KNR6调控玉米雌花序发育的分子机理研究

Kernel number per row (KNR) is an important yield component of maize. However, there is little information on what loci were utilized for improving grain productivity .Previously, through fine-mapping, association mapping, expression analysis and transgenic validation, we establish that KERNEL NUMBER PER ROW6 (KNR6), encodes a serine/threonine protein kinase which controls maize grain yield by determining pistillate floret number, ear length and kernel number per row. Further investigation KNR6 showed protein kinase activity, undergoes auto-phosphorylation and phosphorylation. But, the information on the molecular basis of beneficial alleles not clearly. In this project, our objectives are i) Through the phenotypic comparison of NIL lines and transgenic lines, cytological observation of immature ears, endogenous hormone determination, to reveal the biological function of KNR6 regulating the number of florets in the ear; ii) by in vitro substrate phosphorylation proteomics analysis and proteomic analysis of phosphorylation of different allelic forms of KNR6, screening the key proteins of inflorescence development for KNR6 phosphorylation, validate the interaction by the BIFC, IP-MS LCI-assay, RNA in situ hybridization and so on; iii)compare the differentially-expressed genes between wild-type and KNR6-CRISPR/Cas9 by RNA-seq, Protein phosphorylation omics combine all of the mentioned examinations results to illuminate the regulatory network of KNR6. Our results will help us further understand the genetic basis and molecular mechanisms underlying the natural variation of kernel number of maize, and it is also helpful for guiding the application of KNR6 in maize breeding.

行粒数是重要的产量构成因子，本研究前期证实编码丝氨酸苏氨酸蛋白激酶的基因KNR6 (KERNEL NUMBER PER ROW6)，通过影响雌穗穗长和行粒数进而控制玉米产量。KNR6具有蛋白激酶活性、自磷酸化和磷酸化功能，但KNR6调控行粒数的分子机制仍不清楚，本研究将在已有基础上通过对近等基因系及遗传转化材料幼穗细胞学观察分析，内源激素原位测定，探明KNR6调控雌穗小花数目的生物学功能；通过体外底物磷酸化蛋白组学分析和KNR6不同等位基因型的磷酸化差异蛋白组学分析，结合免疫沉淀-质谱联用（IP-MS）、LCI-assay、原位杂交等实验验证，筛选KNR6磷酸化调控花序发育的关键互作蛋白；结合KNR6-CRISPR/Cas9家系及近等基因系磷酸化蛋白质组学、RNA-Seq分析，探明KNR6参与调控玉米花序发育的表达网络。本研究结果有助于解析穗粒数形成的分子机制及指导KNR6的育实践。

之前的研究中，我们克隆了一个编码丝氨酸苏氨酸蛋白激酶的基因KNR6，调控玉米穗长和行粒数，但是KNR6调控玉米行粒数的分子机制和网络尚不清楚。本研究证实了KNR6磷酸化AGAP，调控AGAP与ARF1参与的细胞内的囊泡运输，维持雌穗花序中生长素含量的平衡，调控花序分生组织发育，同时基于CRISPR/Cas9构建了KNR6和AGAP基因编辑系，将knr6ko1与agapko1杂交，分离出AGAP/KNR6、agapko1/KNR6、AGAP/knr6ko1和agapko1/knr6ko1基因型，两个单突变体(agapko1/KNR6和AGAP/knr6ko1)的穗长和行粒数比野生型AGAP/KNR6的短，而双突变体agapko1/knr6ko1的穗长比两个单突变体更短、行粒数更少，即2个基因突变能够增强单基因突变异常表型，表明AGAP与KNR6遗传互作共同调控穗长和行粒数。最后，在包含508个自交系的不同玉米群体中，评估了KNR6和4个已知开花基因ZmCCT9、ZmCCT10、VGT1和ZCN8对生育期和穗长的影响，推测了通过标记辅助选择或基因组编辑，将ZmCCT9、ZmCCT10和VGT1的早开花等位基因叠加，平衡KNR6的长穗/晚开花TE-等位基因的晚开花效应，从而提高玉米果穗产量，实现开花时间和穗大小之间的权衡平衡。本项研究已发表SCI文章1篇，在投SCI文章1篇，该项目为研究玉米花序发育、穗长和粒数的调控机制提供了新的思路，同时为设计KNR6以实现玉米产量最大化提供了一种知识驱动策略。