大麦黄矮病毒-GAV在燕麦植株体内运动规律的初步研究

 利用RT-PCR方法研究了大麦黄矮病毒-GAV在燕麦植株内的移动规律。先将介体麦二叉蚜(Schizaphis graminum)在BYDV-GAV新鲜病叶上饲毒,再将获毒蚜虫放置到二叶期的健康燕麦植株接种48h,随后分期提取接种植株的第1~6片叶和根组织的总RNA,利用特异引物扩增BYDV-GAV的外壳蛋白(CP)基因以检测病毒在燕麦植株内的复制和移动。结果表明,在接种5d后,接种叶片(第2片叶)呈现阳性,接种7d后,植株新生的第4片叶被侵染,接种9d后,部分的第3片叶呈现阳性,至接种16d,几乎所有的叶片均呈现阳性。仅在接种的第5、7和9d收集的根组织呈现阳性,而所有的第1片叶均为阴性,可能是由于这些组织内病毒含量太低所致。本研究初步揭示了BYDV-GAV长距离运动的规律并且发现该病毒在燕麦根部从接种到系统发病都没有进行大量增殖,为今后进一步研究病毒运动机制选取适当的植物材料提供了基本信息。     

不同交配型球孢白僵菌在玉米植株中的定殖及其交配型基因变异比较研究

本文通过分析两种不同交配型球孢白僵菌菌株(MAT1-1-1,I型;MAT1-2-1,II型)在玉米植株中的定殖效率和回收菌株交配型基因序列的变异,拟揭示球孢白僵菌在玉米植株内生定殖过程中玉米对不同交配型菌株的选择规律、不同交配型菌株对环境的适应性能力。试验分为4个接种组,接种菌株为I型交配型菌株(Bb OFDH1-5)(处理B5)、II型交配型菌株(Bb DPSD2)(处理B2)、两个菌株等量混合(处理B5+B2)、对照组(处理CK),处理组B5、B2、B5+B2接种孢子浓度均为108孢子/m L,对照组接种量为零。结果表明,处理B5、B2、B5+B2三组中均有球孢白僵菌在玉米叶片中定殖,其中,交配型II菌株处理组B2中内生定殖效率高,检测率为33.00%;混合处理组B5+B2中交配型II菌株比I型菌株回收比率高,回收比为12:10。交配型基因序列分析显示,交配型I菌株处理组B5和混合处理组B5+B2的回收菌株中I型交配型基因非编码区序列出现多个位点突变,编码区仅在3个位点出现突变;交配型II菌株处理组B2和混合处理组B5+B2中II型交配型基因序列均没有发生变异。以上结论说明球孢白僵菌在玉米植株定殖过程中,交配型II菌株Bb DPSD2定殖率较I型菌株Bb OFDH1-5高;玉米植株更倾向于选择II型交配型菌株作为内生菌株;交配型基因MAT1-1-1比MAT1-2-1更易发生突变表明I型菌株对自然环境的适应性更强。     

南方根结线虫侵染下罗汉果WRKY转录因子的鉴定分析

为了确定南方根结线虫侵染早期罗汉果植株中与侵染相关的WRKY基因,以接种南方根结线虫7 d后的罗汉果侧根为材料进行转录组测序分析。基于测序序列的Nr、SwissProt注释结果,共鉴定到21个WRKY基因,其中有4个转录因子含有2个WRKY结构域,其余17个转录因子只有1个WRKY结构域。系统发育分析表明,5个WRKY转录因子为第一家族成员,2个为第三家族成员,其余14个为第二家族成员。有8个WRKY转录因子与已报道的根结线虫诱导相关WRKY转录因子同源性较高,初步推测下调表达的SgWRKY2、SgWRKY3和SgWRKY17可能与南方根结线虫的早期侵染有关,它们很可能参与了线虫取食位点与巨细胞形成的复杂调控作用。本研究将为今后阐明根结线虫与罗汉果互作的分子机制提供参考依据。     

