水稻白叶枯病菌致病相关基因筛选系统的建立

水稻白叶枯病菌Xanthomonas oryzae pv.oryzae是水稻生产中最严重的细菌性病害之一。本研究采用Tn5转座子随机插入突变的方法构建水稻白叶枯病菌广西菌株K74的突变体库,Southern杂交显示Tn5随机单拷贝插入基因组。通过水稻致病性检测试验,目前获得15个致病力降低50%以上的突变体,为定位Tn5插入突变基因,经质粒拯救、测序、序列分析后发现:4个突变基因为gum基因簇基因,5个为LPS基因簇基因,2个为metB基因,1个为hrpB1基因,2个是与糖合成转移酶相关基因,1个为编码假定蛋白的基因。其中14个突变基因是已知的与致病相关的基因。实验结果表明本研究成功建立了一个筛选水稻白叶枯病菌致病相关基因的系统,为进一步研究水稻白叶枯病致病相关基因,阐明该病的致病机理奠定基础。     

水稻白叶枯病菌1个毒性相关基因的鉴定

我们曾报道了来自水稻白叶枯病菌真基文库的重级质粒ｐＧＸＮ３０００中含有一个能互补甘蓝黑腐病菌ｒｐｆＧ致病因子调控基因突变全的基因，并通过转座子诱变获得子转座子插在ｐＧＸＮ３０００中的这一基因的转座子插重组质粒，本研究利用这些转座子插入重组质粒，通过标记置换方法构建了不稻白叶枯病菌这一基因的突变菌株，植株试验结果表明，突变株虽然在水稻中仍能正常生长繁殖，但毒性严重降低，说明水稻白叶枯病菌的这一基因在     

水稻对白叶枯病菌抗性与活性氧关系的研究

以 3种水稻白叶枯病菌和 4个抗、感水稻品种为样品 ,研究了水稻对白叶枯病菌抗性与白叶枯病菌细胞保护酶以及氧自由基的关系。研究结果表明 :接种 15d的水稻SOD、POD显示活性 ,而CAT无活性 ;用水稻叶片酶解液培养 3种病原菌 ,菌体显示SOD和CAT活性 ,但未测出POD活性。抗、感水稻对白叶枯病菌的影响不同 ,感病水稻使病原菌SOD和CAT活性升高 ,抗病水稻使白叶枯病菌SOD、CAT活性降低。而抗、感水稻对白叶枯病菌氧自由基的影响 ,正好与对酶的影响相反。     

稻瘟病抗性基因Pita2的克隆及其介导的防御相关基因表达分析

为了克隆位于水稻12号染色体着丝粒区域具有广谱抗性的稻瘟病抗性基因Pita2,研究构建并筛选了该基因供体品种PiNo.4的Fosmid文库,构建Pita2候选区域的物理图谱并互补验证了5个候选基因,克隆得到了稻瘟病抗性基因Pita2,确认了该基因就是Ptr基因.一组基因型接近、对Pita2致病性相反的稻瘟病菌菌株对水稻...     

水稻白叶枯病拮抗菌的筛选、鉴定及发酵液稳定性分析

从稻田土壤、水稻植株、昆虫肠道等环境中分离纯化得到88株菌株,以水稻白叶枯病菌生理小种P6菌株为指示菌,通过琼脂块法和牛津杯法从中筛选得到了对水稻白叶枯病菌具有明显拮抗作用的12号菌株,其抑菌圈直径为6.09 cm.根据12号拮抗菌株菌落和菌丝形态特征、生理生化特征及16S rDNA序列分析结果,鉴定该菌株为Streptomyces gramineus.该拮抗菌发酵液具有较强的热稳定性、酸碱稳定性和光照稳定性,将其作为生物农药具有一定的应用价值和开发前景.     

