红豆杉TcLBDs基因的克隆与表达分析  

Cloning and Expression of Lateral Organ Boundaries Domain Genes (TcLBDs) in Taxus chinensis

作　　者：李艳艳[1] 杨艳芳[1] 王俊青 王帅[1] 刘洪伟[1] 邱德有[1] 

机构地区：[1]中国林业科学研究院林业研究所林木遗传育种国家重点实验室,北京100091 [2]湖北襄阳市林业科学研究所,襄阳441052

出　　处：《林业科学》2015年第10期126-133,共8页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(31170628;31300567)

摘　　要：【目的】LBD(LATERAL ORGAN BOUNDARIES DOMAIN)转录因子在植物次生生长中具有潜在的作用。克隆红豆杉LBD基因,研究LBD在红豆杉剥皮后树皮再生过程中的表达状况,以揭示其调控作用。【方法】在红豆杉剥皮再生组织转录组数据库中搜索LBD基因,根据获得的基因序列设计引物,利用RT-PCR方法克隆得到2个红豆杉TcLBDs基因的cDNA片段,分别命名为TcLBD1和TcLBD6。进一步利用生物信息学工具对其进行分析,最后利用半定量RT-PCR对红豆杉不同组织、利用qRT-PCR技术对其剥皮再生不同时期的TcLBDs的表达进行分析。【结果】TcLBD1的cDNA包含549 bp开放阅读框(ORF),编码182个氨基酸;TcLBD6的cDNA包含687 bp开放阅读框,编码228个氨基酸。TcLBD1和TcLBD6编码的蛋白质分子量分别为19.97,25.13 kDa,序列分析表明二者在N端存在着LBD类转录因子特有的LOB结构域,都属于第1类(ClassⅠ)LBD;二者在核酸序列和氨基酸序列水平上分别有55.2%和57.9%的相似性,且蛋白质二级结构也具有较高的相似性。系统进化树结果显示TcLBD1与北美云杉的蛋白(ABK21479)聚为一簇,亲缘关系最近;TcLBD6与拟南芥的AS2/LBD6聚在一起。组织表达谱分析表明Tc LBD1在茎和木质部(含形成层)中表达量明显高于根、叶片和韧皮部(含形成层)中的表达量;TcLBD6主要在根和茎中表达。2个TcLBDs基因在红豆杉剥皮再生不同时期再生组织间的表达模式结果显示,在其剥皮再生过程中Tc LBD1基因表达量在6天时达到最高,整体呈上调表达;Tc LBD6表达水平在18天后显著上调。【结论】克隆2个红豆杉TcLBDs基因,TcLBD1和TcLBD6,这2个基因在红豆杉剥皮再生过程中均有响应,推测二者在红豆杉剥皮后树皮再生过程中起调控作用。【Objective】LBD（ LATERAL ORGAN BOUNDARIES DOMAIN） transcription factors play a potential role in plant secondary growth. The aims were to clone the cDNA of LBDs from Taxus chinensis and reveal their potential regulatory role in tissues regeneration after bark girdling by investigating the expression profiles of these LBDs. 【Method】According to the sequences of TcLBDs genes obtained from the transcriptome database of the regeneration tissues after bark girdling in T. chinensis,specific primers were designed. The two LBD genes were isolated using reverse transcriptionpolymerase chain reaction（ RT-PCR）. Bioinformatic characteristics of the cloned Tc LBDs were analyzed using online service. Lastly,the expression profiles of these genes in different tissues and at different regeneration stages after bark girdling were analyzed by semi-quantitative RT-PCR and quantitative real-time PCR（ qRT-PCR） respectively. 【Result】The results showed that the cDNA of TcLBD1 contained a 549 bp open reading frame（ ORF） in length which encoded polypeptide of 182 amino acids,and TcLBD6 contained a 687 bp ORF encoding 228 amino acid residues. The molecular weights of TcLBD1 and TcLBD6 encoded protein were 19. 97 kDa and 25. 13 k Da,respectively. The sequences analysis showed that the amino acid sequences of TcLBD1 and TcLBD6 contained one specific conserved LOB motifs in N-terminaland were classified into class Ⅰ of LBD transcription factors,both of them shared 55. 2% and 57. 9% sequence similarity in nucleotide and amino acid,respectively,and had high identity with each other in secondary structure of protein.Phylogenetic tree analysis suggested that TcLBD1 could be clustered together with protein（ ABK21479） of Picea sitchensis and they are closest in genetic relationship,TcLBD6 could be clustered with protein ASYMMETRIC LEAVES2 / LBD6 of Arabidopsis thaliana. The analysis of tissues expression patterns showed that the transcript abundance of TcLBD1 was higher in stems and xylem with cambium than that in

关 键 词：红豆杉 LBD 转录因子 生物信息学分析 基因表达 

分 类 号：S718.46[农业科学—林学]