【作者】 孙婷；

【导师】 程晓非； 栾晓燕；

【作者基本信息】 东北农业大学 ， 农业硕士（专业学位）， 2021， 硕士

【摘要】 老山芹（Heracleum moellendorffii Hance）,为伞形科多年生宿根草本植物,主要分布在我国的东北地区,具有丰富的营养价值。近年来,山野菜以其天然无污染的品质,又能让人们感受大自然的力量备受欢迎。老山芹因是“药食同源”的山野菜,成为最受欢迎的山野菜之一。为满足市场需求,黑龙江省的许多地方开始人工栽培老山芹,现老山芹的常规栽培产业已在全国得到发展,随着种植面积逐年扩大,易受病害影响,严重影响其产量和品质,给农户造成巨大经济损失。尽管它具有重要的经济价值,但对其病害的关注却很少。本研究对黑龙江省常见作物、园艺作物和其他植物进行了植物病毒调查时,许多老山芹表现出典型的被植物病毒侵染的症状,为明确老山芹患病的原因,以患病的老山芹的病叶为研究材料,通过小RNA测序、RT-PCR和RACE扩增等技术进行病原鉴定,并建立两种检测体系,主要的研究结果如下:（1）2019年7月,在黑龙江省望奎县采集植物老山芹的患病样品,通过小RNA深度测序等技术对患病样品进行病毒种类鉴定,证实患病的老山芹中存在伴生豇豆病毒科（Secoviridae）的一种新病毒。然后采用分段克隆的策略,利用RT-PCR和RACE技术进行扩增,克隆了新病毒的全基因组序列。（2）进一步对新病毒全基因组序列进行分析,分析结果表明该新病毒包括2个基因组RNA1和RNA2（Gen Bank登录号分别为MW143070、MW143071）,RNA1全长6616个核苷酸并编码预测分子量为222k Da的多聚蛋白P1,RNA2全长5356个核苷酸并编码预测分子量为184k Da的多聚蛋白P2。其中,RNA1包含5个开放阅读框（Open reading frame,ORF）,分别为蛋白酶辅助因子（Pro-Cofactor）、解旋酶（Helicase）、病毒基因组连接蛋白（viral-linked protein）、蛋白酶（Protease）和RNA依赖的RNA聚合酶（RNA dependent RNA polymerase）;RNA2包含3个开放阅读框（Open reading frame,ORF）,分别为运动蛋白（Movement protein）、大的外壳蛋白（Large coat protein）和小的外壳蛋白（Small coat protein）。与其它伴生豇豆病毒科病毒的核苷酸序列同源性45.36～53.77%,目前还未确定属,且该病毒暂被命名为老山芹黄斑病毒（Mountain celery yellow spot virus,MCYSV）。（3）根据老山芹黄斑病毒建立了RT-PCR和RT-RAA两种检测方法,按照其CP保守位置序列分别设计4对引物进行筛选。其中,引物RTSeco-CPI-F1和RTSeco-CPI-R1作为RT-PCR扩增的引物,最适的退火温度为55℃,灵敏度检测的c DNA稀释极限值浓度为100ng/μl×10^-3;引物RAASecocp S-F4和RAASecocp S-R4作为RT-RAA扩增的引物,最适的退火温度为42℃,灵敏度检测的RNA稀释极限值浓度为700ng/μl×10^-5,高出RT-PCR的100倍,并且两种检测方法均对老山芹黄斑病毒具有较好的特异性。（4）为明确该病毒是否存在其他寄主,将老山芹的病叶机械摩擦接种在10种不同的寄主上,15d后观察发病情况,提取叶片RNA并进行RT-PCR检测,结果未检测到该病毒。更多还原

【Abstract】 Heracleum moellendorffii Hance,a perennial root herb of the Umbelliferae.Mainly distributed in the northeast of China,it has rich nutritional value.In recent years,edible wild vegetables have become popular for their natural pollution-free quality and allowing people to feel the power of nature.Heracleum moellendorffii Hance has become one of the most popular edible wild vegetables because it is a kind of "medicine and food homologous".In order to meet market demand,many places in Heilongjiang Province began to cultivate Heracleum moellendorffii Hance artificially.Nowadays,the conventional cultivation industry of Heracleum moellendorffii Hance has been developed throughout the country.As the planting area expands year by year,it is vulnerable to diseases,which seriously affects its yield and quality.Cause huge economic losses to farmers.Although it has important economic value,little attention has been paid to its disease.In this study,when we conducted a plant virus survey on common crops,horticultural crops and other plants in Heilongjiang Province,we found that many Heracleum moellendorffii Hance exhibited typical virus symptoms.In order to study the cause of this disease,t he diseased Heracleum moellendorffii Hance was used as the research material,and the pathogen was identified through small RNA sequencing,RT-PCR amplification,gene cloning and other technologies,and two detection systems were established.The main research results as follows:(1)From July to September 2019,many Heracleum moellendorffii Hance exhibiting typical virus symptoms were found in Wangkui County,Heilongjiang Province.By using small RNA sequencing technology to identify the type of virus in the diseased samples,a new virus of the associatedsecoviridae family was found from the diseased Heracleum moellendorffii Hance.Then,using the strategy of segmented cloning,using RT-PCR and RACE technology for amplification,the whole genome sequence of the new virus was cloned.(2)Further analysis of the whole genome sequence of the new virus showed that the new virus includes two genomes RNA1 and RNA2(Gen Bank accession numbers are MW143070 and MW143071 respectively).RNA1 has a total length of 6616 nucleotides and encodes a polyprotein P1 with a predicted molecular weight of 222 k Da,and RNA2 has a total length of 5356 nucleotides and encodes a polyprotein P2 with a predicted molecular weight of 184 k Da.Among them,RNA1 contains 5 open reading frames,namely protease cofactor,helicase,viral-linked protein,protease and RNA dependent RNA polymerase;RNA2 contains 3 open reading frames,namely Movement protein),Large coat protein and Small coat protein.The nucleotide sequence homology with othersecoviridae viruses is 45.36～53.77%,and the genus has not yet been determined.And the virus is temporarily named as mountain celery yellow spot virus(MCYSV).(3)Two detection methods RT-PCR and RT-RAA have been established based on mountain celery yellow spot virus.Four pairs of primers were designed for screening according to their CP conservative position sequences.Among them,the primers RTSeco-CPI-F1 and RTSeco-CPI-R1 are used as primers for RT-PCR amplification,the optimal annealing temperature is 55℃,the c DNA dilution limit concentration for sensitivity detection is 100ng/μl ×10-3;RAASecocp S-F4 and RAASecocp S-R4 are used as primers for RT-RAA amplification,the optimal annealing temperature is 42℃,the RNA dilution limit concentration for sensitivity detection is 700ng/μl×10-5,it is 100 times higher than that of RT-PCR,and both detection methods have good specificity for mountain celery yellow spot virus.(4)In order to clarify whether the virus has other hosts,The diseased samples of MCYSV were inoculated on 10 different hosts by mechanical friction.After 15 days,the symptoms of disease were observed,and RNA from the leaves was extracted and RT-PCR was performed,the virus was not detected as a result.更多还原