水稻白叶枯病菌hrp调节基因hrpG的鉴定及其变异分析

根据水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)hrp基因的序列设计引物,通过PCR方法从水稻白叶枯病菌JXOⅢ和PXO99A菌株中扩增得到核苷酸序列完全一致的hrpG基因.以该基因为探针与JXOⅢ/pUFR034基因组文库中含有hrpX克隆pUHRX245(36.8 kb)的4个亚克隆进行southern blot分子杂交,确定在亚克隆pB1中1.3 kb大小的片段和AE4(3.0 kb)片段中存在hrpG同源序列.测序结果表明,在pB1中有586个碱基序列,在AE4中有206个碱基,共同构成完整的hrpG基因,且与已知的hrpX基因是毗邻的.以同样方法从水稻白叶枯病菌PXO99A菌株的hrp-突变体M16中扩增得到hrpG基因对应位置的同源突变序列.序列分析表明其有意义突变为541位的C→T的突变,从而导致亮氨酸变为苯丙氨酸(Leu→Phe).将hrpG基因和突变序列分别导入突变体M16中,转移结合子M16/hrpG(PXO99A)恢复了其在烟草上的过敏反应和在水稻上的致病性,而突变序列的结合子M16/hrpG(M16)则不能使其恢复功能,从而确定M16是由于单个碱基的突变所引起的功能突变.经过对基因及其产物的比对发现,对于第Ⅱ组hrp调节基因hrpG的变异,种间变化明显高于种内不同致病变种的变化.     

孝顺竹内生菌BME17对水稻白叶枯和条斑病生防效果的初步研究

2017年5月从校园内健康孝顺竹叶分离纯化得到1株具有生防潜力的细菌BME17菌株.为研究BME17菌株的生防潜力,对水稻白叶枯病菌PXO99和细菌性条斑病菌RS105的平板抑菌活性以及水稻盆栽的防治效果,以期为水稻白叶枯病和细菌性条斑病的生物防治提供材料.通过形态观察、理化特性和16S rDNA基因测序对其进行鉴定.平板对峙法检测其对PXO99和RS105的拮抗作用,并对该菌的生防效果进行盆栽试验.结果表明,菌株BME17为枯草芽孢杆菌Bacillus subtilis;平板上,内生菌BME17对PXO99的抑菌圈直径(D)/菌落直径(d)为2.18;而对RS105的抑菌效力比值为1.87.盆栽防治试验,内生菌BME17对PXO99引起的水稻白叶枯防治效果为43.06%;而对RS105引起的水稻细菌性条斑病的防治效果为24.32%.内生细菌BME17对水稻细菌病害表现出一定的生防潜力.     

抗虫基因在转基因植株中聚合的初步研究

以质粒 pBUSCK HPT ,pCUSBK HPT和pGNA HPT作抗虫基因供体 ,优良籼型三系恢复系SH5 2 7和广亲合系W 2 0 0 8为受体 ,采用基因枪转化法 ,获得了转修饰的豇豆胰蛋白酶抑制剂基因 (sck)、修饰的Bt毒蛋白基因 (sbk)和雪花莲凝集素基因 (gna)的植株 .通过人工杂交 ,将外源基因聚合 .分子证据显示 ,作者获得了抗性基因聚合的材料 .蛋白活性测定表明 ,聚合基因的表达因载体结构相似而受到影响 ,但抗虫性却得到提高 .作者还讨论了聚合转基因在水稻育种中的作用和及其相关问题 .     

水稻-白叶枯病菌互作中过氧化氢酶的研究

以3种水稻白叶枯病菌和3个抗、感水稻为样品,研究了水稻-白叶枯病菌互作中过氧化氢酶的变化.研究结果表明:接种15d的水稻CAT无活性,CAT同工酶电泳也未显示酶带,对照组CAT则显示活性;在NB培养基中生长的白叶枯病菌显示CAT活性;用水稻酶解液培养了3种病原菌,菌体显示CAT活性,抗、感水稻对白叶枯病菌的影响不同,抗病水稻使病原菌CAT活性降低,感病水稻使CAT活性升高.     

