红麻HcMS1基因的克隆、表达与功能分析

Cloning,Expression and Functional Analysis of HcMS1 Gene from Kenaf

为挖掘红麻雄性不育核基因,解释红麻花蕾败育的分子机理,通过分析转录组数据,利用同源克隆的方法从红麻花药中克隆出拟南芥MALE STERILITY1(MS1)的同源不育基因,并命名为HcMS1基因。利用生物信息学方法对HcMS1蛋白进行结构、理化性质及亲缘关系等分析,并通过qRT-PCR分析HcMS1基因在红麻不育系、保持系不同时期的表达水平;构建过表达载体,利用叶盘法转化本氏烟草并对转基因烟草进行表型观察。HcMS1基因序列开放阅读框(ORF)为1950 bp,编码649个氨基酸。生物信息学分析显示,HcMS1蛋白为亲水蛋白,定位在细胞核上且含有典型PHD-finger结构域,没有信号肽和跨膜区结构,与陆地棉MS1蛋白亲缘关系最近,其次为木槿MS1蛋白。qRT-PCR结果表明,HcMS1基因的表达量在保持系和不育系中均呈现先上升、后下降的趋势,且在红麻花蕾的四分体至单核期表达量最高,这与拟南芥中MS1基因表达模式一致;另外在保持系中基因的表达水平显著高于不育系,推测红麻败育与HcMS1基因的低量表达相关。通过遗传转化试验发现,HcMS1转基因烟草株型较矮,花萼大小不一,花筒长度缩短,并出现自交不结实的现象,说明红麻HcMS1基因的异源表达有影响本氏烟草正常育性的功能。从红麻花药中成功克隆出核不育相关基因HcMS1,并对其进行功能分析,为后续红麻雄性不育育种工作提供了一定的理论依据与基础。

To explore the male sterile gene of kenaf and explain the molecular mechanism of flower bud abortion,the transcriptome data was analyzed and the homologous male sterile gene of Arabidopsis thaliana MALE STERILITY1(MS1)was cloned from kenaf anther by homologous cloning,named HcMS1.The structure,physicochemical properties and genetic relationship of HcMS1 protein were analyzed by bioinformatics method,and the expression level of HcMS1 gene in kenaf male sterile line and maintainer at different stages was analyzed by qRT-PCR.Through the construction of over-expression vector,the transgenic tobacco was transformed by leaf disc method,and the phenotype of transgenic tobacco was observed.The open reading frame(ORF)of HcMS1 was 1950 bp,encoding 649 amino acids.Bioinformatics analysis showed that HcMS1 protein was hydrophilic,located in the nucleus and contained typical PHD-finger domain,without signal peptide and transmembrane domain structure.It had the closest relationship with Gossypium hirsutum MS1 protein,followed by Hibiscus syriacus MS1 protein.qRT-PCR results showed that the expression level of HcMS1 gene in maintainer and male sterile line increased first and then decreased,and the highest expression level was found in the tetrad to mononuclear stage of kenaf buds,which was consistent with the expression pattern of MS1 in Arabidopsis thaliana;In addition,the expression level of HcMS1 gene in maintainer was significantly higher than that in male sterile line,suggesting that the kenaf abortion was related to the low expression of HcMS1 gene.Through genetic transformation experiment,it was found that HcMS1 transgenic tobacco had shorter plant type,different calyx size,shorter anther length and could not produce seeds,which indicated that expression of kenaf HcMS1 heterologous gene could affect the normal fertility of tobacco.We successfully cloned HcMS1 gene from kenaf anther,and analyzed its function,which provided a theoretical basis for subsequent kenaf male sterility breeding.