【作者】 张俊；

【导师】 方守国； 章松柏； 杨金广；

【作者基本信息】 长江大学 ， 植物保护， 2018， 硕士

【摘要】 烟草是我国重要经济作物之一,烟草的产量和品质直接产业经济,国家税收与广大烟农利益,被称为烟草癌症的烟草病毒病成为制约烟草产业发展的一个重要因素。然而目前对于我国不同生态区烟草病毒病的发生分布,种类还缺乏系统了解,针对我国烟草一些常见病毒病的快速检测方式缺乏深入研究。本文通过small RNA深度测序技术对我国滇川高原生态区,武陵秦巴与贵州山地生态区及山东生态区烟草病毒病的发生分布,种类开展了系统研究,针对一些常见烟草病毒病建立了可视化环介导等温扩增检测体系,旨在为烟草病毒病的预测预报与精准绿色防控提供理论依据。本研究主要得出以下结论与创新点:1.明确了滇川高原生态区以PVY为最主要流行病毒,检出率为86.64%,呈大面积流行趋势,该地区应加大力度对PVY展开防控措施。而不同于其他地区的是在滇川高原生态区CMV与TMV危害较轻,而LBVV发生较重,在该地区还检出了ToNSV,PTV,SYSV。2.明确了TMV,CMV,TEV是危害武陵秦巴与贵州山地生态区的主要三大类病毒,检出率分别为35.83%,14.34%,12.32%。此外,TEV在该地区检出率达到12.32%,根据往年中国烟草病虫害预测预报网络数据分析,在重庆,湖北未曾大面积发生的TEV,我们预测TEV在当地已经开始大面积流行。在该地区还检出了MDV,TYLCV,PeVYV,PepMoV,PVCV3.明确了TMV,PVY,CMV仍是危害山东生态区烟草的主要病毒,检出率分别为34.90,23.43%,25.57%,比对到这三个病毒的sRNA占到所有sRNA的83.9%。TVBMV在山东烟区也有大量分布,检出率为8.96%,此外在山东生态区还检出了ReMV,GMMV,ToMV,ToAV,TYLCV4.首次检测到了一些在我国不同生态区烟草上从未报道过的病毒:ToNSV、恩施地区ZYMV分离物、PVCV、PeVYV、四川地区MDV分离物、山东烟区ReMV分离物、GMMV、青岛地区TYLCV分离物。本研究选取了ZYMV、ReMV、MDV、TYLCV作为研究对象进行了分子生物学验证并定位到了原始样本的采集地点。经过设计特异性引物,通过基因扩增,克隆,测序手段,得到了ZYMV湖北恩施分离物ZYMV-HB,山东烟草ReMV分离物ReMV-Shandong CP区域片段以及四川凉山烟草MDV-SCLS分离物,青岛即墨烟草TYLCV-QDJM分离物全基因组序列,经过与国内外不同分离物构建系统发育进化树,分析了其与来自国内外其他分离物的亲缘关系。5.成功建立了PVY、CMV、TMV逆转录可视化环介导等温扩增(RTLAMP)检测体系,通过实验优化了RT-LAMP体系反应条件,可快速、准确、灵敏地检测PVY,CMV及TMV,检测过程无需昂贵的仪器设备可在田间开展,检测结果可用裸眼进行观察、判断,绿色为阳性(病毒感染),橙色为阴性(病毒未感染)。6.针对一种环状DNA病毒,紫云英矮缩病毒(Milk vetch dwarf virus,MDV)建立了可视化环介导等温扩增(LAMP)检测体系。通过反复的优化实验,得到了最佳反应体系与反应条件,实验论证了LAMP检测体系灵敏度是普通PCR检测方式的100倍,以侵烟草的其它DNA病毒(TYLCV,TbCSV,与TbLCV)论证了检测体系的特异性,通过向反应产物中加入SYBR Green I可实现裸眼判断检测结果,在紫外灯下,阳性产生强烈荧光,阴性无法产生荧光,这为该方法在田间的应用创造了价值。综上所述:小RNA深度测序技术依靠其灵敏,大数据分析为植物病毒检测提供了一个强有力的工具。利用该技术明确了我国不同生态区丰富的病毒种类,不仅发现了一些已知的病毒,还检测到了一些少见报道的新病毒及隐症病毒,为我国烟草病毒病精准绿色防控提供了理论依据。针对一些常见病毒病建立的可视化环介导等温扩增检测体系以其良好的灵敏性,便捷性具备十分巨大的田间应用潜力。更多还原