拮抗水稻白叶枯病菌菌核曲霉As-68菌株培养基配方和发酵条件的响应面优化

为提高菌核曲霉(Aspergillus sclerotiorum)As-68菌株发酵液对水稻白叶枯病菌的抑菌活性,用响应面法优化培养基配方和发酵条件.通过单因素实验,筛选出适宜的碳源、氮源及其浓度和适宜发酵条件,利用Plackett-Burman设计,探究碳源浓度、氮源浓度、菌龄和转速对抑制水稻白叶枯病菌影响效果,采用...     

Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker

An F₂ population developed from theXa-4 near isogenic lines, IR24 and IRBB4, was used for fine mapping of the rice bacterial blight resistance gene,Xa-4. Some restriction fragment length polymorphism (RFLP) markers on the high-density map constructed by Harushima et al. and the amplified DNA fragments homologous to the conserved domains of plant disease resistance (R) genes were used to construct the genetic linkage map around the geneXa-4 by scoring susceptible individuals in the population.Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction (PCR) marker M55. The R gene homologous fragment marker RS13 was found co-segregating withXa-4 by analyzing all the plants in the population. This result opened an approach to map-based cloning of this gene, and marker RS13 can be applied to molecular marker-assisted selection ofXa-4 in rice breeding programs.     

Characterization and molecular marker screening of a rice bacteria-resistant gene Xa-min(t)

To test the resistant spectrum of the Xa-min(t) gene introgressed from Oryza minuta, thirty-four isolates of different bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), from 11 countries were used to inoculate the Xa-min(t) introgression line 78-15. Four rice cultivars, IR24, C64 (IRBB21), Nipponbare and Zhonghua 11 were used as controls. The results showed that the Xa-min(t) gene was broad-spectrum and highly resistant to diverse Xoo isolates. The methods of bulk segregant analysis (BSA), randomly amplified polymorphic DNA (RAPD) and sequence characterized amplified regions (SCAR) were used to analyze F2 individuals of the hybrid IR24×78-15 and molecular genetic markers linked to Xa-min(t) gene were identified. A total of 800 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Two RAPD markers, BE05300 and BE061400, produced by primers BE05 and BE06 respectively, were closely linked to the Xa-min(t) gene. Based on the sequences of these two markers, sequence specific primers were designed and used to screen all F2 plants. One RAPD marker, BE05300, was converted into a stable SCAR marker (ScBE05300). Linkage analysis was carried out using markers ScBE05300 and BE061400 on 948 and 719 F2 individuals of the hybrid IR24×78-15. Our results indicate that the genetic distances from Xa-min(t) to ScBE05300 and BE061400 are 2.2 cM and 3.7 cM respectively on the same side. This study may facilitate the construction of the fine physical map of the Xa-min(t) gene.     

4个小麦品种的抗白粉病遗传分析

抗病品种郑315、CP87-26-12和91138-16-2-13-12,与感病品种豫麦49号杂交的F1代表现抗病,F2代抗、感单株的分离比例为31,表明这3个品种均携带1对显性抗病基因.N97189-2和豫麦18号的杂交F1代对白粉病表现高感,F2代抗、感单株的分离比例为13,由此推断N97189-2对白粉病的抗性由1对隐性基因控制.     

