玉米Rubisco全酶复合体的调控机理研究

Maize is an important grain and forage crop in China. As a classical C4 plant, the photosynthetic carbon assimilation pathways in maize have always attracted great interests and attentions from researchers. Our team has been studying photosynthetic carbon metabolism over the years; our previous findings suggest that: the activity and phosphorylation status of pyruvate orthophosphate dikinase (PPDK) and phosphoenolpyruvate carboxykinase (PEPCK), the key enzymes of C4 carbon assimilation pathway in maize, can be regulated by light intensity. However, light-dependent regulation of another vital enzyme for photosynthesis , Rubisco, has seldom been reported. The applicant has previously discovered that the posttranslational modification levels of a number of acetylation and phosphorylation sites in Rubisco are regulated by light and performed relative quantifications on the modification levels using ITRAQ technology. On the basis of the above findings, we intend to: ① isolate the Rubisco complex and its interactive proteins under light conditions of varied intensities and perform both absolute and relative quantification on the isolated proteins; ② explore the regulatory network of the posttranslational modifications of Rubisco and the corresponding interactive factors using the methods of in vitro inhibition and plastome transformation; ③ perform association analysis using populations of various maize inbred lines and identify elite alleles; ④ verify the functions of the identified elite alleles and establish novel maize germplasm with high photosynthetic efficiency using transgenic methods. Upon the above plans, we seek to elaborate the functional and regulatory mechanisms of Rubisco in C4 photosynthetic carbon metabolism , thereby providing useful genetic resources and theoretical bases for maize breeding.

玉米是我国重要的粮饲兼用作物。作为经典的C4植物，其光合固碳途径一直是研究的重点和热点。本课题组长期从事光合碳代谢研究，已有实验结果表明：C4光合固碳途径关键酶丙酮酸磷酸双激酶（PPDK）和磷酸烯醇式丙酮酸羧化激酶(PEPCK) 的酶活性和磷酸化水平受光强调控。然而，另一关键酶Rubisco受光调控的研究较少。申请人前期利用ITRAQ技术，证实Rubisco部分乙酰化和磷酸化位点受光调控，并进行了相对定量分析。在此基础上，我们将①分离不同光强条件下Rubisco复合体和互作蛋白，进行绝对定量和相对定量分析；②利用体外抑制和质体转化等技术验证Rubisco及其互作因子翻译后修饰调控网络；③利用玉米自交系群体进行关联分析，获得优良等位基因；④通过转基因手段，进行功能验证，并获得高光效玉米新种质；以期阐明Rubisco在C4光合碳代谢途径中的作用，为培育玉米新品种提供重要基因资源和理论依据。

与C3植物相比，C4植物的高光效体现在高光和高温条件下Rubisco仍能进行光合作用。通过一天内不同光照和温度条件下及光暗转换条件下的蛋白表达量、磷酸化和乙酰化的定量鉴定，解析了C4玉米在高光和高温条件下Rubisco作用的可能机制。发掘了Rubisco大小亚基、大小亚基组成的十六聚体的组装因子及活性调节因子Raf1、BSD2、BSP1、Cpn20、Cpn60和RCA在高光和高温条件下的磷酸化和乙酰化的变化，而RCA和BSD2的磷酸化变化尤为显著。同时发现了磷酸化调控网络中RAF1的激酶。通过去乙酰化和Rubisco的活性分析，证明了Rubisco大小亚基的乙酰化会影响其活性。此研究中的磷酸化和乙酰化位点调控在C4玉米的高效固碳中发挥着重要的作用。近年来，发现只过表达Rubisco的大亚基和小亚基不能提高玉米的产量。而如果同时表达Raf1可以提高30%的Rubisco含量和15%的碳同化效率。而我们同时转化更多个的基因，将进一步提高玉米的产量。由于受到玉米转基因效率的影响，同时进行多种组合的转基因难度和工作量都很大。因此，我们在大肠杆菌里进行C4植物玉米中不同基因及与其修饰酶的组合，并测定其活性，找出Rubisco固碳效率最优的组合。此研究为下一步玉米体内的Rubisco的人工合成，进一步提高玉米的产量打下坚实的基础。