淹水胁迫下‘中山杉406’ThPDC基因的克隆及功能分析  

Cloning and Functional Analysis of ThPDC Gene in Taxodium’Zhongshanshan406’under Waterlogging Stress

作　　者：裴笑笑 宣磊[1] 华建峰[1] 殷云龙[1] Pei Xiaoxiao;Xuan Lei;Hua Jianfeng;Yin Yunlong(Jiangsu Engineering Research Center for Taxodium Rich,Germplasm Innovation and Propagation,Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing,210014)

机构地区：[1]江苏省中国科学院植物研究所(南京中山植物园)江苏省落羽杉属树木种质创新与繁育工程研究中心,南京210014

出　　处：《分子植物育种》2022年第10期3230-3238,共9页Molecular Plant Breeding

基　　金：国家自然科学基金面上项目(31870592)资助。

摘　　要：以落羽杉属杂交种-‘中山杉406’(Taxodium mucronatum♀×T.distichum♂)(Taxodium‘Zhongshanshan406’)为研究对象,通过RACE技术从‘中山杉406’中克隆出ThPDC基因的cDNA全长,并利用生物信息学手段分析其编码蛋白的结构和功能,进行蛋白序列同源性比对和系统发育进化树的构建;同时,采用PEG介导法进行亚细胞定位;通过RT-PCR技术,检测Th PDC基因在不同部位及不同程度淹水胁迫下的表达量。结果表明:ThPDC基因包含1个长度为1812 bp的开放阅读框(open read frame,ORF),编码603个氨基酸,并获得长度为3927 bp的内含子;ThPDC与鹰嘴豆(Cicer arietinum)和蓝星睡莲(Nymphaea colorata)的PDC蛋白亲缘关系较近,同源性分别为79.93%和80.27%;亚细胞定位显示ThPDC所编码的蛋白质定位于细胞核、细胞膜和细胞质上;水淹胁迫下,Th PDC的相对表达量在根、茎、叶中均显著上升,全淹(TS)植株ThPDC的相对表达量明显高于半淹(HF)植株的表达量,且CK、HF和TS三组中,ThPDC2都在根中表达量最高。可见,ThPDC基因与‘中山杉406’耐淹性密切相关,该基因很有可能参与了‘中山杉406’水淹胁迫响应的调控,研究结果为进一步ThPDC启动子克隆和基因功能的研究提供科学参考。Taking Taxodium’Zhongshanshan406’as research object,cDNA of ThPDC gene was cloned by rapid-amplification of cDNA ends(RACE),the structure and function of the coding protein were analyzed by bioinformatics,and the homologous alignment of protein sequences and phylogenetic tree were constructed;meanwhile,subcellular localization was perfomed with PEG-mediated transformation and the expression levels of ThPDC at different timepoints and under different degrees of flooding stresses were detected by real-time PCR.The results showed that ThPDC contained a intron with length of 3927 bp and an open reading frames(ORF)with length of 1812 bp,which encoded 603 amino acids.The protein sequence of ThPDC exhibited 79.93%sequence identities with Cicer arietinum and 80.27%sequence identities with Nymphaea colorata.The results of subcellular localization showed that the ThPDC protein was located in the nucleus,cell membrane,and cytoplasm.The relative expression of Th PDC increased significantly in root,stem and leaf under waterlogging stress,and the expression levels of ThPDC in totally submerged(TS)plants were significantly higher than that in half flooded(HF)plants.Moreover,the expression levels of ThPDC in roots were the highest in CK,HF and TS groups.It is suggested that ThPDC is closely related to the waterlogging resistance of Taxodium.’Zhongshanshan406’,which may be involved in regulation of waterlogging stress response of Taxodium.’Zhongshanshan406’.These results provide reference for further studies on ThPDC promoter cloning and gene function.

关 键 词：中山杉406 ThPDC基因 亚细胞定位 表达 

分 类 号：S791.34[农业科学—林木遗传育种]