【作者】 张帆；

【导师】 张仲凯； 黄立钰；

【作者基本信息】 云南大学 ， 农艺与种业（专业学位）， 2023， 硕士

【摘要】 正番茄斑萎病毒属(Orthotospovirus)病毒是一类重要的农作物病毒病原,为害蔬菜、花卉及多种经济作物和粮食作物。云南是世界上orthotospoviruses高发生区,其中代表种番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)是云南发生的优势种之一。本实验室前期研究发现TSWV可以通过番茄和辣椒种子传播,但是病毒在花器官中的分布和如何侵入种胚的方式尚未明确。本研究应用TSWV-CD毒株(西藏辣椒斑萎病分离株)接种辣椒,通过实时荧光定量(RT-qPCR)、荧光免疫组化(IHC)、透射电子显微镜(TEM)观察等方法研究TSWV在辣椒花器官中的定位分布,明确TSWV种传的关键花器官部位花粉及胚珠是否有病毒分布;通过人工授粉实验研究携带TSWV的花粉是否可以侵入胚珠并使种子带毒,以及母株携带病毒是否可以不经花粉直接侵入胚珠,得到以下研究结果。1.发现TSWV的基因、蛋白和病毒粒体在辣椒不同花器官中分布。其中,TSWV-N/-NSm复制表达量最高,TSWV-RdRp复制表达量最低,TSWV-N、NSm和Gp的复制表达量在不同辣椒花组织(花瓣、子房、花柱、花药和萼片)中都呈现相似的趋势,且在花药和花瓣中表达量明显高于其它部位。TSWV-RdRp和NSs基因表达量除了在辣椒子房和花柱中出现明显的差异,在其它部位RdRp和NSs的复制表达量都比较接近,各个基因在萼片中的表达量都处于较低的水平;TSWV各功能蛋白在辣椒花组织中皆有分布,其中RdRp荧光亮度较低,与RT-qPCR结果相符;首次在辣椒的花器官(花瓣、子房、胚珠、花柱、花药和萼片)中观察到80～120nm的TSWV成熟的病毒粒体。2.授粉实验证明TSWV不仅可以通过母株直接传入胚珠,同时也可以通过花粉传播到胚珠,通过母株带病毒的得到的种子以及通过花粉带毒得到的种子带病毒率分别为30%和40%。在TSWV系统侵染的辣椒植株中,自花授粉后代种子中TSWV的检出率高达96%。在健康不携带的TSWV辣椒植株自花授粉后代种子TSWV检出率为0%。3.发现TSWV存在于辣椒不同的花器官组织中,TSWV不仅可以系统性的侵染辣椒的花器官组织细胞,同时存在一定的概率进入到辣椒的胚珠和花粉中从而实现种传。上述研究结果表明,TSWV在系统侵染的辣椒花器官组织细胞中分布,通过母株(子房)和花粉传播到种胚中从而实现病毒入侵种子,为后期研究如何阻断病毒入侵种胚以及挖掘病毒入侵过程中寄主的主要效应因子奠定基础。更多还原

【Abstract】 Orthotospovirus were an important pathogens of crop virus diseases that harmful to vegetables,flowers,food and economic crops.Yunnan is a high incidence area of orthotospoviruses in the world,among which the representative species tomato spotted wilt orthotospovirus(TSWV)is one of the dominant species in Yunnan.Previous studies in our laboratory found that TSWV can be transmitted through tomato and pepper seeds,but the distribution of the virus in flower organs and how it invades the embryo is not clear.In this study,TSWV-CD isolate(Tibetan pepper spot wilt isolate)was used to inoculate pepper,and the localization and distribution of TSWV in pepper flower organs were studied by real-time real-time quantitative(RT-qPCR),fluorescence immunohistochemistry(IHC),transmission electron microscopy(TEM)observation and other methods,and whether pollen and ovules were distributed in key flower organ parts of TSWV species were clarified.The following results were obtained by artificial pollination experiments to study whether TSWV-carrying pollen can invade ovules and make seeds toxic,and whether the mother plant can carry viruses directly invade ovules without pollen.1.In this experiment,mature tomato mottle wilt virus particles were observed in the flower tissues(petals,ovaries,ovules,styles,anthers and sepals)of pepper for the first time,proving that the virus can replicate and assemble in different flower tissues to achieve systemic infection.1.It was found that the genes,proteins and virions of TSWV were distributed in different flower organs of peppers.Among them,TSWV-N/-NSm replication expression was the highest,TSWV-RdRp replication expression was the lowest,and the replication expression of TSWV-N,NSm and Gp showed similar trends in different pepper flower tissues(petals,ovary,flower column,anthers and sepals),and the expression in anthers and petals was significantly higher than that in other parts.The expression of TSWV-RdRp and NSs genes was relatively similar in addition to obvious differences in the pepper ovary and flower column,and the expression of RdRp and NSs in other parts was relatively close,and the expression of each gene in sepals was at a low level.All functional proteins of TSWV were distributed in pepper flower tissues,and the fluorescence brightness of RdRp was low,which was consistent with the RT-qPCR results.For the first time,80～120nm TSWV-mature virochondria were observed in the flower organs(petals,ovary,ovules,flower columns,anthers and sepals)of pepper.2.Pollination experiments have proved that TSWV can not only be directly introduced to ovules through the mother plant,but also transmitted to ovules through pollen,and the virus detection rate is as high as 30%and 40%,respectively.In pepper plants infested by the TSWV system,the detection rate of TSWV in the embryos of self-pollinating offspring was as high as 96%.3.For the first time,it is proved that TSWV can be replicated and assembled in different flower tissues of peppers,which can not only systematically infect the flower organ tissue cells of peppers,but also have a certain probability of entering the ovules of peppers to achieve seed transmission.The above results show that TSWV can achieve viral invasion through pollen transmission into the embryo.It was proved that anthers,flower column and ovary were the key tissues of indirect invasion of pepper embryo by virus,which laid a foundation for the later study of how to block virus invasion embryo and mine the main effector factors of the host during virus invasion.更多还原