水稻CSC11介导干热风/干旱诱导的钙信号调控雄蕊发育

doi:10.3864/j.issn.0578-1752.2021.10.001

摘要/Abstract

摘要：

【目的】水稻花期偶遇干热风/干旱,导致脆弱的生殖细胞快速失水,极大地降低产量,这一过程中钙离子作为通用的第二信使传导了干旱或其他逆境信号,但背后的分子机制尚不清楚。分析钙离子透过性胁迫反应阳离子通道家族（calcium-permeable stress-responsive cation channels,CSCs）基因的生理和分子功能,为研究作物干热风的感应机制提供新的理论基础和思路。【方法】采用电生理学和遗传学方法,利用双电极电压钳技术在水稻中鉴定得到一个具有典型特征的受体类-钙通道蛋白,名为OsCSC11,对其蛋白序列进行生物信息学和进化关系分析。运用qRT-PCR和GUS报告基因活性分析确认OsCSC11的表达模式,在拟南芥原生质体细胞和洋葱表皮细胞中瞬时表达OsCSC11-GFP融合蛋白,验证OsCSC11的亚细胞定位;同时利用CRISPR/Cas9基因编辑技术获得OsCSC11的突变体,并通过细胞学等手段分析突变体表型和相关生理功能。【结果】蛋白序列比对发现,OsCSC11具有CSCs家族成员典型的保守结构域DUF221,但与其他成员序列差异大,存在不同于其他成员的特异结构域（motif）属于独立的亚家族。OsCSC11主要在水稻的花药和叶片中表达,进一步分析发现全长OsCSC11处于静息状态,可被高渗透溶液激活;但是删除N端156氨基酸（TM1-3）之后的OsCSC11^ΔTM1-3具有组成型的通道活性,特异选择钙、镁二价阳离子;推测TM1-3是这类通道的受体结构域,感应干热风胁迫,而OsCSC11^ΔTM1-3区域负责钙信号产生。OsCSC11和OsCSC11^ΔTM1-3均定位在细胞质膜上,与其干热风的受体功能相适应。与野生型相比,功能缺失突变体oscsc11-1和oscsc11-2的雄蕊较小、花药表面蹙皱,整体多呈弯曲状态,花粉含水量较低,败育率高达60%—70%。【结论】OsCSC11是水稻感应短期干热风/干旱刺激、介导钙离子内流,调控花药水分状态和花粉发育的受体类钙通道,可能参与了水稻雄蕊应对干热风的原初感应过程。

关键词: 水稻, 干热风/干旱, 受体类通道, 雄蕊异常, 花粉败育

Abstract:

【Objective】When rice is occasionally stressed by dry-hot wind (DHW) or drought at flowering stage, the yield greatly decreases due to the rapid water loss in developing gamete cells. During this process, calcium as a universal second messenger mediates cellular signal transduction in response to drought or other stresses. However, the underlying molecular mechanism remains elusive. This study attempts to dissect the physiological and molecular function of Oryza sativa Calcium-permeable Stress-responsive Cation channels (OsCSCs) which will provide a new strategy for studying the stress responsive mechanism to DHW in crops.【Method】Based on the methods of genetics, electrophysiology and bioinformatics, a typical receptor-like-channel named OsCSC11 was identified. The expression pattern of OsCSC11 were analyzed by qRT-PCR and GUS staining. Further, subcellular localization of OsCSC11-GFP was observed in Arabidopsis protoplasts and onion epidermal cells. Meanwhile, the oscsc11 mutants were generated by CRISPR/Cas9 gene editing tool. Finally, the phenotype and physiological functions were analyzed by cytological method. 【Result】Sequence alignment results in DUF221 family revealed that OsCSC11 includes a typical conserved domain and some unique motifs, which belongs to an independent subfamily. OsCSC11 is mainly expressed in anthers and leaves in rice. Further analysis showed that the activity of full-length protein of OsCSC11 which is in a resting state can be activated by the hypertonic solution. However, OsCSC11^ΔTM1-3(TM1-3 was truncated in OsCSC11) has constitutive channel activity that specifically mediates divalent cations of calcium and magnesium. Thus, we speculated that TM1-3 is a receptor domain in CSCs/OSCAs channel for sensing DHW stresses, and the rest part of OsCSC11 (OsCSC11^ΔTM1-3) generates calcium signal. OsCSC11 and OsCSC11^ΔTM1-3 are both localized in the plasma membrane which may be related to the receptor function. In loss of function mutants oscsc11-1 and oscsc11-2, anthers become smaller and wrinkler compared to wild type, and pollen sterility rate reached to 60%-70% and water content dramatically reduced.【Conclusion】OsCSC11 functions in regulating anther water status and pollen development through mediating calcium influx and possibly involves in the primary sensing step under DHW stresses.

Key words: rice (Oryza sativa L.), dry-hot wind/drought, receptor-like channel, abnormal stamen, pollen sterility

任志杰,李倩,孙钰佳,孔冬冬,刘良玉,侯聪聪,李乐攻. 水稻CSC11介导干热风/干旱诱导的钙信号调控雄蕊发育[J]. 中国农业科学, 2021, 54(10): 2039-2052.

REN ZhiJie,LI Qian,SUN YuJia,KONG DongDong,LIU LiangYu,HOU CongCong,LI LeGong. OsCSC11 Mediates Dry-Hot Wind/Drought-Induced Ca²⁺ Signal to Regulate Stamen Development in Rice[J]. Scientia Agricultura Sinica, 2021, 54(10): 2039-2052.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Purpose
CSC11-BamHⅠ-F	CGGGATCCATGGGGCCGACCGCGCCGCCGCCGGACGCCG	蛙卵表达载体构建 Vector construction for oocyte expression
CSC11-EcoRⅠ-R	GGAATTCTCAGGATTGATACAGGCTCCAATCC
CSC11^ΔTM1-3-BamHⅠ-F	CGGGATCCATGGAGGACGCCCTTCGCA
ProCSC11- Hind Ⅲ-F	CCCAAGCTTTATAGAATGGGTCATCATAGCA	p1300-proCSC11-GUS表达载体构建 Construction of of p1300-proCSC11-GUS vector
ProCSC11- BamHⅠ-R	CGGGATCCCGCCGGGGGACGGGGACGTGAC
CSC11- EcoRⅠ-F	GGAATTCATGGGGCCGACCGCGCCGCCGCCGGACGCCG	GFP融合表达载体 GFP-CSC11 expression vector
CSC11-BamHⅠ-R	CGGGATCCGGATTGATACAGGCTCCAATCC
CSC11^ΔTM1-3-EcoRⅠ-F	GGAATTCATGGAGGACGCCCTTCGCA
Target-F	GCGGCGGGGAGCCGGAGGCG	基因敲除载体 Gene editing
Target-R	CGCCTCCGGCTCCCCGCCGC	基因敲除载体 Gene editing
CSC11-CRI-F	ACCTCGCGTGATCTAGCCCCACC	靶点检测及测序引物 Target detection and sequencing primer
CSC11-CRI-R	GCTTCTCTCAAGCTGGAGCTCC	靶点检测及测序引物 Target detection and sequencing primer
11-qRT-F	GGGCATTCCCAAGACGCT	qRT-PCR检测引物 Primers used for qRT-PCR
11-qRT-R	CCAAGAAATCCTGTTCCGCA
OsACTIN1-F	TCCATCTTGGCATCTCTCAG
OsACTIN1-R	GTACCCGCATCAGGCATCTG