Cloning Auxin-binding Proteins ABP1 Gene CDNA Of Ramie [Boehmeria Nivea (Linn.) Gaud.] And The Preliminary Studies On Its Function

Ramie is one of the important fiber crops in China.Its cortex fibers are the main product that to be harvested and be the important textile material.Furthermore,the annual multi season growths of ramie make it interesting to study on its growth regulation especially in like phytohormones mechanism.The research about mechanism of auxin is our first choice.Recent researches on molecular mechanism of the first founded auxin binding protein(ABP1) in some model plants have made great progress and providing perfect alternative information for understanding the molecular mechanism of auxin binding protein in ramie.In this research the ramie auxin binding protein gene cDNA sequence was cloned by combination the techniques of degenerate PCR and RACE.The cloned cDNA for auxin binding protein of ramie is designated as BnABP1.Sequencing analysis showed the cDNA sequence be cloned is 849 bp,which can be translated into a putative 189 amino acids protein.Bioinformatics analysis ascertained this cDNA sequence is the ramie auxin binding protein gene cDNA and the information was submitted to GenBank with an accession number EU195804.The putative protein shares a highest homologous with the protein in Gossypium hirsutum.In order to investigate the expression model and regulation mechanism of ABP1 in ramie,the nonconservative sequence at 3'terminal of BnABP1cDNA was selected as target and it is amplified by semi-quantitative RT-PCR.The different tissues of shoot, leaf,stem and root are subject to screen.18S rRNA gene was taken as an inner control. The integrate optic density(IOD) of amplified BnABP1 and 18S rRNA molecules in agarose gel were detected with gel analysis software and defined the ratio of IOD as relative expressive quantity.The results indicated that BnABP1 expressing both in stem, leaf and bud but it is not detectable in root.The expression level from high to low is bud, leaf and stem with the relative content 0.755,0.632,0.360 respectively.The expression data presented shows that BnABP1 is activity transcripted in many tissues but is leak in the root in ramie in its third gowth season.BnABP1 gene cDNA encoding sequence was amplified and inserted into the multiple cloning sites of prokaryotic expression vector pET-32a.The prokaryotic expression of BnABP1 are induced in E.coli BL21(DE3) by IPTG.A preditcted pretein band can be induced obviously on SDS-PAGE. The BnABP1 encoding sequence was also ampilified and inserted into a plant transformation vector with the green fluorescent protein(GFP) chiemria expression pCAMBIA1300.The chimera protein is under the control of promoter CaMV 35S.The recombinated pCAMBIA1300-GFP-BnABP1 was used to transform tobacco WS38 via leaf disc infection with Agrobacterium tumefaciens.The fluorescence signal was intensive in plasma membrane and inner membrane system in transgenic cells.It reveals that BnABP1 is more likely located in cell membranes.