超速离心结合Real-time PCR分离纯化枣疯病植原体DNA

分离纯化枣疯病植原体基因组DNA,有助于进一步开展对此病原全基因组测序的研究,CTAB法提取枣疯病叶片的总DNA后,采用氯化铯双苯酰亚胺密度梯度离心法从枣树总DNA中富集纯化枣疯病植原体DNA,并通过Real-time PCR方法对分离纯化效果进行定量检测。在此基础上,探索了不同CsCl初始密度对DNA分离效果的影响。在20℃,初始密度为1.650 0g/cm3,经206 000×g下离心23h后,感染枣疯病样品(IS)的离心管中出现2条DNA亮带,正常枣树样品(NS)离心管中只有1条。Real-time PCR检测结果表明NS管中的条带为枣树基因组DNA;IS管中与NS管中相同位置的条带为枣树基因组DNA,另1条带为枣疯病植原体基因组DNA。在保留其他试验条件下,不同CsCl初始密度,会影响DNA条带的位置,也会影响枣疯病植原体DNA与枣树DNA的分离效果,在1.562 2g/cm3的初始浓度下分离效果最好。采用超速离心法可以有效地从感染枣疯病的枣树中分离得到纯的枣疯病植原体DNA,同时利用Real-time PCR法可以实现分离效果的评价,利用此方法分离得到的DNA可用于枣疯病植原体的全基因组测序。     

小麦蓝矮植原体RNA沉默抑制子互作蛋白的筛选与分析

【目的】揭示小麦蓝矮植原体(WBD phytoplasma)的致病机理及其与寄主的互作关系,为植原体病害防治提供理论依据。【方法】以SWP16为诱饵,将SWP16构建到诱饵载体pBT3-STE和pBT3-SUC上,在小麦酵母双杂交膜系统cDNA文库中筛选与SWP16互作的蛋白,并确定最佳的筛库条件。通过α-半乳糖苷酶活性检测试验对筛选到的互作蛋白进行二次验证,并运用Uniprot对其进行Gene Ontology（GO）注释分析。【结果】以pBT3STE-SWP16作为筛库的诱饵蛋白,确定20 mmol/L 3-AT为筛选文库的最佳浓度,从小麦cDNA文库中筛选到20种互作蛋白,包括Rieske 蛋白、细胞色素b5、CASP蛋白等。经过复筛和α-半乳糖苷酶活性检测试验进一步验证了互作关系,其中14种互作蛋白参与运输、pH调节、光合作用、内质网应激反应、蛋白质泛素化等生理过程;18种互作蛋白存在于线粒体、内质网、细胞膜、细胞质、叶绿体和细胞壁等6种细胞组分中;20种互作蛋白的分子功能主要包括几丁质酶活性、反转运蛋白活性、脱水酶活性、转移酶活性和转录因子活性等。【结论】筛选获得20种与小麦蓝矮植原体效应蛋白SWP16互作的蛋白,有助于推动小麦蓝矮植原体与寄主互作关系的研究。     

中国植原体病害的状况、分布及多样性研究进展

植原体是引起众多植物病害的一类重要原核致病菌,能引起许多重要粮食作物、蔬菜、果树、观赏植物和林木严重病害,造成巨大损失。为了深入了解中国植原体病害的状况、分布及多样性,最终实现植原体病害的科学防控,归纳总结了中国植原体病害的研究历史、经济重要性、症状特点、流行传播、鉴定方法、地域分布及多样性等。提出了今后应从抗性资源筛选、抗病基因鉴定,基因组特征、致病机理、病害流行学、防治方法和昆虫传毒机理等方面开展植原体病害研究的建议。     

植物菌原体检测技术

回顾了植物菌原体的检测从早期的电镜观察,到现在应用分子生物学技术测定的发展历史,概述了各种检测技术的基本原理和特点,以及我国在这个领域的研究现状.     

