Prokaryotic Expression And Purification Of The Auxin Binding Protein (ABP1) And Auxin Receptor (TIR1) In Rice

Auxin(IAA), the first phytohormone discovered in plants, plays a key role in plant growth and development. It is involved in a series of environmental and growth signal transduction, including cell elongation and division, lateral root formation and differentiation, vascular differentiation, gravitropism and phototropism of plants, apical dominance and so on. Auxin signal transduction begins with the specific binding between auxin and auxin receptor. Therefore, the study of auxin receptor is directly related to the exploration of the auxin signaling pathway.In this paper, prokaryotic expression and purification of the auxin-binding protein ABP1 and auxin receptor (transport inhibitor response, TIR1) were studied. The main results are as following:1. Primers were designed according to cDNA sequences of ABP1 and TIR1 in GenBank, next, the ABP1 and TIR1 gene were obtained by RT-PCR from the cDNA of super hybrid rice Zhu 1S. Sequencing and Blast alignment confirmed the consistency is no difference between the above sequences and the corresponding sequences in GenBank.2. Sequenced ABP1 and TIR1 were cloned to the prokaryotic expression vector pET-32a (+) by the double digestion and ligation. Afterwards, we constructed recombinant prokaryotic expression vector pET-32a-ABP1 and pET-32a-TIR1 respectively. After PCR and double digestion identification, the positive clones were screened out.3. The ABP1 and TIR1 prokaryotic expression vectors were transformed into expression host strain BL21 (DE3), then after, IPTG was added to induce expression. By exploring the temperature, time points, IPTG concentration as expression conditions, we found that the optimal expression conditions for getting fusion protein pET-32a-ABP1 was 37℃,5h and 0.8mmol/L IPTG.While there is no noticeable difference of the fusion protein pET-32a-TIR1 induced in different concentrations. Finally, we found that the expression form of ABP1 is soluble, but unfortunately the expression form of TIR1 is inclusion body.4. There are two his-tags which have high affinity with nickel in pET32a, so we can use the nickel column to obtain target proteins. In this study, we found that expression form of ABP1 was soluble so we purified it in natural conditions, at the same time,the TIR1 was purified in inclusion body conditions.The ABP1 and TIR1 protein obtained by prokaryotic expression could facilitate for fellow-on experiments of the auxin signal transduction mechanisms, analysis of protein molecular structure and the development of biosensors for measure auxin detection.