转二价核酶基因马铃薯及抗病性研究

 用克隆的特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)复制酶基因负链RNA的二价核酶基因,构建植物表达载体pROKⅡ/DR,经土壤农杆菌(Agrobacterium tumefaciens)介导叶盘法转化马铃薯外植体,获得再生植株。PCR和Southern-blot检测,证明目的基因已成功地导入马铃薯再生植株,其转化率约为14.5%,并能够在无性繁殖后代植株中稳定存在。RT-PCR检测表明,再生马铃薯植株中的二价核酶基因可以转录表达。经病毒接种的转基因马铃薯株系L5、L7、L8和J-1的无性繁殖后代在继发感染中仍表现出较高的抗病性,为最终获得抗PLRV马铃薯新品系打下了基础。     

瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染

 RNA干扰(RNA interference,RNAi)是与内源性mRNA编码区某段序列同源的双链RNA导入细胞后,该mRNA发生特异性降解,从而导致该基因表达沉默的现象。小干扰RNA(small interfering RNA,siRNA)作为RNAi途径的重要中介,已被广泛应用于动、植物抗病毒治疗研究。本文以烟草花叶病毒(Tobacco mosaic virus,TMV)外壳蛋白基因为靶位,设计合成表达小干扰RNA的寡核苷酸,亚克隆到植物双元表达载体pBI121中,直接转化根癌农杆菌。通过根癌农杆菌介导的瞬时表达法,研究了同源于TMV外壳蛋白的siRNA对TMV侵染的干扰作用。结果表明,瞬时表达的siRNA能够特异性干扰TMV侵染。含有重组表达载体pBI121/siRNA的根癌农杆菌渗入普通烟植株,在TMV接种后14d其上部叶片没有表现典型的花叶症状。对这些叶片进行Northern杂交试验也没有检测到TMV病毒的RNA积累或仅有很少量的积累。在枯斑寄主心叶烟上,siRNA的瞬时表达可使TMV侵染后的枯斑数明显减少,甚至不产生枯斑。此外,同源于TMV外壳蛋白的siRNA瞬时表达对非同源的黄瓜花叶病毒(Cucumber mosaic virus,CMV)没有抑制作用,表明siRNA的干扰作用具有高度的同源依赖性。     

橡胶树白粉菌侵染过程转录组学分析

为明确橡胶树白粉菌Erysiphe quercicola参与致病过程相关基因的表达情况，基于RNA-Seq测序技术对橡胶树白粉菌侵染过程进行转录调控研究，通过对病原菌孢子（0 h）及3个侵染时期（接种1、3和30 d）的转录组进行比较，筛选差异表达基因并对其进行功能注释分析，同时对不同侵染阶段的基因表达趋势进行聚类分析。结果表明，相比于病原菌孢子，3个侵染时期（接种后1、3和30 d）分别有198、458和27个差异表达基因。基因功能富集分析发现氧化还原酶相关基因在侵染1 d阶段显著富集，可能参与病原菌侵染前期对活性氧的防御。基因表达趋势聚类分析显示不同侵染阶段的基因共分为51种表达类型，其中编码候选效应蛋白基因集中分布在侵染1 d后上调表达的6个类型当中。表明橡胶树白粉菌侵染过程相关基因具有明显的功能倾向性和表达趋势特征。     

