云南泡桐丛枝病植原体核糖体蛋白基因片段序列分析

 应用植原体核糖体蛋白基因通用引物对rpF1/rpR1,对采自云南省曲靖市的泡桐丛枝病植原体DNA (PaWB-QJ)进行PCR扩增,得到1.3 kb的特异片段,证明此病株中存在植原体。将此片段与pGEM-T Easy载体连接并转化大肠杆菌JM109感受态细胞,进行PCR鉴定、核糖体蛋白基因部分核苷酸序列测定及分析。结果表明,该株系(PaWB-QJ)核糖体蛋白基因片段长1 244 bp,包含rps19、rpl22和rps3基因。对PaWB-QJ株系的核糖体蛋白基因序列的同源性比较结果显示与16S rI-B亚组的翠菊黄化(Aster yellows,AY)、长春花黄化(Periwinkle yellows,PY)和泡桐丛枝德国株系(Paulownia witches'-broom,PaWB-German)的亲缘关系最近,达到99.0%以上,而与其它组中的株系明显低于97.0%,所以认为该植原体株系属于翠菊黄化组B亚组(16SrI-B)。     

山东省枣疯病植原体的鉴定及分子变异分析

 本研究对山东省11个地区的枣疯病样品进行了鉴定和分子变异分析。以样品总DNA为模板,经扩增和序列测定,分别得到16S rRNA (1 432 bp)、核糖体蛋白基因rp (1 196 bp)、转运蛋白基因secA (836 bp) 和secY (1 421 bp) 的序列,secA基因序列是首次从枣疯病植原体中扩增获得。对获得的序列与NCBI数据库中相关植原体序列进行聚类和核苷酸变异分析,结果显示山东省枣疯病植原体属于16SrⅤ-B、rpⅤ-C、secYⅤ-C亚组,相对于16S rRNA基因,rp,secA和secY变异更大,非同义突变更多,更利于对国内不同来源的枣疯病植原体的精细系统进化分析。     

云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。     

小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

枣疯病植原体TaqMan探针实时荧光定量PCR检测方法的建立

根据枣疯病植原体16S rDNA基因保守区域设计、合成特异性引物和TaqMan探针, 以构建的重组质粒作为阳性标准品, 建立并优化了对枣疯病植原体的TaqMan实时荧光定量PCR检测方法。对优化后的方法进行灵敏度、特异性及稳定性评价, 制作了标准曲线。结果显示, 制作的标准曲线有极好的线性关系, 相关系数( r 2 )达到0.998, 建立的实时荧光定量PCR检测方法能够特异性地检测枣疯病植原体, 能检测到60拷贝的质粒DNA。本研究建立的实时荧光定量PCR检测方法灵敏度、特异性、重复性好, 不仅能够实现对枣疯病植原体的快速检测, 而且为实现从病原定量水平上对枣疯病病情分级奠定了基础。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

桃红叶植原体检测及鉴定

对表现红叶的桃植株进行植原体16SrRNA基因PCR扩增,得到1.2kb的特异片段.将此片段与pGEM T Easy载体连接并转化到大肠杆茵DH5α感受态细胞中.通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行序列测定及同源性比较分析,确定该株系属于翠菊黄化植原体组(Aster yellows group,16SrI).在国内首次报道了翠菊黄化组中的植原体侵染桃树.     

三叶草绿变病植原体的分子鉴定

三叶草(Trifolium pratense Linn.)为车轴草属,蝶形花科多年生草本植物,原产亚洲南部和欧洲东南部,是一种世界性分布与栽培的优良牧草.因其花叶兼优、草姿美、绿期长而具有较高的观赏价值,近几年作为草坪用草被广泛种植.在自然条件下,三叶草很容易受到不同种植原体的侵染,国外已报道的侵染三叶草的植原体有:三叶草绿变植原体( Clover phyllody phytoplasma,CPh)和三叶草增殖植原体(Clover proliferation phytoplasma,CP)等,这些植原体分别属于16SrI组和16SrⅥ组[1,2].     

