棉花PTS2受体基因(GhPex7)的克隆及表达分析

利用cDNA—AFLP差异片段F010，通过RACE延伸、EST、检索等方法获得了一个棉花过氧化物酶体定位信号2受体蛋白基因(peroxisomal targetingsignal type 2 receptor，GhPex7p)的编码序列。该cDNA包含一个954bp的开放阅读框，编码317个氨基酸，推测其等电点为5．603。同源性分析表明：推测GhPex7与拟南芥、酵母、果蝇、小鼠和人的Pex7p基因存在序列相似性，其中与拟南芥的同源性最高，为83％，并具有3段WD-40蛋白家族的保守域，与拟南芥AtPex7的编码蛋白同类。Southern杂交结果表明该基因在陆地棉基因组中存在两个拷贝。Northern blotting和RT-PCR分析表明该基因在棉花根、茎、叶、花、胚珠和纤维中均表达，但茎、叶组织中的表达水平明显高于胚珠和纤维。

Cloning and Characterization of the PTS2 Receptor Gene (GhPex7) from Cotton (Gossypium hirsutum L.)

Peroxisomal targeting signals (PTS),including PTS1 and PTS2,were proposed to play an important role in introducing proteins into the peroxisomal matrix.Pex7,the PTS2 receptor,is crucial to the import of PTS2 proteins.Based on the sequence of a fragment (F010) recovered from cDNA AFLP,the full length cDNA sequence was obtained by RACE and the contig in cotton ESTs,and the coding sequence was further cloned.The GhPex7 cDNA,1314 bp in length,contained 5 non coding (77 bp) upstream,3 complete downstream with polyA signal tail and ORF of 954 bp,which coded for a deduced protein of 317 amino acids.The deduced protein had a predicted MW 35 57 kDa and a pI of 5 603.Homology analysis demonstrated that GhPex7 protein contained three highly conserved domains and three G bata domains of WD 40 proteins family.The sequence of nucleotides and amino acids shared 83% and 76% identity with known Arabidopsis AtPex7,respectively,and its amino acid sequence shared from 28% to 42% identity with those of Drosophila melanogaster,Saccach.cerevisiae,Mus musculus and Homo sapiens. Southern blotting suggested that at least two copies of GhPex7 gene existed in Gossypium hirsutum genome.By Northern blotting and RT PCR analysis,expression of the GhPex7 was detected in roots,stems,leaves,buds,ovules and fibers,however it was stronger in leaves and stems than in other tissues.At the stage of cotton's ovules and fibers development,RT PCR analysis also indicated that expression activity of GhPex7 in ovule at 0DPA mutant plant (no fiber) was stronger than in wild type plant,its expression in wild type fiber at 23DPA was stronger than at 12,16 DPA too.