系统侵染的番茄植株中黄瓜花叶病毒的时序变化

 采用实时荧光定量PCR (FQ-PCR)和DAS-ELISA方法,研究了22~26℃温室条件下番茄幼苗中黄瓜花叶病毒CNA株系(CMV-CNA)各基因组RNA组分及其外壳蛋白(CP)含量的动态变化,同时结合同期感病植株症状发展和病情指数,分析并探讨CMV各基因组RNA、CP以及病症显示程度之间的时间效应及其相关性。以18S rRNA为内参照,FQ-PCR相对定量分析结果显示:接种后5~30 d,CMV三分体基因组RNA在系统侵染的番茄组织中负荷量变化趋势大体一致,但是不同时期含量差异显著,均经历对数增长期、稳定期和回落期。其中,以RNA2负荷量变化情况最为平缓。DAS-ELISA检测结果显示:CP含量随接种时间延长而持续升高,但其对数增长趋势相对滞后于基因组RNA。番茄幼苗发病症状与CMV基因组RNA及CP负荷量的变化趋势大体一致,但症状表现时间相对滞后。CMV-CNA株系在番茄幼苗中以基因组RNA、CP以及病症显示先后次序出现高峰期,显示病毒基因组RNA及其CP在植物组织内负荷量的变化与植株症状表现并不同步。其动态变化规律将为研究CMV侵染机制,病毒与寄主互作及防病控病提供量化依据。     

低温对黄瓜花叶病毒CMV-BG株系2b基因多态性的影响

 本文将黄瓜花叶病毒CMV-BG株系侵染普通烟草的不同单株后,放在不同温度下培养,30d后对接种植株症状进行对比观察,并对2b基因表达进行了检测及其序列测定分析。结果表明:被CMV-BG株系侵染的植株在低温条件下,植株新生的大部分叶片严重畸形,而CMV-BG株系2b基因的表达与不同温度条件没有相关性;CMV2b基因序列与源分离物CMV-BG序列相比发生了较明显的变异,它们作为一组序列与分离物SF/GF/SY等的相似性甚至高于源BG序列;此外,尽管来自2组植物中的病毒序列变化均未表现正选择(positive selection),但来自低温培养的侵染植株的2b基因的多态性明显高于常温条件培养的植株,发生非同义突变的位点数目也高于同义突变的位点数目。因此认为,低温有助于加强CMV-BG株系2b基因的多态性。     

黄瓜花叶病毒RNA1侵染性cDNA克隆构建及病毒致病因子的分析

侵染性克隆是研究植物病毒的基础。黄瓜花叶病毒是目前最重要的植物病毒之一。本文以黄瓜花叶病毒M株系为材料来构建病毒的侵染性克隆。由于RNA1的c DNA对多数克隆菌株有毒性,因而采取了分段克隆的方法来构建RNA1的侵染性c DNA克隆,并进一步通过假重组技术来研究病毒的致病因子。结果表明,采用分段克隆的方法成功获得了CMV-M RNA1的侵染性克隆;M株系侵染烟草引起的黄白化症状与病毒的RNA3有直接相关性。     

Photosynthesis and sugar concentration are impaired by the defective active silicon uptake in rice plants infected with Bipolaris oryzae

The effect of soluble silicon (Si) on photosynthetic parameters and soluble sugar concentrations was determined in leaves of rice cv. Oochikara and mutant plants of Oochikara defective in active Si uptake [low silicon 1 (lsi1)]. Plants were grown in hydroponic culture amended with 0 (?Si) or 2 mm Si (+Si), under either low or high photon flux density (PFD) and with or without inoculation with Bipolaris oryzae, the causal agent of brown spot of rice. Leaf Si concentration increased by 141 and 435% in +Si cv. Oochikara and by 119 and 251% in +Si lsi1 mutant plants under high and low PFD, respectively, compared with ?Si plants. Plant biomass accumulation was improved by Si regardless of PFD, especially plants for cv. Oochikara. Brown spot severity was highest in ?Si plants for cv. Oochikara and lsi1 mutant plants under low PFD. In the presence of Si, disease severity in plants grown under both low and high PFD was reduced, except for lsi1 mutant plants under high PFD. Plant inoculation reduced the photosynthetic parameters measured regardless of plant material or Si supply. A decrease of net carbon assimilation rate (A) of inoculated plants under low PFD compared with non‐inoculated plants was associated with damage in the photosynthetic apparatus, except for +Si cv. Oochikara in which stomatal restriction [low water vapour conductance (g_s)] contributed to A reduction. Under high PFD, damage to the photosynthetic apparatus of inoculated plants was the main reason for the reduction in A for +Si and ?Si lsi1 mutant plants. In addition, for ?Si cv. Oochikara, a reduction in g_s contributed to reduced A. However, for +Si cv. Oochikara, g_s was the limiting factor for A. Inoculated plants of +Si cv. Oochikara had higher A values than +Si lsi1 mutant plants, regardless of environmental conditions. Soluble sugars were not detected in leaf tissues of plants under low PFD. For high PFD, Si improved the hexose concentration in non‐inoculated plants at 144 h after inoculation (hai) for lsi1 mutant plants and from 96 hai onwards for cv. Oochikara compared with ?Si plants. However, plant inoculation reduced hexose concentration compared with non‐inoculated plants, mainly in +Si plants, regardless of plant material. Sucrose concentration increased in leaves of cv. Oochikara in the presence of Si whether inoculated or not. For +Si lsi1 mutant plants, sucrose concentration increased only at 48 hai compared with ?Si plants, whether inoculated or not. The results of this study show that a minimum Si concentration is needed in leaf tissues of rice plants to avoid the negative impact of B. oryzae infection on photosynthesis and sugar concentration. High leaf Si concentration resulted in an increased soluble sugar concentration and together, but in independent ways, soluble sugar and Si reduced brown spot severity of rice.     

