落叶松LkERF-B2基因的克隆及表达载体的构建

Cloning of LkERF-B2 Gene in Larch(Larix kaempferi) and Construction of its Expression Vector

AP2/ERF转录因子在植物的生长发育过程中具有非常重要的作用,包括调节花分生组织的形成,花器官的发育和参与胁迫反应。为探究落叶松（Larix kaempferi（Lamb.）Carr）中Lk ERF-B2基因的功能,本研究以日本落叶松为材料,利用逆转录PCR（reverse transcription-PCR,RT-PCR）技术从落叶松扩增获得了1个ERF类转录因子基因的全长编码序列,命名为Lk ERF-B2,该基因全长编码序列长度为1 344 bp,编码447个氨基酸,具有1个AP2结构域。同源性分析和系统进化分析结果表明,Lk ERF-B2与云杉（Picea sitchensis ABK25082.1）的亲缘关系最近,同源性达79.0%。生物信息学分析结果显示,该基因编码的蛋白质为亲水性蛋白,无跨膜区域,具有5.59%的α-螺旋,3.58%的β-折叠,90.83%的环肽链。最后将落叶松Lk ERF-B2基因连接到植物表达载体Pri201-AN-GUS上,成功构建了植物表达载体Pri201-AN-GUS-Lk ERF-B2。以上研究结果表明,成功克隆的落叶松的Lk ERF-B2基因属于AP2/ERF家族,具有明显的保守性和特异性。该基因的克隆及分析为揭示落叶松Lk ERF-B2基因的功能特征提供了理论依据。

AP2/ERF transcription factors play a very important roles in the growth and development of plants,including the regulation of flower meristem formation,the development of floral organs and the participation of stress.To investigate function of Lk ERF-B2 in Larch（Larix kaempferi（Lamb.） Carr）,in this study,Japanese larch was used as the material.The full-length encoding sequence of of ERF transcription factor gene was amplified from Larch by reverse transcription-PCR（RT-PCR） technique,named Lk ERF-B2.The length of the full length encoding sequence of Lk ERF-B2 was 1 344 bp,encoding a protein of 447 amino acids and had one AP2 domain.Phylogenetic analysis and homology analysis showed that,Lk ERF-B2 protein had the highest sequence similarity（about 79.0%） and closest genetic relationship to spruce（Picea sitchensis ABK25082.1）.Results of bioinformatics analysis showed that Lk ERF-B2 was a hydrophilic protein,and it had no transmembrane region.Lk ERF-B2 contained 5.59% alpha-helix,3.58% beta-sheet and 90.83% loops.Finally,Lk ERF-B2 gene was ligated into the plant expression vector Pri201-AN-GUS,and the plant expression vector Pri201-AN-GUS-Lk ERF-B22 was successfully constructed.All the results demonstrated that Lk ERF-B2 gene identified from Larix kaempferibelonged to AP2/ERF family,and this gene had obvious conservation and specificity.Cloning and analysis of this gene would provide a theoreticalbasis for revealing the functional characteristics of Larix kaempferi Lk ERF-B2 gene.