不同MHC-B单倍型SPF鸡对禽白血病的抗性研究

目的探索不同MHC-B单倍型SPF鸡对禽白血病(Avian leukosis,AL)的抗性差异,为建立AL抗性和易感鸡系提供实验依据。方法选择纯合G1、G5、G6鸡系及杂合G8鸡系雏鸡进行人工感染白血病病毒A亚群(Avian leukosis virus,ALV-A)RAV-1毒株实验,G8系雏鸡(6羽)作为对照。攻毒后对各鸡系的死亡率、病变率、血液中抗体水平以及外周血淋巴细胞的病毒载量等与抗性相关指标进行动态评估与统计学分析,最终确定抗性较强和较弱的鸡系。结果 G5系病变率和死亡率均明显高于其它攻毒鸡系,G1系最低,G6和G8系居中;G1系在攻毒后第5周开始,抗体效价一直高于其它鸡系,而G5系则最低,并且在攻毒后第8周至11周和第15周至18周,G1系的抗体效价显著高于G5系(P0.05);G1系的病毒载量在攻毒后第7周至15周,低于其它鸡系,而G5系自攻毒后第9周至13周,极显著高于G1系(P0.01)。结论 G1系在感染ALV后,死亡率较低,病变轻微,抗体保护力较强,且病毒的增殖较慢,因而其对AL具有较强的抗性,而G5系则相反较为易感。本研究不仅为建立AL抗性较强和较易感的鸡系提供了实验依据,而且对进一步阐明鸡主要组织相容性复合体(Major histocompatablity complex,MHC)多态性与AL抗病性的关系有重要作用。     

转多个抗真菌蛋白基因水稻植株的获得及其抗稻瘟病菌的初步研究

用基因枪法将水稻碱性几丁质酶（ＲＣ２４）基因、苜蓿葡聚糖酶（βＧｌｕ）基因、大麦核糖体失活蛋白（ＢＲＩＰ）基因和潮霉素（ｈｐｔ）基因同时导入籼稻品种（七丝软占）中,获得了７个潮霉素抗性再生系,Ｓｏｕｔｈｅｒｎｂｌｏｔ证明２～３个抗真菌蛋白基因已整合到水稻基因组中．初步抗性鉴定表明Ｒ０代转基因水稻植株对稻瘟病菌的抗性有所提高     

水稻白叶枯病菌无毒新基因的克隆和序列分析

从菲律宾水稻白叶枯病菌9a生理小种的代表菌株PX0339中克隆到一个新的无毒基因,全长2118bp,编码705个氨基酸,分子质量和等电点分别约为74．7ku和6．585．NCBI网站BLAST表明,该基因属于avrBs3基因家族,命名为arp3．与所有avrBs3同源基因的Alignment分析发现,arp3的5’端缺少了两个长度分别是144bp和45bp的片断,但在水稻白叶枯病菌中,arp3基因的5’端的特征很普遍．全长的arp3基因中间区域是一个特异的只有5．5次102bp的重复区,是已克隆的avrBs3基因家族中重复次数最少的一个,C-端有3个核定位信号NLSs和1个酸性转录激活域AAD．     

Proteomic analysis of blast-resistant near-isogenic lines derived from japonica rice,var. Yunyin,infected with Magnaporthe oryzae

Rice blast is one of the most devastating dis- eases in the world and outbreaks occur frequently. The differences in protein expression between blast resistant and susceptible near-isogenic lines （NILs） of japonica rice var. Yunyin infected with Magnaporthe oryzae were ana- lyzed using proteomics, indicated that 67 different proteins were identified from 75 obtained proteins using the matrix- assisted laser desorption/ionization time-of-flight mass spectrometry technique. Seven specific expression proteins, 43 up-regulated proteins and 17 down-regulated proteins were identified among the 67 proteins. The bioinformatical analysis demonstrated that these 67 different proteins were involved in many biological physiological processes including five proteins related to photosynthesis, 25 pro- teins related to metabolism, six proteins related to anti- oxidants, 10 proteins related to protein synthesis and modification, five proteins related to signal transduction,four proteins related to adversity stress and 12 non-func- tional proteins. These identified proteins were directly or indirectly related to stress. Five proteins related to different physiological processes were selected. Their cDNA sequences were predicted and their expression patterns were analyzed using real-time PCR, demonstrated that the genes would response to M. oryzae and the response blindingly different between blast resistant and blast sus- ceptible NILs.