Preliminary Study Of Salvia The Smtctp Cloning And Function

The translationally controlled tumour protein(TCTP),which is also named as 'P21','P23'. 'Q23',is a highly conserved protein and widely expresses in all eukaryotic organisms.It was initially described as a growth-related protein in mouse ascites and erythroleukemia cells,but recenl studies demonstrated that TCTP is a multifunctional protein in animals.A series of reports highlighted the importance of TCTP for an extracellular function as a histamine release factor and for intracellular function as a microtubule-stabilizing protein,a calcium-binding protein,an antiapoptotic protein,a guanine nucleotide dissociation inhibitor in protein synthesis.However,each of these designations emphasizes only a particular function of this interesting protein and does not fully appreciate its wide-ranging biological importance.In addition,there were few reports about the biological functions of TCTP in plants.In this study,the translationally controlled tumor protein gene was cloned and the primary biological function was analyzed in Salvia miltiorrhiza Bunge.The main results were as follows:1.An entire cDNA sequence was cloned using RT-PCR technology from Salvia miltiorrhiza,and it was 591 bp long(named as SmTCTP,Genebank accession number:EU182720).Its open reading fragment(ORF) was 507 bp long,which encoded 168 amino acids.Sequence alignments of TCTP sequences revealed a high degree of conservation over a long period of evolution.SmTCTP(putative amino acid sequence of the SmTCTP) shared more than 80%identities with the TCTP sequences reported in other plants,such as Arabidopsis thaliana,Ipomoea nil,Fragaria x ananassa,Hevea brasiliensis,Cucurbita maxima,Brassica oleracea,Triticum aestivum,Zea mays,Elaeis guineensis, Oryza sativa.SmTCTP contained two TCTP's signature motifs,TCTP1 and TCTP2,and had two conserved domains,the tubulin-binding region and the Ca²⁺-binding area.The DNA sequence of SmTCTP was also obtained,which was 1619 bp long and consisted of five extrons and four introns. There was consensus sequence in the intron / extron border.2.SmTCTP and other TCTP sequnces,which were registered in GenBank,were analyzed by the tools of bioinformatics such as TargetP,TMHMM,Protscale,SOPMA and SWISS-MODEL in the following aspects:the composition of nucleotide acid sequence and amino acid sequence,transit peptides,transmembrane topological structure,hydrophobicity or hydrophilicity,secondary and tertiary structure of protein,and so on.The results showed that SmTCTP is a hydrophilic, non-transmembrane protein without transit peptides,and included aβ-stranded core area and anα-helical core region. 3.Real-time quantitative PCR technique was performed to assess the specific expression profiles of SmTCTP.The results showed that the order of organ expression from large to small was leaf,stem, root,and that TCTP levels were highly regulated by various environmental factors such as mechanical wounding,NaCl,CuSO₄,In which,500 mg/L CuSO₄ reduced the transcription level of SmTCTP gene in a short period of time,the expression reduced to a minimum when dealing 4h,after which the expression rebounded,and the expression was in the manner of wave when exposed to wounding.These results revealed that SmTCTP involved in stimulating response to various environmental factors.4.We constructed the overexpression vector pSmTCTP1 and RNA inference vector pSmTCTP2, which were introduced into S.miltiorrhiza by Agrobacterium tumefaciens-mediated transformation, and 9 overexpression transformated plants and 21 RNAi transformated plants were obtained by PCR analysis.The later transformation efficiency was about 67.7%.The expression level of SmTCTP gene was markedly down-regulated in all detected RNAi transgenic plants,whereas it was not overexpressed in all deceted overexpression transgenic plants.Compared with the control plants, most of the transformed plants did not showed abnormal phenotypes in normal culture conditions, but when they were cultured in 1/2 MS media with 40 mmol/L NaCl,the roots of all cultured transformed plants growed earlier and longer than control,and when they were cultured in the condition of 50 mg/L CuSO₄,the most leaves of transformed plantlets appeared perished or etiolated spots.