基于RNA-Seq技术解析藏红花侧芽成花机制研究

All the medicinal ingredients of saffron (Crocus sativus L.) were focused on the three stigmas of pistils. Improving the number of flowers provides an efficient approach to double the yields of saffron, which can resolve the serious contradiction between less supply of saffron and more demand of the pharmaceutical markets. The saffron has one to three buds and six to ten lateral buds. However, lateral buds cannot flower generally. During long-term practice of harvesting saffron cultivation, we collected the mutants of flowering at lateral bud. This project is intended to take the mutant/wild type bulbs as research materials, and the differentially expression genes (DEGs) were obtained by using Illumina RNA-Seq technologies. We selected the genes that are homologous to key genes of flowering in Arabidopsis thaliana as the candidate key genes of flowering in saffron. At the same time, DEGs that do not express in the non-flowering phenotype and express in the flowering phenotype were selected as the candidate key genes of flowering in saffron, similar to the DEGs that express in the non-flowering phenotype and do not express in the flowering phenotype. Further, we plan to regulate the number of flowers by controlling light, temperature and gibberellins. When the numbers of flowers were changed, we detected the expressions of candidate key genes of flowering in saffron and selected the key genes of flowering in saffron which were changed with the trends of the numbers of flowers, or opposite to the trends of the numbers of flowers. The net of regulation which control the number of flowers was established according to the expression characteristics of key genes of flowering in saffron. In the transgenic experiments, key genes of flowering in saffron were cloned into Arabidopsis thaliana and the functions of flowering genes were validated. Therefore, we can explore the mechanism of flowering at lateral buds in saffron. This research can provide genes resources for the gene engineering to improve the number of flowers, which have important theoretical significances and practical values.

藏红花以雌蕊3根柱头入药，增加开花数量可以成倍提高藏红花产量，彻底解决藏红花日益严重的供需矛盾。藏红花有顶芽1-3个，侧芽6-10个，但是罕见侧芽开花。课题组在长期的藏红花栽培采收实践中，收集了侧芽开花的突变体。本项目拟以突变体/野生型藏红花球茎为材料，利用Illumina二代高通量测序技术进行转录组测序，获得差异表达基因库；以拟南芥开花相关基因的同源基因和在不开花表型中不表达/表达且在开花表型中表达/不表达的差异表达基因作为候选藏红花开花关键基因；进一步验证其在光照、温度和赤霉素等开花数量调控途径作用下、响应外部条件诱导的表达特征，从中挑选出开花关键基因，构建藏红花开花数量调控网络；在拟南芥遗传转化体系中验证藏红花开花关键基因的功能，解析藏红花侧芽成花机制。该项目的开展，不仅为培育藏红花多花高产的新品种提供理论支撑，也为生产上进行开花调控提供理论指导。

藏红花以花入药，如能解析藏红花成花机制使通过调控开花关键基因表达而控制藏红花的开花数量，可以成倍增加藏红花产量、彻底打破产量不足瓶颈。课题组首先通过三代测序技术分别建立了高质量的开花藏红花和不开藏红花全株的全长转录组数据库，同时获得了开花/不开花藏红花组织之间特异存在的unigene、AS、lncRNA和SSR，为阐明藏红花花芽生长发育的分子机制提供了多种新的研究思路。然后，以侧芽开花（突变体）/ 侧芽不开花（野生型）球茎为材料，结合二三代测序技术和定量PCR技术筛选并验证出了5个参与侧芽开花突变体花芽形成和发育的新基因PB.69153.1、PB.29894.1、PB.74967.1、PB.1664.2、PB.3985.7。其中3个基因（PB.29894.1、 PB.74967.1、PB1664.2）同时还响应温度和光照对藏红花开花过程的调控，是藏红花开花关键基因。通过拟南芥转基因研究证明了PB.29894.1（CsFT-like）基因参与了植物开花过程的调控，解析了藏红花侧芽成花的分子机制，为培养藏红花的多花高产新品种奠定了基础。进一步结合二三代测序技术和定量PCR技术筛选了在温敏和内源途径中可能调节开花过程的差异基因，发现了62个开花相关基因，构建了藏红花开花调控网络，揭示藏红花的开花过程在一系列开花基因，例如CsVRN、CsFCA、CsSPLs、CsG3OX、CsGASA6、CsSUS2、CsAP1-like等的作用下，通过春化、赤霉素、光周期、自主和年龄途径进行调控。最后证明PB.20221.2和PB.38952.1是与藏红花开花数量性状最为密切的新基因，可用以作为分子指导藏红花生产实践中的开花数量调控。