Characterization And Phylogenetic Analysis Of Kr(u|")ppel-Like Transcription Factor (KLF) Gene Family In Tree Shrews (Tupaia Belangeri Chinensis)

Research Objectives:Kriippel-like factors (KLFs) are a family of three tandem zinc finger transcriptional factors which regulate cell proliferation, differenciation, apotosis and carcinogenesis. The phylogenetics and expression patterns of KLFs have not yet been studied in tree shrews, a novel animal model with much closer relationship to primates than rodents. So we need to identify 17 TS-KLFs, and phylogenetically analyse the whole CDSs and zinc finger domains among different species. The N-terminus also should be identified for CtBP and Sin3A binding motifs and NLS. Meanwhile, it’s meaningful to distinguish the posttranscriptional modification sites of TS-KLF5, including ubiquitination, phosphorylation, acetylation and sumolytion. At last, the expression pattern of TS-KLFs should be confirmed on mRNA or protein level respectively.Research Methods:PCR primers for amplification of undetermined sequences of TS-KLF2,5,9 were designed based their orthologs. Amplified target sequences were cloned into T vector, then the sequences were identified by sequencing. Other 14 TS-KLFs were accessed from NCBI and tree shrew database. Then Clustal X was used to align the whole anmino acids and zinc finger proteins among different species or not. The phylogenetic trees were performed by MEGA5 using NJ method. The online BoxShade was utilized to conduct multiple alignments to determin some important motifs or modification sites. In addition, Q-PCR, western blot and immunohistochemistry were manipulated for identifing the TS-KLFs expression pattern.Results:Gene identification confirmed the CDS of TS-KLF2,5,9. Sequence deposited to genebank gained the following accession numbers:KT948683, KT948685, KT948684. Tree shrew has 17 different KLFs, the whole amino acids phylogenetic analysis indicates that KLFs are evolutionary conserved among different species. Tree shrew highly conserved zinc finger domains have an average of over 97% homology with human, slightly higher than mouse. The zinc finger amino acids phylogenetic tree shows TS-KLF1,2,5,17 sharing closer evolutionary relationship with their orthologs in human compared to those in mouse. To high variant N-terminus motifs analysis, TS-KLF3,8,12 have conserved PVDL/T motif for CtBP binding, and TS-KLF9,10,11 contain Sin3A bing motif with AA/VXXL. Multiple sequences of N-terminus alignment revels that TS-KLF9,10,11 own high conserved NLSs. Further analysis of TS-KLF5 demonstrates some important posttranslational modification sites exist, for example, CPD and PY domains for ubiquitination, S153 phosphorylation, K369 acetylation, K162 and K209 sumolytion. Q-PCR, western blot and IHC revealed that TS-KLFs had different expression patterns in different tissues, even various between mRNA and protein levels.Conclusions:Tree shrew KLFs is more evolutionary conserved than mouse. There exists many similarities between tree shrew and human on KLFs structure and function. Tree shrew owns great potential to be outstanding animal model for human diseases. This article provides some fundamental information for future studies regarding structure and function of the TS-KLF gene family.