Rice Research Institute, Sichuan Agricultural University, Wenjiang, 611130, China; State Key Laboratory of Hybrid Rice, Sichuan Agricultural University, Wenjiang, 611130, China.
Rice Research Institute, Sichuan Agricultural University, Wenjiang, 611130, China; Station of Foundation and Improved Seed-breeding of Sichuan Province, Shuangliu, 610200, China.
J Genet Genomics. 2019 Jan 20;46(1):41-51. doi: 10.1016/j.jgg.2019.01.003. Epub 2019 Jan 23.
Grain weight and grain number are two important traits directly determining grain yield in rice. To date, a lot of genes related to grain weight and grain number have been identified; however, the regulatory mechanism underlying these genes remains largely unknown. In this study, we studied the biological function of OsSPL18 during grain and panicle development in rice. Knockout (KO) mutants of OsSPL18 exhibited reduced grain width and thickness, panicle length and grain number, but increased tiller number. Cytological analysis showed that OsSPL18 regulates the development of spikelet hulls by affecting cell proliferation. qRT-PCR and GUS staining analyses showed that OsSPL18 was highly expressed in developing young panicles and young spikelet hulls, in agreement with its function in regulating grain and panicle development. Transcriptional activation experiments indicated that OsSPL18 is a functional transcription factor with activation domains in both the N-terminus and C-terminus, and both activation domains are indispensable for its biological functions. Quantitative expression analysis showed that DEP1, a major grain number regulator, was significantly down-regulated in OsSPL18 KO lines. Both yeast one-hybrid and dual-luciferase (LUC) assays showed that OsSPL18 could bind to the DEP1 promoter, suggesting that OsSPL18 regulates panicle development by positively regulating the expression of DEP1. Sequence analysis showed that OsSPL18 contains the OsmiR156k complementary sequence in the third exon; 5' RLM-RACE experiments indicated that OsSPL18 could be cleaved by OsmiR156k. Taken together, our results uncovered a new OsmiR156k-OsSPL18-DEP1 pathway regulating grain number in rice.
粒重和粒数是直接决定水稻产量的两个重要性状。迄今为止，已经鉴定出了许多与粒重和粒数相关的基因，但这些基因的调控机制在很大程度上仍然未知。在这项研究中，我们研究了 OsSPL18 在水稻籽粒和穗发育过程中的生物学功能。OsSPL18 的敲除（KO）突变体表现出粒宽和粒厚减小、穗长和粒数减少、分蘖数增加的特征。细胞学分析表明，OsSPL18 通过影响细胞增殖来调节小穗颖壳的发育。qRT-PCR 和 GUS 染色分析表明，OsSPL18 在发育中的幼穗和幼颖壳中高表达，与它在调节籽粒和穗发育中的功能一致。转录激活实验表明，OsSPL18 是一种具有功能的转录因子，其 N 端和 C 端都具有激活结构域，这两个激活结构域对其生物学功能都是不可或缺的。定量表达分析表明，DEP1，一个主要的粒数调节因子，在 OsSPL18 KO 系中显著下调。酵母单杂交和双荧光素酶（LUC）测定均表明，OsSPL18 可以与 DEP1 启动子结合，提示 OsSPL18 通过正向调控 DEP1 的表达来调节穗发育。序列分析表明，OsSPL18 在第三外显子中包含 OsmiR156k 的互补序列；5' RLM-RACE 实验表明，OsSPL18 可以被 OsmiR156k 切割。总之，我们的结果揭示了一个新的 OsmiR156k-OsSPL18-DEP1 途径，该途径调节水稻的粒数。
