羊草LcMADS12基因的克隆及原核表达分析  

Cloning and prokaryotic expression analysis of LcMADS12 from Leymus chinensis

作　　者：贾俊婷 赵品苍[1] 刘祝江[1] 袁光孝 杨伟光[1,3] 刘书 陈双燕[1] 李晓霞[1] 刘公社[1] JIA Jun -ting;ZHAO Pin -cang;LIU Zhu -jiang;YUAN Guang- xiao;YANG Wei- guang;LIU Shu;CHEN Shuang- yan;LI Xiao -xia;LIU Gong she(Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;Graduate School of the Chinese Academy of Science, Beijing 100049, China;Institute of Animal Science of Heilongjiang Province, Qiqihar 161005, China)

机构地区：[1]中国科学院植物研究所北方资源植物重点实验室,北京100093 [2]中国科学院大学,北京100049 [3]黑龙江省畜牧研究所,黑龙江齐齐哈尔161005

出　　处：《草业学报》2018年第7期64-72,共9页Acta Prataculturae Sinica

基　　金：国家重点基础研究发展计划(973计划)项目(2014CB138704)资助

摘　　要：显花植物中,E类MADS-box基因在调控花分化及花器官发育过程中发挥重要作用。为初步了解羊草花发育的分子机制,依据实验室前期雌蕊转录组数据,以羊草花序cDNA为模板,克隆获得了一个MADS-box基因,命名为LcMADS12。LcMADS12基因的开放阅读框全长为741bp,编码246个氨基酸。生物信息学显示,LcMADS12基因编码氨基酸序列中无信号肽,不存在跨膜区。多重序列比对及功能结构域分析表明,LcMADS12具有保守的MADS-box结构域和半保守的K区,属于E类功能基因SEP亚家族成员。系统进化分析表明LcMADS12与小麦TaAGL16同源性为99%,与水稻基因OsMADS7同源性为86%。组织表达模式分析表明,LcMADS12基因在羊草营养器官中不表达;而在成熟雄蕊、雌蕊、内稃中表达较高,在外稃中低表达,说明LcMADS12基因的表达具有组织特异性。推测LcMADS12基因可能在羊草生殖发育过程中发挥重要作用。酵母单杂交实验及原核表达结果表明LcMADS12蛋白具有转录激活活性,且获得融合蛋白高效表达体系。LcMADS12基因的克隆和融合蛋白的获得为深入开展该基因功能研究奠定了基础。In flowering plants,the E-function genes,which belong to the MADS-box gene family,play central roles in floral meristem and floral organ development.To understand the molecular mechanisms of floral development in Leymus chinensis,a MADS-box gene,LcMADS12,was cloned according to RNA-sequencing data obtained in this research.The LcMADS12 gene contained a 741 bp open reading frame（ORF）which encoded246 amino acids.Bioinformatics analysis showed that the LcMADS12 encoded an amino acid sequence which did not include signal peptide and the transmembrane region.Multi-sequence alignment and functional domain analysis showed that LcMADS12 had a conserved MADS-box domain and a semi-conserved K region,which was a member of the E-class functional gene SEP subfamily.Phylogenetic analysis showed that LcMADS12 had a99% homology with wheat TaAGL16,and 86% homology with OsMADS7.Tissue-specific analysis showed that no expression of LcMADS12 gene was detected in the vegetative organs（root,stem,leaf）of L.chinensis.In the floral organ,the expression of LcMADS12 was detected higher in the mature stamen,pistil and palea,and was lower or not expressed in lemma and glume.These results indicate that expression of the LcMADS12 gene is tissue-specific.We suggest that LcMADS12 could play an important role in the reproductive development of L.chinensis.A yeast one-hybrid experiment showed that LcMADS12 had a transcriptional activation function.In addition,aprokaryotic expression vector was constructed and the fusion protein could be expressed highly.The cloning of LcMADS12 and the acquisition of fusion proteins has laid the foundation for further study of the function of this gene.

关 键 词：羊草 MADS-BOX基因 LcMADS12 表达分析 原核表达 

分 类 号：S543.9[农业科学—作物学]