Disease development and bacterial multiplication in the susceptible Ler accession depend on functional hypersensitive response and pathogenicity (hrp) genes, key elements for bacterial pathogenicity. Genetic analysis using Ler x Col-0 recombinant inbred lines showed that resistance is governed by at least three loci: QRS1 (Quantitative Resistance to R. solanacearum) and QRS2 on chromosome 2, and QRS3 on chromosome 5. These loci explain about 90% of the resistance carried by the Col-0 accession. The ERECTA gene, which encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) and affects development of aerial organs, is dimorphic in our population and lies close to QRS1. Susceptible Ler plants transformed with a wild-type ERECTA gene, and the LER line showed increased disease resistance to R. solanacearum as indicated by reduced wilt symptoms and impaired bacterial growth, suggesting unexpected cross-talk between resistance and developmental pathways.     

A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-NBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes activation of defense responses and hypersensitive cell death

In this study we characterized the sensitive to low humidity 1 (slh1) mutant of Arabidopsis ecotype No-0 which exhibits normal growth on agar plate medium but which on transfer to soil shows growth arrest and development of necrotic lesions. cDNA microarray hybridization and RNA gel blot analysis revealed that genes associated with activation of disease resistance were upregulated in the slh1 mutants in response to conditions of low humidity. Furthermore, the slh1 mutants accumulate callose, autofluorescent compounds and salicylic acid (SA). We demonstrate that SA is required for the slh1 phenotype but not PAD4 or NPR1. SLH1 was isolated by map-based cloning and it encodes a resistance (R)-like protein consisting of a domain with Toll and interleukin-1 receptor homology (TIR), a nucleotide-binding domain (NB), leucine-rich repeats (LRR) and a carboxy-terminal WRKY domain. SLH1 is identical to the R gene RRS1-R of the Arabidopsis ecotype Nd-1, a gene which confers resistance to the bacterial pathogen Ralstonia solanacearum GMI1000 and also functions as an R gene to this pathogen in No-0. We identified a 3 bp insertion mutation in slh1 that results in the addition of a single amino acid in the WRKY domain; thereby impairing its DNA-binding activity. Our data suggest that SLH1 disease resistance signaling may be negatively regulated by its WRKY domain in the R protein and that the constitutive defense activation conferred by the slh1 mutation is inhibited by conditions of high humidity.     

青枯菌诱导的花生基因表达谱SSH分析

以抗青枯病花生种质‘J4’和‘中花6号’、感青枯病花生品种‘中花12号’为材料,用强产青枯菌毒菌株(Ralstonia solanacearum)对其根系分别接种,采用抑制差减杂交(SSH)技术检测花生根系应答侵染的基因表达谱变化,并对文库中差异基因进行Real-time PCR分析。结果表明:经菌液PCR检测对挑选出的1 036阳性克隆片段进行测序及片段整合分析,获得162条花生基因,有功能注释的基因58条,其中44条基因参与了细胞结构(6%)、信号转导(12%)、抗病防御(5%)、转录调控(12%)等生理过程。用Real-time PCR技术对7个基因在‘中花6号’和‘中花12号’中的表达模式分析结果表明,6个基因在青枯菌侵染早期在抗病材料‘中花6号’中呈上调表达,可能与青枯病抗性直接相关。     

Prediction of protein-protein interactions between Ralstonia solanacearum and Arabidopsis thaliana

Ralstonia solanacearum is a devastating bacterial pathogen that has an unusually wide host range. R. solanacearum, together with Arabidopsis thaliana, has become a model system for studying the molecular basis of plant-pathogen interactions. Protein-protein interactions (PPIs) play a critical role in the infection process, and some PPIs can initiate a plant defense response. However, experimental investigations have rarely addressed such PPIs. Using two computational methods, the interolog and the domain-based methods, we predicted 3,074 potential PPIs between 119 R. solanacearum and 1,442 A. thaliana proteins. Interestingly, we found that the potential pathogen-targeted proteins are more important in the A. thaliana PPI network. To facilitate further studies, all predicted PPI data were compiled into a database server called PPIRA (http://protein.cau.edu.cn/ppira/). We hope that our work will provide new insights for future research addressing the pathogenesis of R. solanacearum.     

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum.

Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum.     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.