金莲花绿变植原体的分子鉴定

本研究对河北省大面积发生的金莲花绿变病的病原进行检测和鉴定。以金莲花叶片的总DNA为模板,使用植原体16S rDNA和核糖体蛋白(ribosomal protein)基因rp的特异性引物进行PCR扩增,在感病金莲花样品中扩增到植原体的16S rDNA(1 432 bp)片段和rp基因(1 240 bp)片段。序列分析发现,获得的16S rDNA和rp基因片段与洋葱黄化植原体Onion yellows phytoplasma(GenBank登录号:AP006628)的相似度最高,分别为99.9%和99.3%,确定金莲花绿变病的病原为植原体,暂命名为金莲花绿变植原体Trollius chinensis virescence phytoplasma。对金莲花绿变植原体的16S rDNA进行虚拟RFLP分析,发现其酶切图谱与16SrⅠ-B亚组的洋葱黄化植原体的参照图谱完全一致,相似系数1.00。16S rDNA和rp基因的系统发育进化树显示,金莲花绿变植原体与16SrⅠ-B亚组的植原体聚为一支,属于植原体16S rⅠ-B亚组。     

广东花生丛枝病植原体的分子鉴定

2020年在广东省湛江市遂溪县田间发现表现明显丛枝?小叶, 类似植原体感染症状的花生病株?本研究利用分子生物学技术对其病原进行鉴定?以花生病叶的总DNA为模板, 利用植原体16S rRNA和SecY基因通用引物进行PCR扩增, 获得广东花生丛枝病植原体(PnWB-GDSX-2020)16S rRNA基因片段(1 430 bp, GenBank登录号为MZ427281)和SecY基因片段(1 709 bp, GenBank登录号为MZ437794)?序列一致性和系统进化分析显示, PnWB-GDSX-2020的16S rRNA序列与16SrⅡ-A?16SrⅡ-D和16SrⅡ-V亚组植原体一致性最高, 亲缘关系最近; 进一步利用iPhyClassifier对16S rRNA序列进行在线虚拟RFLP分析, 结果显示, PnWB-GDSX-2020的虚拟RFLP 图谱与16SrⅡ-V亚组的参照株系‘Praxelis clematidea’ phyllody phytoplasma (GenBank登录号:KY568717) 酶切图谱一致, 相似系数为1.00?因此, PnWB-GDSX-2020属于16SrⅡ-V亚组成员?所获得的PnWB-GDSX-2020 Sec Y基因序列与花生丛枝植原体的一致性最高, 亲缘关系最近?本文确定了广东花生丛枝病相关植原体的分类地位, 为当地病害诊断?检测以及防控提供科学依据?     

Identification of genes associated with phytoplasma resistance through suppressive subtraction hybridization in Chinese jujube

Jujube witches' broom (JWB) is a destructive disease for Chinese jujube caused by phytoplasma. A suppression subtractive hybridization library of resistant cultivar ‘Xingguang’ was constructed under phytoplasma stress to identify genes related to JWB resistance. 77 of 200 unique expressed sequence tags had significant sequence homologies and were classified into 10 functional groups. The most abundant group was disease/defense (20.8%), which was consistent with the phytoplasma stress. These differentially expressed genes provide the groundwork for addressing the plant–phytoplasma interaction. Meanwhile, the expression of five selected genes (TLP, PR10, HSP70, ERF, kinase-related protein) was confirmed to upregulate at different infection periods.     

Identification of wheat blue dwarf phytoplasma effectors targeting plant proliferation and defence responses

The identification of effectors from pathogenic microbes is one of the most important subjects for elucidating infection mechanisms. Wheat blue dwarf (WBD) phytoplasma causes dwarfism, witches' broom, and yellow leaf tips in wheat plants, resulting in severe yield loss in northwestern China. In this study, 37 candidate effector proteins were transiently expressed in Nicotiana benthamiana. Plants expressing the SAP11‐like protein SWP1 exhibited typical witches' broom. Interestingly, another protein, SWP11, induced both cell death and defence responses, including H₂O₂ accumulation and callose deposition. Analysis by qRT‐PCR was used to show that a marker gene of the hypersensitive response, HIN1, and three pathogenesis‐related genes, PR1, PR2 and PR3, were significantly up‐regulated in leaves of N. benthamiana expressing SWP11. In addition, SWP12 and SWP21 (TENGU‐like) were shown to suppress SWP11‐, BAX‐, and/or INF1‐induced cell death. These results indicated that SWP21 has a distinct role in virulence compared with TENGU and that WBD phytoplasma possesses effectors that target plant proliferation and defence responses. The ability of these effectors to trigger or suppress plant immunity provides new insights into the phytoplasma–plant interaction.     

