基于RNA-seq的寒兰SSR标记开发和开花基因挖掘

发布时间：2018-09-03 06:52

【摘要】：兰科植物是最进化的单子叶植物类群,分布区域广,生态环境多样,形态、结构和生理特性均高度特异,是进行花发育研究的理想材料。寒兰(Cymbidium kanran)隶属于兰科(Orchidaceae)兰属(Cymbidium),是国兰家族的重要成员,具有唇瓣形态变异多样,四季开花和花期长等特点,被誉为“兰中之王”。然而,包括寒兰在内的兰科植物从种子到开花所需时间非常长,严重地制约了花发育的相关研究以及资源开发。目前试管开花的体系在很多兰科植物中已建立起来,但花芽诱导率及后续生殖生长情况还有待改善,并且试管开花的诱导增加了操作的成本和技术难度,所以寒兰开花分子机制的研究对其发展具有重要意义。本文拟对寒兰进行转录组De Novo测序,获得参考序列,然后对寒兰开花各个阶段的顶芽进行数字化基因表达谱(Digital gene expression profiling;DGE)分析,筛选差异表达的基因,再利用实时荧光定量PCR(Quantitative real-time reverse-transcription polymerase chain reaction;RT-qPCR)对所得的差异基因进行验证,初步揭示了寒兰开花的分子机制。研究结果为寒兰开花的分子调控机制奠定了基础,也为兰科植物的花期调控提供了依据。主要结果包括:1.利用Solexa/Illumina二代高通量测序平台,对寒兰根、茎、叶和花四部分混合样品进行转录组测序,得到有效序列108,927,860 nt,经过组装获得Unigene 68,699条,平均长度是867 nt。2.利用寒兰转录组文库中68,699条Unigene进行SSR位点分析,共获得位点11,646个,占16.95%。随机选取141对SSR引物,其中79对可以扩增出特异条带,扩增率达到56.03%,可以扩增出具有多态性条带的引物共15对,达10.64%。3.对寒兰三个时期的花芽进行DGE分析,通过对三个转录组数据库的比较,共获得23,720个差异表达基因,从中筛选出与成花过程有关的9个基因,分别为:CkFPA,CkGA2OX,CkGID1,CkCO,CkPHYB,CkELF3,CkFRI,CkFLC和CkAP1。4.通过RT-qPCR技术,验证9个差异表达基因在寒兰成花过程中的表达模式,发现其中CkFPA,CkGA2OX,CkCO,CkFLC和CkAP1 5个基因在寒兰开花过程呈现显著差异及规律的表达模式,表明其与寒兰开花密切相关。综上所述,寒兰成花是由自主途径、赤霉素途径、光周期途径和春化途径共同调控的,其中CkFPA,CkGA2OX,CkCO,CkFLC耦合CkAP1在成花过程中的高表达,诱导寒兰开花。本研究构建了高质量的转录组数据库,获得了寒兰开花候选功能基因,为进一步揭示寒兰开花时间调控机制奠定了基础,并可为寒兰早花分子育种提供丰富的基因资源,具有重要的理论和实践意义。
[Abstract]:Orchidaceae is the most evolved monocotyledonous group with wide distribution, diverse ecological environment, highly specific morphology, structure and physiological characteristics, and is an ideal material for flower development. (Cymbidium kanran) is an important member of the Guolan family, which belongs to the (Orchidaceae) genus (Cymbidium),. It is known as the "king of orchids" because of the variety of labial morphology, the flowering of four seasons and the long flowering period. However, it takes a long time from seed to flowering for the orchids, including cold orchids, which seriously restrict the research of flower development and the exploitation of resources. At present, the system of in vitro flowering has been established in many orchids, but the rate of flower bud induction and subsequent reproductive growth still need to be improved, and the induction of in vitro flowering increases the cost and technical difficulty of operation. Therefore, the study on the molecular mechanism of flowering is of great significance to its development. In this paper, the transcriptome De Novo sequence of cold orchid was sequenced, and the reference sequence was obtained. Then, the terminal buds of cold orchid were analyzed by (Digital gene expression profiling;DGE (digital gene expression profile), and the differentially expressed genes were screened. The differential genes were verified by real-time fluorescence quantitative PCR (Quantitative real-time reverse-transcription polymerase chain reaction;RT-qPCR, which revealed the molecular mechanism of the flowering of cold orchid. The results laid a foundation for the molecular regulation mechanism of the flowering of the orchid, and also provided the basis for the regulation of the flowering period of the orchids. The main results include: 1. Using Solexa/Illumina second generation high-throughput sequencing platform, four mixed samples of cold orchid root, stem, leaf and flower were sequenced by transcriptome sequencing. The effective sequence of 108927860 nt, was assembled to obtain 68699 pieces of Unigene, with an average length of 867 nt.2.. A total of 11 646 loci (16.95%) were obtained from 68699 Unigene loci in the cold orchid transcriptional library. One hundred and forty-one pairs of SSR primers were randomly selected, of which 79 pairs could amplify specific bands, the amplification rate was 56.03 and 15 pairs of primers with polymorphic bands could be amplified, up to 10.64.3. By DGE analysis of flower buds in three stages of cold orchid, a total of 23 720 differentially expressed genes were obtained by comparing the three transcriptional databases. Nine genes related to flower formation were screened out, namely: CkFPAA CkGA _ 2OXCkGID1, CkPHYBCkELF3CkFRICkFLC and CkAP1.4.. The expression patterns of 9 differentially expressed genes in the flowering process of cold orchid were verified by RT-qPCR technique. It was found that 5 genes of CkFPA,CkGA2OX,CkCO,CkFLC and CkAP1 showed significant differences and regular patterns in flowering process of cold orchid, which indicated that they were closely related to the flowering of cold orchid. To sum up, orchid flower is regulated by independent pathway, gibberellin pathway, photoperiod pathway and vernalization pathway, in which CkFPA,CkGA2OX,CkCO,CkFLC coupled with CkAP1 is highly expressed in the process of flower formation, which induces the flowering of cold orchid. In this study, a high quality transcriptome database was constructed, and candidate functional genes were obtained, which laid a foundation for further revealing the mechanism of flowering time regulation of cold orchid, and could provide abundant gene resources for molecular breeding of cold orchid early flower. It has important theoretical and practical significance.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.31

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