基于Maxent的椰子致死性黄化植原体在中国的适生性分析

利用Maxent生态位模型和Arc GIS软件,并结合椰子树(以下简称椰树)在中国的种植分布,对椰子致死性黄化植原体在中国及中国椰树种植区的适生性进行了预测。结果表明:中国主要的椰树种植区均为椰子致死性黄化病的潜在适生区,其中华南沿海地区及海南大部分地区属于中风险区和高风险区。经ROC曲线分析法验证,Maxent模型的AUC值为0.992,表明预测结果具有较高的可信度。     

A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group

A novel phytoplasma was detected in a cherry plum（Prunus cerasifera Ehrh） tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies（PLBs） were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 μm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16 S ribosomal RNA（rRNA） gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16 S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows（AY） group（16SrI group）. Virtual and actual restriction fragment length polymorphism（RFLP） analysis of the 16 S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group（16SrI） by four restriction sites, Hinf I, Mse I, Sau3 A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16 S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf（CPYL） symptoms is probably a distinct and novel subgroup lineage in the AY group（16SrI）. In addition, the novel phytoplasma was experimentally transmitted to periwinkle（Catharanthus roseus） plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder（Cuscuta odorata） connections.     

泡桐丛枝植原体Sec分泌蛋白转运系统3个亚基基因的克隆及蛋白特性分析

采用PCR方法扩增Sec分泌蛋白转运系统3个亚基基因,并进行了生物信息学分析。通过SWISS MODEL同源建模与3D PSSM折叠子识别法构建蛋白质的三维结构,并通过PROCHECK程序验证构建的三维结构的可靠性。首次从泡桐丛枝(PaWB)植原体基因组中分离出Sec分泌蛋白转运系统3个亚基基因,各基因全长依次为2 508、1 242 bp和411 bp,分别编码835、204个及136个氨基酸的蛋白。SecA、SecY和SecE均为脂溶性稳定蛋白,SecA无明显跨膜区,SecY和SecE分别含有10个和3个明显的疏水跨膜区。二级结构主要为螺旋,其次为折叠和无规则卷曲,没有转角。构建的三维结构符合立体化学原则。泡桐丛枝(PaWB)植原体中存在的Sec分泌蛋白转运系统作为最主要的运输途径,可能直接转运菌体蛋白如毒素等直接到寄主细胞质中或介体昆虫细胞中,引起寄主病症。研究植原体的Sec蛋白转运系统对于了解植原体的致病机理及预防这类病害的发生提供了理论依据。     

桃黄化病植原体昆虫介体的验证及其带毒部位PCR检测

 利用植原体16S rDNA通用引物对采集的北京和天津黄化病桃树总DNA进行巢式PCR检测,证明发病样本的病原为桃黄化病植原体。经过检测昆虫总DNA和经取食过的人工培养液DNA中桃黄化病植原体的16S rDNA,结果表明桃黄化病植原体的有效传播媒介昆虫为桃一点叶蝉。将带毒桃一点叶蝉个体的头部、胸部以及腹部分离,分别在这些部位检测到桃黄化病植原体的16S rDNA,说明桃一点叶蝉的头部、胸部以及腹部都可带毒,表明植原体可从植物汁液进入叶蝉的口针、食道和肠道。     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.     

A Leafhopper (Hishimonus sellatus) Transmits Phylogenetically Distant Phytoplasmas: Rhus Yellows and Hovenia Witches' Broom Phytoplasma

Phytoplasmas causing a severe decline of three tree species, i.e., Rhus javanica, Hovenia tomentella and Zizyphus jujuba, in Japan were examined for their transmissibility by a leafhopper species Hishimonus sellatus, and for their phylogenetic relatedness. By H. sellatus, Rhus yellows (RhY) phytoplasma was transmissible to white clover and periwinkle seedlings, causing typical symptoms in these plants. Jujube witches' broom (JWB) phytoplasma was also transferred to the host plant, Z. jujuba, by the leafhopper. Because JWB phytoplasma was transmitted to Hovenia tomentella and caused the same symptoms as Hovenia witches' broom (HWB), JWB phytoplasma may be very closely related to HWB phytoplasma. RFLP analysis of the PCR products of 16S rDNA revealed that RhY phytoplasma belongs to the Aster yellows (AY) group, and JWB and HWB phytoplasmas belong to a different group (possibly Elm yellows group). Thus, we found that one species of leafhopper can carry phylogenetically distant phytoplasmas. Received 23 April 2001/ Accepted in revised form 29 October 2001