The Cloning And Characterization Of Auxin-binding Protein TaABP1 In Wheat

Auxin as the most important hormones in plant growth and development,it can promote cell growth,organ development and fruit ripening.Therefore,cloning and studying of auxin-related genes is of great significance to understand the mechanism of auxin regulation in plant growth and development.There are many functional genes that related to plant development have been isolated and identified,however,the auxin receptor protein gene TaABP1 which has been combined with auxin to participate almost all the growth and development of plants remains unclear.Moreover,discovering new proteins that interact with TaABP1 helps to explain its multiple functions.In this study,the TaABP1 gene was isolated from the wheat for the first time and analyzed by bioinformatics,subcellular localization and prokaryotic expression,furthermore,the functional analysis of TaABP1 was conducted in Arabidopsis thaliana via transgenic technology.Moreover,the psaO gene,one of the candidate gene that expressed the Photosystem I subunit O,was screened by the Yeast Two Hybrid system and analyzed for molecular characterization.The results of this study are as follows:1.Auxin binding protein gene TaABP1(1)A novel wheat ABP1 gene was isolated and characterized by RT-PCR and RACE from callus tissue,the complete TaABP1 cDNA sequence is 621 bp and encodeing a 206 amino acids protein.(2)The deduced TaABP1 protein is belong to cupin gene family and is highly conserved across a broad range of plant species.(3)TaABP1 protein was expressed in E.coli and its identity was confirmed by mass spectroscopy.(4)TaABP1 is mainly localized in the cytoplasm and plasmalemma in wheat protoplasts.(5)Real-time PCR showed that TaABP1 transcripts are more abundant in developing floral organs,particularly in actively dividing calli,pistils,glumes,stamens and spikelets,than in stems,leaves and roots.(6)To elucidate the function of TaABP1 in plant responses to auxin,TaABP1-overexpressing Arabidopsis thaliana lines were generated and compared with mutant abp1-c1 and wild-type Arabidopsis.When grown in the presence of auxin,the overexpression lines had slightly delayed growth,but the germination rate of these lines was slightly higher than for the abp1-c1 mutant and WT.When cultured vertically to observe root elongation,the length of the primary root was shorter while the lateral root number increased dramatically in seedlings overexpressing TaABP1.When grown in the absence of auxin,seedlings overexpressing TaABP1 had auxin-related physiological responses: leaf margins bending to the abaxial surface,petioles tilted upward,decreased leaf area and increased number of rosette leaves.In contrast,these responses were little altered in the abp1-c1 mutant.Moreover,the expression levels of early auxin-regulated genes in seedlings overexpressing TaABP1 were very sensitive to auxin application;transcription of these genes was substantially and quickly up-regulated.These results suggest that TaABP1 is a multifunctional regulator of plant growth and development that acts through the auxin-induced signaling pathway.2.Photosystem I subunit O TapsaO(1)The full-length TapsaO is 659 bp and which encodes a 144 amino acids protein was isolated via Yeast Two Hybrid system.(2)There are two transmembrane domain in PsaO which is a secretory membrane protein,the phylogenetic analysis showed the protein is homologous conversed across a broad range of plant species.(3)Real-time PCR showed that the expressions of PsaO are tissue-specific and more abundant in leaf organs compared with calli and roots.(4)The TapsaO has responded to the stress treaments of ABA,drought,salt,hydrogen peroxide and dark,which indicated that it might be involved in photosynthesis and stress response of wheat.