Hypersensitive reaction in alfalfa (Medicago sativa) induced by alfalfa mosaic virus

Alfalfa (Medicago sativa) cv. Beaver was screened for resistance to alfalfa mosaic virus (AIMV). A1MV infection in clonal plants was tested by double-antibody sandwich ELISA 4 and 8 weeks after inoculation with purified A1MV (1 mg/ml in 0025 M phosphate buffer, pH 7 0). Twelve clones showed a hypersensitive reaction 3–4 days after inoculation and the infection was restricted to the inoculated leaves. All the plants of hypersensitive clones consistently produced local lesions when inoculated with purified AIM V. In contrast, plants inoculated with AIMV in crude sap remained symptomless, although AIMV was detectable in inoculated leaves. The remaining 16 clones were susceptible to AIMV and showed systemic infection.     

Phenylalanine ammonia lyase activity in chilli CM-334 infected by Phytophthora capsici and Nacobbus aberrans

We tested the hypothesis that PAL activity in chilli plants CM-334 inoculated with Nacobbus aberrans (Na) alone or in combination with Phytophthora capsici (Pc), is lower than in those inoculated only with Pc. At 21 days after nematode inoculation, inoculated plants showed a significant (P < 0.01) reduction of 48% in PAL activity compared to those non-inoculated in two separate experiments. In two other tests, where plants were inoculated with the oomycete 21 days after inoculation with the nematode, PAL activity at 2, 4, 6, 8 and 24 h after inoculation with Pc was significantly higher (Tukey, P < 0.01) in plants inoculated only with Pc than in plants inoculated only with Na or both pathogens (Na+Pc).     

Rhizoctonia solani Kühn AG 2-1 on kohlrabi in Italy

Rhizoctonia solani AG 2-1 was recorded in Central Italy on kohlrabi plants showing root and stem rot. After artificial inoculation the fungus caused damping-off of 7-day-old seedlings and root and stem rot of 4-month-old plants developed after 15 days of incubation. This seems to be the first record ofR. solani AG 2-1 on kohlrabi.     

转Pti5-VP16基因小麦抗白粉病鉴定及可溶性蛋白分析

对PCR呈阳性的转基因小麦植株(T₀)及T₁代植株接种小麦白粉病菌,鉴定结果表明:T₀代接种7 d,病害表型多数为抗病;接种14 d,病害表型为抗病、中抗及中感,病害严重度明显轻于对照。T₁代接种7 d后,多数表现为中抗、抗,少数为中感;接种28 d后,仅有2株仍表现抗病,其余表现为感病,但病害严重度轻于对照。对T₁代植株可溶性蛋白分析表明T₁代植株与对照相比多一条新谱带。