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。     

泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用

 根据报道的泡桐丛枝植原体(Paulownia witches’-broom phytoplasma,PaWB)抗原膜蛋白(antigenic membrane pro-tein,AMP)基因的核苷酸序列设计引物,提取发病泡桐总DNA,经PCR扩增并成功克隆泡桐丛枝植原体amp基因。序列分析表明,amp基因由696个核苷酸组成,编码231个氨基酸残基,与GenBank中登录的2个泡桐丛枝植原体的膜蛋白核苷酸序列同源性为100%。将amp基因91-604 nt部分序列(命名为ampd)克隆到原核表达载体pGEX-4T-3,诱导后,经SDS聚丙烯酰胺凝胶电泳分析,表明融合蛋白在大肠杆菌BL21(DE3)中得到了高效表达。以纯化的带GST标签的AMPD融合蛋白为抗原免疫德国大白兔,制备了PaWB-AMPD抗血清。利用该血清,通过Western印迹、点印迹、ELISA、间接免疫荧光和免疫捕获PCR试验均能在发病泡桐组织中特异检测到泡桐丛枝植原体。     

杏褪绿卷叶植原体新疆分离物16S rDNA基因克隆与序列分析

利用植原体16S rDNA基因通用引物对新疆轮台县疑似杏褪绿卷叶病植株总DNA进行巢氏PCR检测,扩增出大小约1.2 kb的特异性条带。对扩增产物克隆和测序,确定特异片段大小为1248 bp。序列同源性比较和系统进化分析表明,新疆杏褪绿卷叶植原体不同分离株16S rDNA基因序列同源性极高,达到99.8%~100%。与16SrⅤ组成员的同源性达到98.2%以上,其中与16SrⅤ-B亚组的枣疯病植原体山东宝山分离株,甜樱桃绿化植原体山东分离株同源性最高,达到99.4%~99.6%。进一步虚拟RFLP分析,结果表明该植原体属于榆树黄化组(16SrⅤ)的一个新的亚组,与其相似性最高的是16SrⅤ-B亚组,相似系数为0.94。本研究首次报道了新疆杏褪绿卷叶植原体16S rDNA的序列,确定了其分类地位,为杏褪绿卷叶病的早期诊断和检测提供了基础。     

安徽桑黄花型萎缩病植原体16S rDNA序列分析及分子检测

 Mulberry yellow dwarf(MYD)disease is an quarantine disease and the causal agent is a phytoplasma.Two pairs of published universal primer, P1/P7 and Rm16F2/Rm16R1, based on the 16S-23S rDNA sequence of phytoplasma and total DNA extracted from infected mulberry tissues were employed for PCR and nested-PCR detection.The results revealed that a phytoplasma-specific 1 830 bp fragment with a G+C content of 46.01% was sequenced(GenBank accession No.GQ249410).The sequence shared 99.7% and 99.8% identity with aster yellows, the representatiive phytoplasma in 16SrI group, and mulberry dwarf phytoplasma classified into subgroup B in 16SrI group and named as the MYD phytoplasma strain Anhui(MYD-Anh).A phylogenetic tree based on 16S rDNA sequences was constructed and showed that MYD-Anh was clustered into 16SrI group.Identity of 16S rDNA sequence between MYD-Anh and mulberry yellow dwarf phytoplasma strain Zhenjiang(MD-zj) was nearly 100%, and they might belong to the same strain.Nested-PCR was used to detect the pathogenic phytoplasma from the differential tissues of mulberry infected with MYD-Anh.The results showed that a phytoplasma-specific 1.4 kb fragment was amplified with total DNA extracted from bark and vein.Nested-PCR was more sensitive than PCR for detecting MYD phytoplasma.     

