美洲黑杨木质素生物合成转录因子PdLim1基因的克隆、表达及植物表达载体构建

Isolation,expression and construction of plant expression vector of PdLim1,a transcription factor involved in the lignin biosynthesis in Populus deltoides

在植物体内,Lim1基因与木质素生物合成酶基因启动子区域富含AC的Pal盒结合,转录调控木质素的生物合成过程。该研究组合利用生物信息学和realtime PCR方法,首次从美洲黑杨形成层cDNA中分离出PdLim1cDNA全长,并进行了测序和序列分析。结果表明,克隆的美洲黑杨PdLim1cDNA片段总长为952bp,基因内部含有完整的开放阅读框架,大小为594bp,可编码长度为197个氨基酸残基的蛋白质,所推导的蛋白质氨基酸序列与拟南芥AtLim1和水稻OsLim1蛋白的同源性分别为82.1%和78.2%。组织特异性realtime PCR结果显示,PdLim1基因在杨树根、茎、叶片和顶端分生组织中均有表达,但其表达模式却不同:PdLim1在成熟叶片和成熟木质部中表达丰度最高,在树皮、根部、韧皮部和形成层表达丰度较高,在未成熟木质部有少量表达,在顶端分生组织中表达丰度最低。在此基础上,以pBI121为表达载体,构建了在CaMV35S启动子驱动下,PdLim1基因的正义(pBI121-CaMV35S-sense PdLim1)和反义(pBI121-CaMV35S-antisense PdLim1)类型的双元植物表达载体。该研究为杨树PdLim1的基因工程改良木材纤维品质性状提供了重要的理论依据,具有潜在的应用价值。

In plants,the Lim1 gene regulates the lignin biosynthesis by binding the AC-rich motif,the Pal box,which is an important cis-acting element located in the promoter region of genes encoding enzymes in the lignin biosynthesis. A full-length cDNA clone encoding Lim1 was isolated from the cDNA prepared from the cambium zone of Populus deltoides by the approach of biological informatics and realtime PCR method. The cDNA was 952 bp in length with an open reading frame （ORF）,which is capable of encoding a protein of 197 amino acids. The deduced protein sequence of the PdLim1 shared 82.1% and 78.2% identity with those of Arabidopsis thaliana and Oryza sativa Lim1,respectively. Realtime PCR results indicated that PdLim1 was expressed in root,stem,leave and apical shoot meristems,but different in expression level. The PdLim1 transcripts were the most abundant in mature leaf and xylem,with higher expression in bark,root,phloem and cambium,with some expression in the immature xylem,but a low-abundance was detected in apical shoot meristems. The plant binary vectors,in which the PdLim1 cDNA was introduced in sense or antisense orientation to the CaMV35S promoter,i.e. pBI121-CaMV35S-sense PdLim1 and pBI121-CaMV35S-antisense PdLim1,were constructed. The results,therefore,provide important foundation for the improvement of wood fiber property by the strategy of gene engineering of PdLim1 and have potential application in tree breeding.