莱茵衣藻MAPKKK激酶Femu9p介导的缺铁信号转导途径研究

Iron is a very important and essential element of organisms. The study of iron-deficient signal transduction pathways helps to understand iron metabolism. A mutant named mu9 was screened from iron-deficient library of Chlamydomonas, in which the candidate gene Femu9p contains a MAPKKK domain of kinases, a highly homology to both human MAPKKK3 and Arabidopsis AtMAPKKK1. Yeast two hybrid will be applied to find a Femu9p-binding CrMAPKK and a CrMAPKK-binding CrMAPK, followed by identification of proteins interactions through bimolecular fluorescence complementation and Co-immunoprecipitation. Co-transformation, together with immunoprecipitation and in vitro enzyme acitivity analysis, will be used to test the activation of Femu9p to CrMAPKK and of CrMAPKK to CrMAPK. Study of genes expression and enzymes activity analysis of above kinases will reveal MAPK cascade phosphorylation of iron-deficient signal transduction pathway in Chlamydomonas.

铁是生物体十分重要且必不可少的元素，对缺铁信号转导途径的研究对于了解机体铁代谢机制有着十分重要的意义。本课题组从莱茵衣藻缺铁应答突变体库中筛选到一个突变体mu9，其候选基因Femu9p含MAPKKK激酶功能域，与人类MAPKKK3和拟南芥AtMAPKKK1有较高同源性。本研究拟通过酵母双杂交技术，筛选与Femu9p互作的CrMAPKK,以及与CrMAPKK互作的CrMAPK。利用双分子荧光互补或免疫共沉淀验证蛋白间的作用。通过共转化结合免疫沉淀和体外激酶活性分析，检测Femu9p对CrMAPKK以及CrMAPKK对CrMAPK的激活作用。继而分析上述激酶基因敲除对MAPK级联途径相关基因表达及酶活影响，以及对Femu9p,CrMAPKK,CrMAPK生物学、细胞学功能研究，初步揭示以莱茵衣藻为代表的单细胞光合真核生物MAPK级联磷酸化缺铁信号转导机制。

铁的代谢调控机制一直是多年来科研人员探讨和研究的重要课题，但目前的研究主要集中于铁吸收和铁运输方面，有关细胞铁信号应答及信号转导体系相关的功能基因报道很少。丝裂原活化蛋白激酶是存在于真核生物中的丝氨酸/ 苏氨酸型蛋白激酶，它与MAPKK,MAPKKK 组成级联信号通路，在真核生物分化、生长、发育和凋亡等多方面发挥重要的作用。本研究首先通过RNA‐seq 转录组测序，分析衣藻MAPK、MAPKK、MAPKKK 基因家族在‐Fe,‐N,‐P 和16℃低温下，RNA 水平表达的变化，结果显示：-Fe 和16℃低温下，多数MAPK 级联途径基因表达降低；而‐P 和‐N 条件下，多数MAPK 级联途径基因表达升高。MAPK、MAPKK、MAPKKK 基因家族在‐Fe,‐N,‐P 和16℃低温下转录表达的显著改变，说明它们可能参与‐Fe,‐N,‐P 和16℃低温信号应答和调控。随后，我们通过RNAi 干涉的技术对CrMAPK3，CrMAPK4，CrMAPK5，CrMEK1，CrMAPKKK5，CrMAPKKK13 基因进行敲除，结果显示：对CrMAPK3，CrMAPK4，CrMAPK5, CrMEK1基因的敲除（沉默），造成铁代谢相关基因FOX1，FTR1，ATR1，FEA1，NRAMP2 转录表达的下降。说明CrMAPK3，CrMAPK4，CrMAPK5， CrMEK1与铁代谢相关基因表达相关, 正向调节它们的表达。而对CrMAPKKK5，CrMAPKKK13基因的敲除（沉默），造成铁代谢相关基因转录表达的上升。说明CrMAPKKK5，CrMAPKKK13 反向调节铁代谢相关基因表达。