【作者】 顾沛雯；

【导师】 吴云锋；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2007， 博士

【摘要】 小麦蓝矮病是陕西乃至西北冬麦麦区的一个重要病害,由介体条沙叶蝉专化性传播。利用植原体16S rDNA基因保守序列通用引物对Rm16F2/Rm16R1,应用PCR技术从小麦蓝矮病株叶片中扩增到1.4 kb的特异片段。通过对16S rDNA基因片段序列同源性比较,结果表明小麦蓝矮病病原与三叶草绿变、翠菊黄化、绣球花绿变、草莓矮化和番茄巨芽植原体亲缘关系较近,其同源率为99.2%～99.9%。据此可以判定小麦蓝矮病植原体是属于植原体16SrⅠ组。利用引物fTufu/rTufu对小麦蓝矮病植原体延伸因子tuf基因进行PCR扩增得到约850bp的特异片段,结果表明小麦蓝矮病植原体tuf基因片段长842bp,编码280个氨基酸。通过对小麦蓝矮病植原体和其它14种植原体延伸因子tuf基因的同源性比较分析,结果表明小麦蓝矮病植原体与三叶草绿变(KVF)亲缘关系最近,核苷酸同源性为99.9%,氨基酸同源性为100%,从亚组水平上确定小麦蓝矮植原体的分类地位。采用介体接种植物,症状观察和应用植原体16S rDNA基因通用引物对R16mF2/R16mR1进行PCR扩增,在接种小麦和传毒介体中均扩增出1.4kb的特异片段,鉴定出小麦蓝矮病植原体新寄主7种。用巢式PCR方法对小麦蓝矮病田自然发病的20种杂草进行分子检测,确定了9种杂草和自生小麦自然带毒,分别是米米蒿、婆婆纳、小花唐芥、野燕麦、麦加公、节节麦、反枝苋、三叶草和大画眉草,成为重要的中间寄主和侵染源。利用6种植原体特异性限制性内切酶AluⅠ、EcoRⅠ、HpaⅡ、Sau3AⅠ、RsaⅠ和TaqⅠ,对9种杂草和自生小麦的巢式扩增片段进行RFLP(Restriction Fragment Length Polymorphism)分析表明:扩增片段的RFLP图谱与目前已知的16S r I组翠菊黄化植原体的RFLP图谱相近,说明病原基本一致,无寄主生物学分化现象。通过电镜超微结构观察,在接种小麦、长春花和带毒条沙叶蝉体内有大量植原体,而在健康植物组织、无毒条沙叶蝉和带毒条沙叶蝉所产卵中未见植原体的存在。通过介体传毒试验和PCR检测发现,条沙叶蝉最适获毒期为7 d,植原体在虫体内的潜育期为15～17 d,接毒期为2～3 d。条沙叶蝉一旦获毒可终生持毒和传毒。不同虫态的条沙叶蝉带毒率没有明显的差异。寄生植物的种类影响其带毒率。为探讨小麦蓝矮植原体免疫膜蛋白在介体-病原-寄主互作的分子机理获得基础数据。通过植原体免疫膜蛋白基因序列两侧的保守区设计引物对Imp1051/Imp2265,用PCR方法扩增小麦蓝矮植原体免疫膜蛋白基因。从小麦蓝矮病病株和接种长春花中均扩增到约1.0kb的特异片段,对扩增片段的最大开放阅读框和基因的同源矩阵、系统发育树分析;对克隆基因所编码蛋白进行跨膜区、亲疏水区和前导信号序列分析。其中小麦蓝矮植原体免疫膜蛋白基因长495bp,推测的编码蛋白含有164个氨基酸。与10种植原体的免疫膜蛋白基因进行序列同源性分析,小麦蓝矮与三叶草绿变植原体同源性最高,核苷酸和编码的氨基酸序列的同源率分别为98.4%和95.1%。蛋白质结构分析结果表明:小麦蓝矮免疫膜蛋白N端有一个跨膜的前导信号序列,C端为跨膜锚定区,中间为膜外亲水区。与三叶草绿变、翠菊黄化、洋葱黄化和泡桐丛枝植原体的免疫膜蛋白为同型蛋白。更多还原

