稻瘟菌蛋白激发子基因pemG1植物表达载体的构建及其转化烟草的研究

设计含MluⅠ酶切位点的接头,将其连接入经KpnⅠ和BamHⅠ酶切后的质粒pCAMBIA2300-U-bi-Ocs,得到pCAMBIA2300-Ubi-adaptor-Ocs。设计含有KpnⅠ和MluⅠ酶切位点的引物,以稻瘟菌基因组DNA为模板扩增得到pemG1序列。将其连接入pMD18-T得到pMD18-T-pemG1。最后,用KpnⅠ和MluⅠ从pMD18-T-pemG1切下pemG1基因片段,将其连接入pCAMBIA2300-Ubi-adaptor-Ocs的KpnⅠ-MluⅠ酶切位点,构建成稻瘟菌蛋白激发子基因pemG1的植物表达载体体pCAMBIA2300-Ubi-pemG1-Ocs。用冻融法将pCAMBIA2300-Ubi-pemG1-Ocs导入根癌农杆菌菌株AGL-1,再通过根癌农杆菌介导转化获得了转基因烟草株系。用PCR,Northern和Western杂交验证了pemG1基因在受体烟草基因组中的整合,转录和表达。此研究为下一步通过稻瘟菌蛋白激发子基因pemG1的表达来提高转基因烟草的抗病性打下了基础。     

Diversity and Complexity of Erysiphe graminis f.sp. hordei Collected from Barley Cultivar Mixtures or Barley Plots Treated with a Resistance Elicitor

Powdery mildew populations were analysed to determine the effects of a resistance elicitor and cultivar mixtures on genetic complexity and diversity. Isolations were made from a range of spring barley monocultures and mixtures in a field trial, and characterised for virulence and RAPD profile. In a second trial, isolates were taken from a single mixture from untreated and resistance elicitor-treated areas and from the components of the mixture in monoculture. The mildew population was not only highly heterogeneous for virulence characteristics, but also proved heterogeneous within pathotypes for molecular markers, indicating the major impact of sexual recombination on population structure and the lack of clonal dominance. Various diversity measurements were compared and the value of dissimilarity measurement for revealing genetic distance within a population was highlighted. There was a trend towards increasing complexity as the season progressed, but there was no consistent relationship between cultivar or mixture, disease control treatment, fertiliser treatment, replicate or position in trial, and pathogen genotype. Whilst the resistance elicitor did reduce mildew by 78% in the first trial, and there was no interaction with fertiliser level in its expression, control was substantially less in the second trial. There were no differences between mildew isolates from elicitor and control treatments. It was felt that more effective and consistent resistance elicitors need to be developed before it can be stated that they are unlikely to be eroded by selecting resistant or adapted mildew genotypes.     

棉疫病菌90kD胞外蛋白激发子生物活性与稳定性研究

 对棉疫病菌(Phytophthora boehmeriae Saw.)90 kD胞外蛋白激发子的生物活性与稳定性进行了研究。用不同浓度的激发子处理烟叶测定其诱发过敏反应的有效浓度,结果是所测定的0.5~100 nmol/L各浓度均可诱发过敏反应,但100 pmol/L不能诱发过敏反应,表明该激发子诱导烟草产生过敏反应的最低有效浓度在100 pmol/L~0.5 nmol/L之间。该激发子可诱导不同烟草(Nicotiana tabacum L.)品种产生过敏反应,但不能诱导辣椒(Capsicum annuum L.)和茄子(Solanum melongena L.)等茄科作物及棉花(Gossypium arboreum Linn.)发生过敏反应,初步表明该激发子诱导植物发生过敏反应具有一定的专化性。以10 nmol/L激发子溶液处理烟叶后分别于第0、24、48和72 h接种烟草疫霉(Phytophthora nicotianae),结果证明该激发子可以诱导烟草产生抗性反应,其抗性以处理后立即接种烟草疫霉为最强,接种后48h防效达68%,以后随时间的延长逐渐减弱。激发子分别经p H值2~14的水溶液25℃处理30 min,或分别经4、25、60和100℃处理5 min仍能诱发过敏反应,但是经蛋白酶K处理后不能诱发过敏反应。表明该激发子对酸、碱和温度不敏感,但对蛋白酶K敏感。     

水杨酸诱导下人参培养物差异表达基因片段的克隆

本研究以人参愈伤悬浮细胞为材料,在其生长的第28天添加1×10^-3mg／L水杨酸,测定水杨酸添加后,过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶等3种酶在72h内的变化及皂苷含量,结果表明：水杨酸添加后对过氧化物酶和苯丙氨酸解氨酶的活力影响最大,分别在24h和48h达到最大峰值,在18h开始影响多酚氧化酶的活力,培养物生长的第28天添加水杨酸可以明显提高人参愈伤组织中皂苷的合成。确定添加水杨酸后24h提取总RNA,进行cDNA-RDA分析,筛选差异片段。确定差异基因并在GenBank中注册,注册号为FE900130。为探讨水杨酸作为诱导子对人参次生代谢的影响奠定基础。     

