矮牵牛重金属转运蛋白PhHMA5铜解毒机理研究

Copper is an essential micronutrient for all living organisms as a cofactor for fundamental metabolic processes such as respiration, photosynthesis, oxidative stress resistance, ethylene signaling and pigmentation. Copper cycles between two oxidation states, because of its redox properties, copper could be strongly toxic when present in excess. Due to the over application of fertilizers and fungicides and the industrial activities, soils in China have been seriously contaminated. Growing flowers on Cu heavily contaminated soils can reduce the entry of copper into the food chain, meanwhile, could produce economic benefits. The cultivated garden petunia, with its diverse morphology and colorful flowers is one of the most popular bedding flowers in the world. Petunia also has a long history as a model plant for scientific research. Therefore, petunia could be an ideal candidate plant grown on heavily contaminated soils. Heavy metal transporting P-type ATPase 5 (HMA5) has been shown to play a key role in transmembrane Cu transport and Cu detoxification in Arabidopsis, rice and Silene vulgaris. However, the up-regulator and function of HMA5 in petunia are poorly understood. In this work, the function and up-regulator of PhHMA5 in petunia will be studied. The expression level of PhHMA5 shows significant difference in wild-type petunia and ph7 mutant by RNA-seq analysis. To further identify this result, yeast one-hybrid and transient expression of pPhHMA5:GFP together with 35S:PH7-RFP in ph7 mutant will be performed. We are going to use real-time RT-PCR to study the expression pattern of PhHMA5 in various tissues of petunia. In order to investigate the role of PhHMA5 in transmembrane Cu transport and and Cu detoxification, the root length and Cu concentration in various organs of wild-type petunia, Phhma5 mutant, Phhma5 mutant complemented line and PhHMA5 over-expressing line under different Cu treatments will be measured. To further unravel the copper stress response mechanisms of PhHMA5, the mRNA expression level and the sub-cellular localization under different Cu treatments will be studied. This study will contribute to our understanding of Cu resistance mechanism in petunia, moreover, it will provide the evidence for Cu tolerant plants selection and breeding.

土壤铜污染日益严重，危害农产品安全。在污染较重土壤用花卉等经济作物代替粮食作物种植是合理利用土壤的可行措施。重金属转运蛋白HMA5在拟南芥、水稻和白玉草中被证实与铜解毒有关，但其上游调控转录因子未知。此外，拟南芥缺失HMA5I亚家族蛋白，白玉草稳定转化等研究技术还未建立，这些限制了对HMA5家族蛋白功能和调控网络的研究。矮牵牛是草本花卉，经济价值巨大，申请者前期发现矮牵牛含有两种HMA5蛋白，其中一个PhHMA5的表达受WRKY蛋白影响。以此为切入点，本项目拟通过酵母杂交和原生质体瞬时表达明确此调控关系；获得PhHMA5转座子突变株和过表达植株，测定各株系不同器官铜离子含量及铜胁迫下的生长状况，结合不同铜供应对基因表达量及蛋白亚细胞定位的影响，阐明PhHMA5的铜解毒功能及作用机制。本项目以期丰富对植物铜胁迫应答网络的认知，为后续研究奠定基础，并以期为筛选铜耐受植物提供理论依据和候选植株。

铜是植物必须的微量元素，但是过量的铜能危害植物的生长。矮牵牛是重要的观赏植物和分子生物学研究的模式植物，其铜转运机理和模式植物拟南芥等存在不同，且尚不完全清楚。本研究以矮牵牛W115株系为研究材料，研究了PhHMA5在矮牵牛不同器官中的表达模式，分析了铜胁迫处理对其表达的调控作用；利用CRISPR/Cas9基因编辑技术成功获得了矮牵牛phhma5突变体，铜胁迫处理发现phhma5突变体根部的伸长量显著低于野生型，证明phhma5突变体对铜胁迫更敏感；进一步通过原生质体瞬时表达发现PhHMA5-GFP定位在液泡膜上，因此我们推测PhHMA5定位在液泡膜上通过将铜离子泵入液泡进行区隔化来进行铜解毒。通过分析RNA-seq数据发现，PhHMA5的表达受到AN1-PH3-PH4-AN11（MBWW）转录复合体的调控，其在突变体中其表达量显著降低；进一步通过分析PH4-GFP的Chip-seq数据发现PhHMA5在PH4-GFP稳定转化矮牵牛中高度富集，证明AN1-PH3-PH4-AN11通过MYB蛋白PH4结合PhHMA5的启动子来调控其表达；利用CRISPR/Cas9基因编辑技术成功获得了矮牵牛phph3突变体，铜胁迫处理发现突变体叶片失绿，光合作用相关参数显著下降，证明phph3突变体对铜胁迫比野生型敏感。综上，通过本项目的实施进一步完善了植物铜解毒机理的作用网络，丰富了对MBWW转录复合体功能的认知。