病毒诱导的花叶滇山茶的花叶呈色及调控机理

Recently, the virus induced ornamental plants is on the increase, while the basic research is weak. Camellia reticulata is the Chinese endemic species, the second-class national protected plant, the key of "eight famous flowers" in Yunnan Province, and the city flower of Kunming. It was found that some precious cultivars of C. reticulata were virus induced mosaic germplasms. However, the scientific cultivation and propagation of the virus induced mosaic C. reticulata was limited seriously, as the coloring and regulation mechanism of mosaic leaf has yet remained unclear. Therefore, the virus induced mosaic C. reticulata will be used as material in this project. The SSH cDNA library for the coloring process of mosaic leaf will be constructed to enrich the mosaic leaf related genes. Then the coloring mechanism of the virus induced mosaic leaf will be clarified by virus characteristics, cell morphology, pigment content, and these above genes' expression profiles using microarray technology. Meanwhile, the mild strain and attenuated strain of the virus will be selected for mosaic leaf regulation in C. reticulata by bark-paste method, which is invented by ourselves. Then, the regulation mechanism of the virus induced mosaic leaf will be revealed by virus characteristics, cell morphology and pigment content, and the temporal and spatial expression characteristics of the leaf color key genes. This project will lay the theoretical foundation for scientific cultivation and propagation of the virus induced mosaic C. reticulata, and be benefit for deeply understanding of the virus induced mosaic ornamental plants.

当前，病毒诱导的花叶观赏植物日益增多，其基础研究薄弱。滇山茶为中国特有种、国家二级保护植物、云南省八大名花之首、昆明市花。前期调查发现，滇山茶的一些名贵品种为病毒诱导的花叶种质，但花叶呈色及调控机理尚不明确，极大地限制了病毒诱导的花叶滇山茶的科学栽培与繁育。为此，本项目以病毒诱导的花叶滇山茶为材料，通过构建花叶呈色过程的抑制差减杂交cDNA文库，富集花叶呈色相关基因，采用microarray技术分析花叶呈色的基因表达谱，并结合病毒特性和花叶呈色的细胞形态、色素含量，阐明病毒诱导的花叶呈色机理。同时，筛选病毒的温和毒株、弱毒株，利用自主发明的贴皮法展开花叶调控，检测花叶调控中叶色关键基因的时空表达特性，并结合病毒特性及花叶调控的细胞形态、色素含量，揭示病毒诱导的花叶调控机理。项目的实施，不仅可为病毒诱导的花叶滇山茶的科学栽培与繁育提供理论依据，还有助于深入认识病毒诱导的花叶观赏植物。

滇山茶为中国特有种、国家二级保护植物、云南省八大名花之首、昆明市花。前期调查发现，滇山茶的一些名贵品种为病毒诱导的花叶种质，但花叶呈色及调控机理尚不明确，不利于病毒诱导的花叶滇山茶的科学栽培与繁育。为此，本项目利用电子显微镜技术和小RNA测序及RT-PCR技术对花叶病毒进行鉴定；利用转录组测序分析对叶色呈色机理进行研究，研究结果如下：.1. 利用小RNA高通量测序得到初步鉴定为病毒的序列276条。通过比对病毒数据库，分别与花生顶芽坏死病毒、西瓜银斑病毒、番茄褪绿病毒、辣椒褪绿病毒以及南方番茄病毒具有同源性。使用病毒特异性引物为引物，进行RT-PCR验证并通过BLAST比对分析，结果均为假阳性。.2. 花叶呈色机理。使用转录组测序技术对绿叶和花叶的RNA构建cDNA文库，通过测序、组装拼接获得Unigene。分析表明，绿叶与花叶共有4851个基因发生差异性表达。在GOG、GO、KEGG、KOG、Pfam、Swiss-part、NR数据库中被注释的基因数分别为1826个、2291个、1302个、2356个、2356个、2948个、2610个、3969个。对差异表达基因进行直系同源性分析，结果显示获得25个功能类别。富集分析差异基因表达途径，有131条代谢通路被富集，其叶色呈色机理相关基因为途径为3条：卟啉和叶绿素合成途径、类胡萝卜素合成途径以及类黄酮合成途径。分析表明，黄斑区花叶病毒诱导的叶绿素合成相关基因的沉默可能是导致花叶的主要原因。.3. 花叶调控。根据花叶症状的叶片比例，确定了温和毒株25个和弱毒株4个。并采用注射或贴皮法进行不同毒株之间的传染和交叉保护实验，获得了花叶滇山茶的繁育和调控方法。.项目的实施，不仅可为病毒诱导的花叶滇山茶的科学栽培与繁育提供理论依据，还有助于人们对病毒诱导的花叶观赏植物的深入认识。.