【作者】 林文力；

【导师】 廖晓兰； 朱水芳；

【作者基本信息】 湖南农业大学 ， 生物安全与检疫， 2010， 硕士

【摘要】 植原体是一类能引起多种植物病害的植物病原原核生物。本研究利用分子生物学技术对我国部分地区的枣疯病植原体以及河南地区杨树黄叶病进行了检测和鉴定,主要结果如下：1)利用植原体通用引物对R16F2n/R16R2、fTufu/rTufu和rp(v)F1A/rp(v)R1A对北京和河北地区枣疯病植原体的16S rDNA基因,延伸因子tuf基因,核糖体蛋白基因(rp)进行PCR扩增,分别得到1.2 kb,0.8 kb以及1.2 kb的特异片段。经序列测定和同源性比较表明：北京地区枣疯病植原体16S rDNA基因与其它地区枣疯病植原体株系差异不大；tuf基因与16SrV组的葡萄黄叶病(FD)同源性最高,为92.84%,而与已经公布的其它地区(陕西杨凌)的枣疯病植原体tuf基因同源性较低,为57.29%；北京地区枣疯病植原体rp基因与16SrV组的枣疯病泰山株系(JWB-Taishan)以及大麻丛枝病植原体(HFWB)同源性最高,均为99.83%,与16SrV组的成员同源性均在96%以上。2)利用植原体16S rDNA基因保守序列通用引物对P1/P7与R16F2n/R16R2对河南地区表现叶片黄化症状的杨树总DNA进行巢式PCR扩增,结果得到约1.2 kb的特异性DNA片段；利用植原体rp基因检测引物对rpF1/rpR1对河南地区表现叶片黄化症状的杨树总DNA进行直接PCR扩增,再运用rp(Ⅰ)F1/rp(Ⅰ)R1引物对进行巢式PCR扩增,得到大小约1.2 kb的特异片段。经序列测定和同源性比较表明此植原体归属于16SrI组。通过对北京及河北地区枣疯病植原体PCR扩增,发现其tuf基因与其它地区枣疯病tuf基因存在较大的差异,从而为枣疯病在亚组水平上的分类提供了新的参考依据；用植原体检测引物对从河南地区杨树黄叶病症的样品中扩增到特异性条带,表明河南杨树黄叶病株中存在植原体,这是在国内首次报道植原体侵染杨树。更多还原

【Abstract】 Phytoplasma is a plant pathogenic prokaryotes that can cause many kinds of plant diseases. By use of molecular biology techniques, the JWB phytoplasma in some parts of China and poplar yellows pathogen in henan province were detected and identified.The main results read as follows:1) By use of PCR, the 16S rDNA,elongation factor Tu (tuf gene) and ribosomal protein (rp) gene of phytoplasma associated with JWB in Beijing and Hebei districts were amplified separately with universal primer pairs R16F2n/R16R2, fTufu/rTufu and rp(v)F1A/rp(v)R1A. We obtained 16S rDNA gene of 1.2 kb, tuf gene of 0.8 kb and rp gene of 1.2 kb from the diseased samples. The results of sequencing and homologous comparison with other phytoplasmas showed that JWB in Beijing shared a high identity with other JWB phytoplasmas in 16S rDNA gene; JWB in Beijing shared 92.84% similarity with Flavescence doree (FD) phytoplasma (Candidatus Phytoplasma vitis) in tuf gene. The tuf gene of JWB in Beijing shared a low identity of 57.29% with JWB tuf gene in Shaanxi district which had been already reported.The homology analysis for sequences of rp gene showed that a high identity with members of the 16SrV group phytoplasmas, with homology rate higher than 96 %. It shared mostly identity of 99.83% to the 16SrV group of JWB strain Taishan and Hemp fiber witches’-broom phytoplasma (HFWB).2) By use of universal primers P1/P7 and R16F2n/R16R2 for 16S rDNA gene, a specific fragment of 1.2 kb in length was amplified with nested PCR from poplar showing typical phytoplasma symptom of yellowing in henan province. By use of universal primers pairs rpF1/rpR1 and rp(Ⅰ)F1/rp(Ⅰ)R1 for rp genes, a specific fragment of 1.2 kb was amplified with nested PCR from poplar diseased sample.The results of sequencing and homologous comparison with other phytoplasmas showed that the phytoplasma strain is one of the members of 16Sr I group.According to the PCR with JWB phytoplasmas in Beijing and Hebei districts, we discovered the JWB tuf gene in Beijing and Hebei had a high difference between with other districts. The results provided the basis for the classification in subgroup of the 16SrV group phytoplasmas. By use of universal primers for dectection of phytoplasma, DNA fragments were obtained from the total DNA of poplar yellows sample.The results indicated existence of phytoplasm aassociated with poplar in Henan province. This is the first report of phytoplasma associated with poplar yellows disease in China.更多还原