武丽芬1,2 夏川2 张立超2 孔秀英2 陈景堂1,3,* 刘旭1,2,*
摘要 Abstract
1 材料与方法
1.1 试验材料及生长条件
1.2 小麦总RNA提取及TaEMF2基因的克隆
按照庄盟RNAprep Pure植物总RNA提取试剂盒(离心柱型)提取小麦各组织的总RNA,通过ABM公司5×All-In-One RT MasterMix反转录试剂盒合成第一链cDNA。以水稻OsEMF2b (Os09g0306800)序列为参考,在Triticeae-gene Tribe (http://wheat.cau.edu.cn/TGT/) 中查找在小麦中的同源基因,然后通过Ensemble Plants数据库获得其基因组序列。利用Primer 5设计基因组特异引物(引物序列见表1),以KN199小麦叶片的cDNA为模板进行PCR扩增,PCR反应体系(20 μL):0.4 μL KOD-FX-Neo、10 μL 2×buffer、4 μL dNTPs (2 mmol L‒1)、2 μL上下游引物(2 μmol L‒1)混合物、2 μL cDNA (60 ng)、1.6 μL ddH2O;PCR反应程序如下:94℃预变性5 min;98℃变性10 s,62℃退火1 min 30 s,68℃延伸30 s,35个循环;68℃延伸10 min;10℃ 保存。扩增结束后进行琼脂糖凝胶电泳,用庄盟的DNA凝胶回收试剂盒将目的片段回收,按照操作说明将目的片段连接到pEASY-Blunt Zero克隆载体上,转化大肠杆菌Trans-T1。挑取单克隆菌块至500 mL含有卡那霉素(Kan)抗生素的LB液体培养基中,于28℃摇床,220转 min-1,过夜培养。经测序获得正确的质粒并放于-20℃保存大肠杆菌阳性菌株。
1.4 过表达载体构建与转化
1.5 转基因材料的检测和抽穗期的统计
1.6 转基因小麦的RT-qPCR分析
1.7 TaEMF2的亚细胞定位
2 结果与分析
2.1 TaEMF2基因的结构
根据已报道的水稻OsEMF2b(Os09g0306800)基因序列,在网站Triticeae-gene Tribe (http://wheat.cau.edu.cn/TGT/) 中查找得到该基因在小麦中的3个直系同源基因(TraesCS2A02G000100、TraesCS2B02G023900和TraesCS2D02G000600),通过Ensemble Plants数据库得到3个同源基因的序列,并将其分别命名为TaEMF2-2A、TaEMF2-2B和TaEMF2-2D。序列分析发现这3个基因均由20个外显子和19个内含子组成(图1),其cDNA全长分别为1815 bp、1803 bp和1812 bp,分别编码605、601和604个氨基酸。
图2 EMF2在高等植物中的进化分析
2.3 小麦TaEMF2的亚细胞定位
图3 TaEMF2蛋白在小麦原生质体中的亚细胞定位
mCherry: 核定位Marker; Merged: TaEMF2与mCherry信号融合; 标尺为10 μm。
2.4 敲除TaEMF2导致小麦延迟抽穗
为了验证TaEMF2是否具有调控小麦抽穗期的功能,我们在KN199背景下对TaEMF2的3个直系同源基因(TraesCS2A02G000100、TraesCS2B02G023900和TraesCS2D02G000600)进行了CRISPR/Cas9介导的基因编辑。分别在TaEMF2的5′UTR、第一外显子和第七外显子的位置设计了2个靶位点,构建了基于CRISPR/Cas9系统的基因编辑载体(图4-A, B),利用农杆菌介导的方法转化受体材料KN199,T0代共获得19个单株,并且19个单株均发生纯合或双等位杂合突变。