黄花蒿谷胱甘肽过氧化物酶AaGPX1介导2-茨醇影响不定根发生的分子机理研究

The adventitious root formation (ARF) is very important to plant clonal propagation and production of plant secondary metabolite. And ARF is a significant scientific topic attracted more and more interest in the field of plant development and organogenesis. Our previous work showed that plant monoterpene borneol enhanced adventitious root formation mediated by AaGPX1 under an unclear mechanism. The present proposed work aimed to unclose the mechanism. Firstly, the recombinant protein of AaGPX1 would be obtained by isolation and purification of heterogeneous expression product of AaGPX1 in yeast, and then the enzyme would be characterized in vitro by supplement with different substrates. Secondly, AaGPX1 gene would be over-expressed in Arabadopsis thaliana in order to investigate the relationship between adventitious root formation and AaGPX1 expression. Thirdly, AaGPX1 gene would be knocked down in Artemisia annua by RNA interference, and the ARF difference between the wild-type and transgenic plant would be compared for identification of gene function. Furthermore, the promoter of AaGPX1 would be cloned by using TAIL-PCR and the subcellular location of AaGPX1 could be performed by observation of gene expression level of GFP driven by promoter of AaGPX1 under fluorescence microscope. In addition, the induction factors of AaGPX1 transcription would be identified in transgenic tobacco by application of borneol, SNP, RB and H2O2 alone or in a combination way. Moreover, the induction factors would be further analyzed through quantify the changes of AaGPX1 gene expression level using real-time RT-PCR and the difference of enzymatic activity of GPX in A. annua treated with the induction factors. Besides, through application of one of induction factors and blocking the others, coupled with comparison of the gene expression level of AaGPX1, the interaction among borneol, ROS and AaGPX1 expression would be analyzed. Finally, the ARF of A. annua cutting treated with different induction factors would be addressed. The proposed work is very significant for application and basic research, which should provide theory support for stabilization of elite phenotype of A. annua and should put more insight into the plant ARF regulation mechanism.

植物不定根发生问题，既是植物无性繁殖和天然药用物质生产实践的核心问题，又是植物发育和形态建成等方面的重要理论问题。申请者前期研究表明，2-茨醇在谷胱甘肽过氧化酶AaGPX1的介导下显著促进植物不定根的发生，但作用机理尚不明确。本项目拟通过体外酶实验、超表达和RNA干扰技术验证AaGPX1的确切功能；采用TAIL-PCR法扩增AaGPX1的启动子并通过报告基因分析其表达特性，筛选其诱导物质；通过体外施加和内源阻断诱导成分，结合荧光定量RT-PCR和Western blot技术分析AaGPX1表达水平的变化，深入探讨2-茨醇、活性氧（ROS）和AaGPX1的交叉调控及其对植物不定根发生的影响。通过本项目的实施，有望阐明2-茨醇促进黄花蒿ARF的形态学和分子生物学基础，为解决黄花蒿优良品种（群体）的稳定繁殖问题提供新思路，且将进一步丰富植物ARF的调控机制，具有重要的学术和应用价值。

不定根的生物学研究是发育生物学的重要领域，它不仅有助于阐明调节植物生长发育和次生代谢的机制，而且会促进优良品种的无性繁殖，有效降低药用活性物质生产成本，但目前对其形成的调控机制了解非常有限。黄花蒿是我国重要的中药资源，在发展我国生物产业、提高农民收入等方面发挥了重要作用。然而，由于黄花蒿具有自交不亲和的特性，通过种子繁殖遗传变异大，很难得到稳定的高产群体。而采用扦插、微繁、组培等无性繁殖时，存在不定根形成难的问题。本项目联合体细胞、化学和物理突变技术获得了一个黄花蒿易生根突变株icr。该突变株不定根数目较野生株提高了2-5倍，根发生率是野生株的两倍，两周内约95% icr突变株产生了不定根。体外实验发现， 2-茨醇明显促进植物不定根的发生，并有谷胱甘肽过氧化物酶AaGPX1的参与。在此基础之上，我们克隆了对AaGPX1和2-茨醇脱氢酶AaBDH基因，并通过原核表达、过表达、基因沉默、蛋白组学和转录组学等技术，对2-茨醇促进黄花蒿不定根形成的生理生化和分子生物学基础进行了系统深入的研究。结果表明，AaGPX1与植物抗氧化剂如2-茨醇以及植物过氧化物酶之间存在交互作用， 2-茨醇在及时转变ROS“双重角色”中发挥了重要作用。离体器官因受伤后而爆发出大量的NO、H2O2等ROS，这时过量的ROS充当信号分子，触发生长素、CTK、营养物质等向生根区运输，启动2-茨醇过程。然而，过量的ROS会对植物细胞产生损伤，严重时将导致细胞坏死。2-茨醇协同谷胱甘肽过氧化物酶AaGPX1，在ROS一旦完成其信使任务时，及时清除过量ROS，防止细胞坏死，从而提高不定根发生率和单株不定根数目。基于本项目的系统深入研究，我们提出了AaGPX1影响植物不定根形成的分子机理：2-茨醇通过微调NO、H2O2和1O2等ROS诱导AaGPX1的表达，激化AaGPX1以及其他过氧化物的活性，通过协同作用清除细胞体内过剩的ROS，抑制ROS对细胞的伤害，从而促进植物不定根的形成。本项目为解决黄花蒿优良品种的稳定繁殖提供新思路，同时进一步丰富了植物不定根形成的调控机制。