【Abstract】 Tobacco is one of the important economic crops in China,the quantity and quality of tobacco directly affects the economic benefits of tobacco industry.Tobacco virus disease has become an important factor restricting the development of tobacco industry.However,there is a lack of systematic understanding of the distribution of tobacco virus diseases in different ecological areas.In addition,the rapid detection of some common viral diseases in China lacks intensive study.In this paper,the small RNA deep sequencing technology was used to systematically study the occurrence and distribution of tobacco virus diseases in the ecological area of Yunnan Sichuan Plateau、Shandong ecological area as well as Hubei,Chongqing and Guizhou ecological zone in China.Finally,a LAMP/RT-LAMP detection system was established for some common viral diseases.This study mainly draws the following conclusions and innovations:1.The PVY is the most popular virus in Yunnan Sichuan plateau ecological area.The detection rate is 86.64%,and the epidemic trend is large area.In this area,the prevention and control measures for PVY should be strengthened.In contrast to other regions,CMV and TMV are less dangerous in this area,while LBVV is more serious.Besides,ToNSV,PTV and SYSV are also detected in the area.2.TMV,CMV and TEV are the three main types of viruses that harm Hubei,Chongqing and Guizhou ecological zone.The detection rate is 35.83%,14.34%and12.32%respectively.In addition,the TEV detection rate of 12.32%in this region,according to the usual China tobacco plant diseases and insect pests forecasting network data analysis,The TEV have not widespread occurrence in Chongqing and Hubei,So we predict that TEV has already started to be popular in this region.Besides,MDV,TYLCV,Pe VYV,PepMoV and PVCV are also detected in this area.3.TMV,PVY,CMV is still the main virus that damages the tobacco in shandong ecological area,the detection rate are 34.90,23.43%and 25.57%respectively.TVBMV also has a large distribution in Shandong province with a detection rate of 8.96%.Besides,ReMV,GMMV,ToMV,ToAV and TYLCV are also detected in shandong ecological district.4.In this study,we have detected some viruses that have never been reported in tobacco in different ecosystems in China for the first time,such as ToNSV,ZYMV-HB,PVCV MDV-SCLS,PeVYV,Re MV-Shangdong,GMMV and TYLCV-QDJM.We selected ZYMV,ReMV,MDV and TYLCV as the research objects to verify the molecular biology.First,the coat protein gene sequence of ZYMV-HB and ReMV-Shandong as well as the the genome sequence of MDV-SCLS and TYLCV-QDJM were obtained by gene amplification,cloning and sequencing.Finally,phylogenetic tree was constructed to analyze the phylogenetic relationship between these isolates with other isolates from home and abroad.5.Successfully established a sensitive,rapid and convenient RT-LAMP detection system to detect PVY,CMV and TMV.6.In this study,loop-mediated isothermal amplification（LAMP）was developed and used to detect MDV for the first time.The Mg²⁺and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM,1.8 mM,65°C,and 60 min,respectively.The best ratio of the inner primers（FIP and BIP）to the outer primers（F3 and B3）was 2:1.The detection limit of the method was 100 times greater than that of polymerase chain reaction（PCR）.Thus,we established a visual detection system using LAMP.The nucleotide amplification could be clearly observed by adding SYBR Green I,and the positive and negative reactions exhibited distinctly different colors in daylight;however,the positive reactions can present as green fluorescence under a UV lamp.To sum up,small RNA depth sequencing technology relies on its sensitivity,and big data analysis provides a powerful tool for plant virus detection.In this study,we have used this technology to clarify the distribution of tobacco virus diseases in different ecological areas in China.In addition,LAMP/RT-LAMP detection system was established for some common viral diseases.Therefore,these studies provide a theoretical basis for the prevention and control of tobacco virus diseases in China.更多还原