中山杉406ThSHR3基因的克隆、表达及蛋白互作研究  

Cloning,Expression and Protein Interaction of ThSHR3 Genes in Taxodium hybrid‘Zhongshanshan406’

作　　者：宣磊[1] 王芝权[1] 殷云龙[1] 华建峰[1] XUAN Lei;WANG Zhi-quan;YIN Yun-long;HUA Jian-feng(Jiangsu Engineering Research Center for Taxodium Rich.Germplasm Innovation and Propagation,Institute of Botany,Chinese Academy of Sciences,Nanjing 210014,Jiangsu,China)

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

出　　处：《林业科学研究》2021年第4期32-39,共8页Forest Research

基　　金：国家自然科学基金面上项目(31870592);江苏省中国科学院植物研究所自主创新项目(JSPKLB202048);江苏省青年基金(BK20200291)。

摘　　要：[目的]本研究在前期中山杉(Taxodium hybrid‘Zhongshanshan’)不定根转录组及蛋白组研究的基础上,克隆获得中山杉ThSHR3(SHORT-ROOT 3)基因,对其进行表达特性检测及相关功能分析,为深入研究ThSHR3基因在中山杉不定根发育过程中的调控机理提供理论基础。[方法]以中山杉406扦插苗不定根为材料,利用RACE技术克隆获得ThSHR3基因全长,并对其进行生物信息学分析。采用半定量PCR和荧光定量PCR分别对ThSHR3进行表达特性检测。通过原生质体瞬时表达检测ThSHR3蛋白的亚细胞定位情况,进一步使用双分子荧光互补(BiFC)技术验证ThSHR3的蛋白互作情况。[结果]ThSHR3基因的全长为2019 bp,其中,开放阅读框(ORF)为1446 bp,共编码482个氨基酸残基。ThSHR3蛋白C端较保守,具有LHRI、VHIID、LHRII、PFYRE和SAW等几个典型的蛋白结构域。系统进化分析表明:ThSHR3属于GRAS转录因子家族中的SHR亚家族。ThSHR3基因在中山杉不定根发育过程中呈逐渐上升的表达模式。原生质体瞬时表达实验表明:ThSHR3蛋白定位于细胞核。BiFC实验证明:ThSHR3蛋白和GRAS家族另一成员ThSCR蛋白存在着明显互作。[结论]中山杉ThSHR3和不定根发育密切相关,且在中山杉不定根的发育过程中发挥着重要作用。[Objective]To obtain the SHORT-ROOT 3 gene(ThSHR3)from Taxodiumhybrid'Zhongshanshan 406'by cloning and to identify and analyze its characteristics and related functions based on proteome and transcriptome data of adventitious roots of Taxodium hybrid'Zhongshanshan406'.[Method]The full length of ThSHR3 gene was cloned by RACE technology and the bioinformatics characteristics were analyzed by bioinformatics software.Semiquantitative PCR and real-time quantitative PCR were used to detect the expression characteristics of ThSHR3.The subcellular localization of ThSHR3 protein was confirmed by transient expression of protoplasts,and the protein interaction of ThSHR3 was verified by bimolecular fluorescence complementation(BiFC)technique.[Result]The authors assembled a full-length of ThSHR3 gene,which consists of 2019 bp nucleotide sequence,containing a 1446 bp open reading frame(ORF)encoding 482 amino acid proteins.ThSHR3 protein has conserved GRAS domains in the C-terminal,such as LHRI,VHIID,LHRII,PFYRE and SAW.Phylogenetic analysis showed that ThSHR3 belongs to the SHR subfamily.ThSHR3 gene showed a gradually increased expression pattern in the development of adventitious root.Transient expression analysis of protoplasts showed that ThSHR3 protein was located in the nucleus.Further BiFC experiment revealed that ThSHR3 protein and ThSCR protein which also belongs to GRAS protein family can interact with each other in nucleus.[Conclusion]This study indicates that ThSHR3 plays an important role in regulating the development of adventitious roots of Taxodium hybrid'Zhongshanshan 406'.

关 键 词：中山杉 SHR 基因克隆 基因表达 蛋白互作 

分 类 号：S722[农业科学—林木遗传育种]