杨树PeHAM1和PeHAM2基因的克隆、表达及亚细胞定位

Cloning, Expression and Subcellular Localization of PeHAM1 and PeHAM2 Genes in Poplar

GRAS蛋白是一类植物特有的、古老的转录因子家族,在植物整个生长发育过程中都发挥着调控作用。以‘南林895’杨(Populus deltoides×Populus. euramericana cv.'Nanlin895')不定根为材料,通过RACE技术克隆鉴定了两个GRAS转录因子,Pe HAM1和PeHAM2。Pe HAM1基因cDNA全长为2 032 bp,包含一个1 647 bp的开放阅读框,推测编码548个氨基酸;PeHAM2基因cDNA全长为2 856 bp,包含一个2 106 bp的开放阅读框,推测编码701个氨基酸。Pe HAM1和PeHAM2蛋白C端都具有LHRI、VHIID、LHRII、PFYRE和SAW等几个典型的保守结构域;系统进化分析表明两者均属于GRAS转录因子家族中的HAM亚家族。PeHAM1基因在杨树不定根发育过程中具有动态的表达模式,在茎和叶中几乎不表达;PeHAM2基因在根、茎、叶中均有表达。原生质体瞬时表达分析表明,Pe HAM1和PeHAM2蛋白均定位于细胞核。进一步分析发现,PeHAM1和PeHAM2基因序列均存在ptc-miR171的剪切位点。本研究表明PeHAM1和PeHAM2可能参与了杨树不定根的生长和发育,这为进一步探究HAM亚家族基因在杨树不定根发育过程中的调控机理提供了坚实的基础。

GRAS protein is a kind of ancient family of plant-specific transcription factor, which can play a regulatory role in the whole process of growth and development of plants. The PeHAM1 and PeHAM2 genes of GRAS transcription factors were cloned and identified by RACE technique using adventitious roots of ‘Nanlin895’poplar(Populus deltoides×Populus. euramericana cv.‘Nanlin895’). The full-length cDNA of PeHAM1 gene was2 032 bp, which contained an open reading frame(ORF) of 1 647 bp, suggesting that encoding 548 amino acids. The full-length of PeHAM2 gene was 2 856 bp and contained an ORF of 2 106 bp, inferring that encoding701 amino acids. Both the PeHAM1 and PeHAM2 proteins had typical domains in the C-terminal, such as LHRI,VHIID, LHRII, PFYRE and SAW. Phylogenetic analysis showed that both PeHAM1 and PeHAM2 belonged to the HAM subfamily of the GRAS transcription factor family. PeHAM1 gene had a dynamic expression pattern in adventitious root development of poplar and was hardly expressed in stem and leaf. PeHAM2 gene was expressed in roots, stems and leaves. Transient expression analysis of protoplasts showed that both PeHAM1 and PeHAM2 proteins were located in the nucleus. Further analysis revealed that there were ptc-miR171 cleavage sites in the PeHAM1 and PeHAM2 gene sequences. This study indicated that PeHAM1 and PeHAM2 might be involved in the growth and development of adventitious roots of poplar, which would provide a solid foundation for further exploration of the regulation mechanism of HAM subfamily genes during the development of adventitious roots of poplar.