十倍体长穗偃麦草雄性育性基因ThMs1的克隆、表达及功能分析  

Cloning,Expression and Functional Analysis of a Male Fertility Gene ThMs1 in Bread Wheat

作　　者：衡燕芳 李健 王峥 陈卓 何航 邓兴旺 马力耕[1] HENG YanFang;LI Jian;WANG Zheng;CHEN Zhuo;HE Hang;DENG XingWang;MA LiGeng(College of Life Sciences,Capital Normal University,Beijing 100048;Peking Univer Sciences,Weifang 261325,Shandong)

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]北京大学现代农业研究院,山东潍坊261325

出　　处：《中国农业科学》2020年第23期4727-4737,I0001-I0005,共16页Scientia Agricultura Sinica

基　　金：北京市自然科学基金委-北京市教委联合资助重点项目(IDHT20170513)。

摘　　要：【目的】从小麦隐性核雄性不育突变体ms1与十倍体长穗偃麦草异附加系中,克隆雄性育性恢复基因ThMs1,解析该基因的表达模式、编码蛋白的生物化学活性和生物学功能,鉴定新的小麦ms1育性恢复基因,从而更好地应用于小麦杂交育种。【方法】通过基因组原位杂交(genomic in situ hybridization,GISH),鉴定ms1与十倍体长穗偃麦草异附加系中小麦外源的染色体类型;通过同源克隆法在小麦-十倍体长穗偃麦草异附加系中克隆雄性育性基因ThMs1,并稳定转化小麦ms1进行基因功能互补验证;利用RT-PCR及实时荧光定量PCR分析检测基因的表达模式;进一步通过RNA原位杂交分析基因的组织细胞特异性表达特性;利用特异性抗体进行Western blot检测ThMs1蛋白在体内的表达情况;经SignalP软件分析预测蛋白结构;通过蛋白-脂质结合活性分析检测ThMs1蛋白是否具有脂类分子结合活性。【结果】证实小麦ms1与十倍体长穗偃麦草异附加系细胞中含有偃麦草4Ag染色体;从小麦-十倍体长穗偃麦草异附加系中克隆了雄性育性基因ThMs1,遗传转化功能互补试验证实ThMs1能够完全恢复小麦ms1突变体的雄性不育表型,基因的聚类分析表明MS1只存在于禾本科植物中;通过RT-PCR和实时荧光定量PCR检测发现ThMs1在减数分裂期的花药中特异表达,RNA原位杂交分析证实ThMs1在小麦小孢子发生过程中特异表达;Western blot检测发现ThMs1在小麦减数分裂期的花药中表达;对ThMs1蛋白氨基酸序列进行结构预测发现,该蛋白具有推测的脂类结合分子结构域,ThMs1蛋白脂类分子结合试验表明ThMs1蛋白特异结合磷脂酸和磷酸化的磷脂酰肌醇。【结论】克隆了一个新的来自十倍体长穗偃麦草、可恢复小麦隐性核雄性不育突变体ms1雄性育性的ThMs1,该基因与小麦Ms1具有相似的分子结构、时空表达模式和脂结合活性,导致2个蛋白在小麦中也具有相�【Objective】To clone a new male fertility gene,ThMs1,from the ms1g-4Ag wheat-Thinopyrum ponticum alien addition line,and characterize the expression pattern and biological function of ThMs1 in bread wheat,with the hope that ThMs1 will be useful in the application in the preparation of wheat hybrid breeding system.【Method】4Ag chromosome from Thinopyrum ponticum in wheat-Thinopyrum ponticum alien addition line was identified by GISH.ThMs1 was cloned from the wheat-Thinopyrum ponticum alien addition line by homology-based cloning,and the analysis of the function of ThMs1 in the control of wheat male fertility was performed through stable genetic transformation.RT-PCR and qRT-PCR were performed to detect the expression pattern of ThMs1,further analyzing its tissue-specific expression by RNA in situ hybridization.The expression of ThMs1 protein in vivo was detected by Western blot using specific anti-Ms1 antibodies.ThMs1 protein structure were predicted by SignalP software.The detection of lipid-binding activity of ThMs1 was performed by dot blot.【Result】The wheat-Thinopyrum ponticum alien addition line harbored 4Ag chromosome from Thinopyrum ponticum.ThMs1,the homolog of bread wheat male fertility gene Ms1,was cloned from the wheat-Thinopyrum ponticum alien addition line.The functional complementation analysis confirmed that the ThMs1 can completely restore the male fertility phenotype of the wheat ms1.The phylogenetic analysis suggested that the MS1 only exists in the Poaceae family.RT-PCR and qRT-PCR analysis confirmed that ThMs1 gene was expressed in anthers during meiosis.RNA in situ hybridization analysis revealed that ThMs1 was specifically expressed in microspore during wheat microsporogenesis.Western blot showed that ThMs1 was detected in wheat anther.The structure prediction analysis revealed that it has a predicted lipid binding domain,and protein lipid-binding experiments indicated that ThMs1 protein specifically binds PA and several phosphatidylinositols.【Conclusion】A new male fertility gen

关 键 词：普通小麦 小麦-十倍体长穗偃麦草异附加系 隐性核雄性不育 ThMs1 MS1 小麦杂交育种体系 分子设计育种 

分 类 号：S512.1[农业科学—作物学]