甜菜夜蛾三个60S核糖体蛋白基因的获得与序列分析

核糖体是生物细胞内的蛋白质合成场所,核糖体蛋白除了参与组装核糖体的大小亚基之外,在生物体内还行使其它功能。本研究以甜菜夜蛾Spodoptera exigua幼虫整体的cDNA为模板,扩增得到了甜菜夜蛾60S核糖体蛋白L6、L7和L10的cDNA序列SeRPL6、SeRPL7和SeRPL10。SeRPL6、SeRPL7和SeRPL10的开放读码框分别为819bp、807bp和660bp,分别编码272、268和219个氨基酸。其中SeRPL7中包含一段由27个氨基酸构成的信号肽序列,SeRPL10中有一个预测的抗生素结合位点。通过NJ进化树构建及与其它昆虫相应的核糖体蛋白比较,发现这三个基因都具有高度的保守性,SeRPL7和SeRPL10氨基酸序列与许多昆虫相应蛋白的同源性都超过了70%。     

Homogeneity analysis of streamflow records in arid and semi-arid regions of northwestern Iran

Homogeneity analysis of the streamflow time series is essential to hydrological modeling, water resources management and climate change studies. In this study, five absolute homogeneity tests and one clustering approach were used to determine the homogeneity status of the streamflow time series(over the period 1960–2010) in 14 hydrometric stations of three important basins(i.e., Aras River Basin, Urmia Lake Basin and Sefid-Roud Basin) in northwestern Iran. Results of the Buishand range test, von Neumann ratio test, cumulative deviation test, standard normal homogeneity test and Pettitt test for monthly streamflow time series detected that about 42.26%, 38.09%, 33.33%, 39.28% and 68.45% of the streamflow time series were inhomogeneous at the 0.01 significance level, respectively. Streamflow time series of the stations located in the eastern parts of the study area or within the Urmia Lake Basin were mostly homogeneous. In contrast, streamflow time series in the stations of the Aras River Basin and Sefied-Roud Basin showed inhomogeneity at annual scales. Based on the overall classification for the monthly and annual streamflow series, we determined that about 45.60%, 11.53% and 42.85% of the time series were categorized into the 'useful', 'doubtful' and 'suspect' classes according to the five absolute homogeneity tests. We also found the homogeneity patterns of the streamflow time series by using the clustering approach. The results suggested the effectiveness of the clustering approach for homogeneity analysis of the streamflow time series in addition to the absolute homogeneity tests. Moreover, results of the absolute homogeneity tests and clustering approach indicated obvious decreasing change points of the streamflow time series in the 1990 s over the three basins, which were mostly related to the hydrological droughts.     

云南芝麻丛枝和花变叶植原体16S rRNA和rp基因序列分析

 本研究通过对表现出丛枝和花变叶症状的芝麻感病植株总DNA进行植原体16S rRNA和rp基因的PCR扩增、克隆、测序及序列分析,明确了两种病株的病原均为植原体,并将其命名为云南元谋芝麻丛枝植原体(SEWB-YNym)和云南元谋芝麻花变叶植原体(SEP-YNym)。两个株系的16S rRNA基因片段长度均为1 248 bp,并且碱基序列完全一致。通过与其他地区报道的芝麻植原体株系16S rRNA基因序列比对后发现这两个株系与来自缅甸的株系不存在位点差异,而与泰国、中国台湾、印度的株系分别存在3~6个位点差异。同时,还从两个株系中获得了长度均为1 171 bp并且碱基序列也完全一致的SEWB-YNym和SEP-YNym的rp基因序列。此rp基因序列包括全部rpl22基因(nt90-476)和部分rps3基因(nt550-1170),分别编码128和206个氨基酸。通过对16S rRNA和rp基因的序列进行同源性比对、构建系统进化树等分析,表明两个株系与候选种‘Candidatus Phytoplasma aurantifolia'相关,为16SrII-A亚组成员,并归属于植原体rp-iii亚进化支。