转基因水稻中β-1,3-葡聚糖酶基因启动子缺失体P3的激发子诱导活性

 选择合适的启动子是植物抗病基因工程的关键性因素,病原菌诱导型启动子的获得将为植物提供更多的启动子选择。将大麦β-1,3-葡聚糖酶同工酶GⅢ基因启动子的缺失体片段P3与报告基因gus (β-葡聚糖酸醛苷酶基因)偶联,构建植物表达载体,通过农杆菌介导法转化水稻。PCR结果表明,所获得的10株潮霉素抗性水稻植株均呈PCR阳性;DNA印迹法结果显示,9株含P3/gus的融合基因已整合到水稻基因组DNA中。GUS组织化学染色及荧光法结果显示,P3缺失体驱动的gus在激发子诱导后,获得了高水平表达。T₁代种子的GUS组织化学染色结果也表明,激发子可以诱导高水平的P3活性。     

稻瘟病菌培养液活性物质CFS对玉米抗病性的诱导作用

液体培养的稻瘟病菌菌丝经过滤，减压浓缩，乙醇沉淀，透析，离心，获得滤液活性物质CFS。以CFS为激发子，研究其对玉米抗病性的诱导作用。结果表明，CFS能够诱导玉米对大斑病菌和弯孢叶斑病菌产生抗病性。CFS的浓度与诱抗效果呈显著正相关关系，在0．01到0．5μg／μl葡萄糖当量的浓度范围内，随着激发子浓度的增加，其诱导的抗病性增强；当CFS浓度高于0．5μg／μl，其诱导的抗病性不再增强。CFS的最高诱抗效果（50％左右）出现在处理后的2～3d，之后逐渐下降，到10d降至20％左右。     

Induced distal defence potentiation against Phytophthora sojae in soybean

Soybean phenylpropanoid defence responses to the wall glucan elicitor (WGE) fromPhytophthora sojae include the accumulation of phenolic polymers and glyceollin in cells immediately proximal to the point of treatment and the accumulation of conjugates of the isoflavones, daidzein and genistein, in distal cells. Since daidzein is a glyceollin precursor and genistein is both toxic to P. sojae and implicated in local potentiation of competency for the glyceollin response, it has been hypothesized that the distal cell response might raise the defence potential of cells distant from the infection court. In this paper, it is demonstrated that the WGE is indeed highly effective in protecting cells distal to the point of treatment from infection by P. sojae. Mycolaminaran, jasmonic acid (JA), methyl jasmonate and the ethylene precursor, 1-amino-cyclopropane carboxylic acid (ACC), are also effective, while salicylic acid (SA) is not. Methyl jasmonate, WGE and mycolaminaran are most effective, resulting in nearly complete protection against the pathogen even in the universally susceptible line, Williams. Dose response data revealed two distinct types of protection shared by all treatments. The first contains the pathogen to the point of inoculation, while the second is lesion limiting. Lesion limiting protection correlates very well with pre-infectional levels of genistein conjugates and accumulation of a specific peroxidase isozyme, P 2-4, and with post-infectional accumulation of glyceollin, suggesting that this type of protection may be the result of a distal potentiation of glyceollin elicitation competency. The mechanism underlying local containment protection is unknown, but it does not correlate well with any of the established phenylpropanoid responses.     

Some Responses of Two Nicotiana tabacum L. Cultivars Exposed to Vanadium

The effect of exposure to different vanadium (V) concentrations was studied in Nicotiana tabacum L. (cv. ‘Burley’ and cv. ‘Virginia’). In both cultivars, the vanadium did not affect the percentage of germination but root and shoot growth was inhibited, especially with the 80 μM dose. An altered root morphogenesis in vanadium growth plants was observed at the end of the experiment. The highest V accumulation was found in roots and the results showed a direct correlation between V content and V supply (P?≤?0.01). The V concentrations did not modify nicotine content in the cv. ‘Burley’, but in the cv. ‘Virginia’ the highest nicotine content was found in vanadium exposed plants (P?≤?0.05). The results suggest that these V concentrations may have a possible elicitor effect on alkaloid synthesis in N. tabacum L. cv. ‘Virginia’, and that this cultivar has a higher V sensitivity than the cv. ‘Burley’.     

7种杀菌剂和5种植物抗病激活剂对油茶炭疽病病菌活性研究

胶孢炭疽菌引起的油茶炭疽病是油茶的主要病害之一,试验通过室内毒力测定及活体测定,分别筛选出防治油茶炭疽病的高效杀菌剂和植物抗病激活剂。采用菌丝抑制法测定杀菌剂及植物抗病激活剂对胶孢炭疽菌的毒力;采用离体叶片法,通过病斑减小率评价植物抗病激活剂对油茶炭疽病的诱抗效果。供试7种杀菌剂中以咪鲜胺抑菌活性最强,EC₅₀为0.129 mg/L;嘧菌酯抑菌活性最低,EC₅₀为52.110 mg/L;植物抗病激活剂对菌丝生长无直接抑制作用,但在2年生的油茶苗上喷施后,对病斑在叶片上的扩展均有一定抑制作用,以水杨酸100 mg/L剂量处理的叶片诱导抗病性最佳,诱抗效果为50.13%,芸苔素内酯、茉莉酸甲酯,井岗霉素,苯并噻二唑处理组的最大诱导防病效果依次为36.82%、26.93%、19.67%、17.73%。该试验结果为杀菌剂和植物抗病激活剂的复配制剂提供参考依据,为油茶炭疽病的防治提供新思路。