【Abstract】 Wheat blue dwarf (WBD) is an important disease of winter wheat disseminated by Psammotettix striatus L. in Shaanxi and Northwest area. Using primers (Rm16F2/Rm16R1) for phytoplasma 16S rDNA, a 1.4 kb DNA fragment was amplified by polymerase chain reaction (PCR) in DNA samples extracted from infected wheats but not in those extracted from healthy plants. Homology analysis of nucleotide sequences showed that the amplified 16S rDNA fragment from WBD was closely related to those of clover phyllody (CPh), aster yellows(AY), hydrangea phyllody (HP), blueberry stunt (BBS) and tomato big bud (TBB), with homology rate 99.2%～99.9%. Our results suggest that WBD is caused by a phytoplasma in 16SrⅠg roup.Using primers (fTufu/rTufu) for phytoplasma tuf gene (EF-Tu) to amplify DNA samples extracted from infected wheats, a 850bp DNA fragment was amplified in DNA samples from infected wheats but not in those extracted from healthy plants. The sequence result revealed that the tuf gene of phytoplasma from wheat blue dwarf consists of 842bp, which encoding 280 amino acids. Homology analysis for sequences of tuf gene from WBD and other 14 kinds of phytoplasmas showed that the tuf gene of WBD was closely related to those of clover phyllody (KVF), homology rate of nucleotide sequences and amino acids is 99.9% and 100%, respectively. Homology analysis for sequences of tuf gene can differentiate and classify phytoplasma of wheat blue dwarf from subgroup.Inoculating test plants by vector, observing symptom and using universal primers (R16mF2/R16mR1) for phytoplasma 16S rDNA, 1.4 kb DNA fragments were amplified by polymerase chain reaction (PCR) in DNA samples extracted from inoculated wheat and viruliferous P. striatus L. respectively. Seven new host plants of WBD were discovered. Nine kinds of weed and wheat(Triticum aestivum L.) were determined to be naturally infected WBD plants by nested-PCR for 20 naturally infected kinds of weed in the field. They are Descurainia sophia L., Veronica didyma Tenore, Erysimum cheiranthoides L., Avena fatua L., Lithospermum arvense, Aegilops squarrosa, Amaranthus retroflexus L., Tritolium repens L., Eragrostis cilianensis (AII) Link., and Triticum aestivum L., which become important mid-host and infected resources. Restriction fragment length polymorphism (RFLP) analysis of nested-PCR products of 16S rDNA with six special restriction enzymes, which are AluⅠ、EcoRⅠ、HpaⅡ、Sau3AⅠ、RsaⅠand TaqⅠ, revealed that most of the digestion patterns by restriction endonucleases coincided with those of Aster yellows (AY) phytoplasma ,which belong to the same 16Sr I group phytoplasma. It was showed that WBD phytoplasma exits in 9 naturally infected kinds of weed and wheat, which had not differentiation of host biology.Phytoplasma was observed in the phloem sieve elements of wheat and periwinkle leaf tissues and hind intestine of P. striatus L. under the electron microscope but no phytoplasma was found in healthy plants, viruliferous P. striatus L. and eggs of viruliferous P .striatus L. Using transmission experiments and PCR assys, the desirable acquisition access period (AAP) of the vector is 7 days. The latent period (LP) in the vector ranged from 15 to 17 days after the start of the acquisition and inoculation access periods (IAPs) is 2 to 3 days. In general, infectivity of the vector lasted for life. The different life stage of the vector is not very critical for WBD acquisition. Host-plant species influenced WBD acquisition.The aim of the experiment is to get the basic molecular information for the mechanism of vector-pathogen-host plant relationships. DNA was extracted from infected wheat and periwinkle samples and the target fragments were amplified using primers Imp1051/Imp2265 designated based on conserved regions of immundominant membrane protein gene from phytoplasmas. A 1.0kb DNA fragment was amplified by PCR in DNA samples extracted from infected wheats and periwinkles but not in those extranted from healthy plants. The amplified fragment is analysed for open reading frame, homology matrix and phylogentic tree; Encoding protein is anlysised for transmembrane region, hydrophilic and hydrophobic region and leader signal sequence. It was showed that immundominant membrane protein gene from wheat blue dwarf (WBD) phytoplasma was 495 bp in length, encoding a predicted protein of 164 amino acids. Homology analysis of sequence from WBD and 10 phytoplasmas showed that immundominant membrane protein gene of WBD phytoplasma was closely related to those of clover phyllody (CPh) phytoplasma, with homology rate of nucleotide and amino acids sequences, 98.4% and 95.1%. Prediction of protein structure showed the immunodominant membrane proteins from WBD phytoplasma possess N-terminal hydrophobic transmembrane regions with export leader signal sequence, a C-terminal hydrophobic transmembrane anchor, and a central hydrophilic region, possibly external to the phytoplasma cell. Immundominant membrane protein of WBD phytoplasma belong to the same type with those of CPh, aster yellow (AY), onion yellow (OY) and paulownia witches’-broom (PaWB) phytoplasmas.更多还原