草原龙胆MIXTA1蛋白调控表皮蜡质合成的分子机制研究

Eustoma grandiflorum is a very important horticulture plants, in which the fresh durations of cut flower is a key market traits. Plant cuticular wax plays an important role in plant self-protective barrier, avoidance of nonstomatal water loss, abiotic and biotic stresses tolerances and protections of UV Radiation. Although it’s reported that enzymes and transcription factors are involved in the cuticular waxes synthesis, the detailed molecular mechanism remains unclear. Previously, we idnentified a MIXTA1， an MYB-type transcription factor， from Eustoma grandiflorum and found that over-expression of MIXTA1 resulted in obvious increase of total wax loads in leaves, and significant decrease of leaves water loss. In this project, we plan to investigate the involvements of MIXTA1 in the regulation of waxy synthesis and response to drought stress using MIXTA1 over-expression transgenetic lines and knock out mutants through phenotypic, physiological, biochemical and molecular approaches. Furthermore, we will figure out the downstream target genes by Chromatin immunoprecipitation (ChIP) sequencing and RNA-seq analysis, and identify interaction proteins of MIXTA1 by IP-MS assays. Finally, we will confirm the function of candidate genes about wax biosynthetic pathway through CRISPR-cas9 editing technology. These results not only reveal novel molecular mechanism of MIXTA1 in cuticular waxes biosynthesis and drought tolerance, but also provide a new idea for extending the durations of Eustoma grandiflorum cut flowers.

草原龙胆是非常流行的鲜切花种类，切花寿命是非常重要的观赏品质。植物表皮蜡质能够防止植物非气孔性水分流失，抵抗干旱胁迫，避免病虫害，抵御辐射，对植物建立自身防护系统有着重要的生物学意义。近年来研究报道，除了一些合成酶类基因外，一些转录因子也参与了蜡质合成，而涉及的调控机制尚不明确。在前期研究中发现草原龙胆的转录因子MIXTA1基因，过量表达导致叶片表面蜡质合成量显著增加，叶片失水率明显下降。本项目拟利用MIXTA1基因过量表达及功能缺失突变体，从表型、生理、生化及分子水平证明MIXTA1参与调控蜡质合成及应答干旱胁迫的功能；从转录水平筛选MIXTA1调控蜡质合成的下游靶基因；从蛋白水平筛选与MIXTA1协同调控蜡质合成的相互作用蛋白，最终通过CRISPR-Cas9编辑技术对筛选获得候选基因进行功能验证，揭示草原龙胆中MIXTA1调控表皮蜡质合成、抵抗干旱胁迫的分子机制。

前期的研究发现EgMIXTA1基因过表达，可以增加草原龙胆叶片表面的表皮蜡质，减少叶片的失水，提高植株抗逆能力。为了进一步验证EgMIXTA1参与草原龙胆表皮蜡质生物合成的途径，该项目以EgMIXTA1基因转基因植株为研究材料，通过转录组测序及生物信息学分析，筛选出了可能受EgMIXTA1调控的候选基因九个，在草原龙胆基因组数据库中获得候选基因序列信息，并进行候选基因表达水平分析及组织特异性表达分析。通过对候选基因启动子序列分析，选取含有重要功能元件及MYB转录因子结合位点的2000 bp启动子序列进行启动子克隆，并利用染色质免疫共沉淀（ChIP）及实时定量PCR的方法对筛选出的候选基因进行初步验证，然后通过双荧光素酶报告系统进一步验证，最终确认受EgMIXTA1转录因子调控的基因为蜡质合成重要基因EgCER3。通过构建EgCER3基因的过表达及CRISPR-Cas9载体，遗传转化草原龙胆及拟南芥中进行基因功能验证。该项目的研究结果揭示了EgMIXTA1调控蜡质合成的下游靶基因，为草原龙胆建立自身防护系统，抵御病虫害及改良品种提供理论基础，创造出具高抗逆性的草原龙胆新品种，将对园艺植物抗性的改良提供依据。