对T1代基因编辑材料的测序和表型鉴定表明,在靶点发生不同突变的株系均表现为晚抽穗表型,然后选择了5个株系种植T2代,获得2个稳定的株系(KO-L1和KO-L4)进行基因型鉴定(图4-C),在KO-L1敲除系中A基因组的第1和第2靶点都发生A碱基的插入,在B基因组的第1靶点未发生编辑,在第2靶点发生A碱基的插入,在D基因组的第1靶点发生了24个碱基(5'-TTTTTGTACATCCAGCCAAAATTT-3')的缺失,在第2靶点发生8个碱基(CAGACCAC)的缺失。在KO-L4敲除系中A基因组的第1靶点缺失T,第2靶点发生4个碱基(CACA)的缺失,在B基因组的第1靶点未发生编辑,在第2靶点发生C插入,在D基因组第1靶点发生A碱基插入,第2靶点发生T/A碱基插入,最终造成TaEMF2蛋白功能缺失。对KO-L1和KO-L4敲除系的抽穗期表型鉴定表明2个突变体的抽穗期比野生型(WT)晚3~4 d (图4-D, E)。
图4 TaEMF2的CRISPR/Cas9基因敲除系的基因型和表型
2.5 过表达TaEMF2-2D基因促进小麦早抽穗
2.6 开花相关基因VRN1和VRN3在TaEMF2转基因株系中的表达分析
3 讨论
植物从营养生长向生殖生长的转变受到许多外部条件和内源基因的调控,EMF基因决定着植物营养生长阶段的发育,在调控植物抽穗和开花中起重要作用。本研究在小麦中克隆了OsEMF2b的直系同源基因,为研究TaEMF2的功能,我们利用CRISPR/Cas9系统在KN199背景下构建了TaEMF2-2A、TaEMF2-2B和TaEMF2-2D的三敲突变体,观察发现TaEMF2三敲突变体表现晚抽穗表型,而过表达TaEMF2-2D植株表现早抽穗表型。已有研究表明EMF2在不同的物种中的功能相对保守,但也有所差异。拟南芥与水稻中EMF2的功能相反,EMF2缺陷促进了拟南芥的开花[19,29],但推迟了水稻的开花[23-24]。在棉花[25]和西蓝花[26]中EMF2沉默后都能导致植株提前开花。在银杏[27]和绿竹[28]中过表达EMF2都能导致植株晚开花。本研究结果与水稻中OsEMF2b调控抽穗功能相同,TaEMF2在小麦中也能促进小麦抽穗开花,这也印证了小麦与水稻序列同源性较高,可能存在相似的生物学功能这一结果。
EMF基因在拟南芥中调控开花的机制主要通过以下途径来实现,EMF2与CLF、FIE和MSI1的PRC2蛋白复合体能够直接与FT和FLC相互作用通过表观遗传抑制调控靶基因的转录来介导拟南芥开花的抑制,或直接靶标FT和FLC,从而抑制这些开花基因的表达,进而抑制拟南芥开花[30]。EMF2也能够负调控晚开花基因LFY和AP1的表达来调节拟南芥花的启动[31]。另外,拟南芥中DNA复制蛋白RPA2A能与多梳蛋白CLF、EMF2和MSI1相互作用形成复合体,募集PRC2到靶基因染色质位点,促进靶基因的H3K27me3水平,进而抑制开花转型[32]。水稻中,OsEMF2b通过介导OsLFL1和OsMADS4的组蛋白甲基化H3K27me3的积累,来调节水稻成花转变[24]。本研究中,在TaEMF2敲除突变体中VRN1和VRN3的表达量显著下调,说明TaEMF2能够调控VRN1和VRN3的表达。因此,推测TaEMF2可能通过靶基因染色质组蛋白的修饰水平调节VRN1和VRN3基因的表达,进而调控小麦的抽穗期,但这一推测还有待进一步研究。另外,本研究中TaEMF2基因除了调控小麦的抽穗期外,也注意到敲除突变体的籽粒大小和千粒重等农艺性状也发生了变化,但还需要多年多点的表型数据调查分析进行验证。
4 结论
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