Sequence Analysis, Prokaryotic Expression And RNAi Vector Construction Of ABP Gene Of Oryza Sativa

Auxin indoleacetic acid(IAA) which ubiquitously exists in higher plants plays a key role in manipulating growth and development of plants.Auxin binding protein(ABP) is the pioneer in signal transduction of auxin manipulation.Its concentration in cells impacts the growth of cells and the sensitivity to auxin directly.Rice is one of the most alimentary crops and model plants as well.Thorough researches of the function and regulation of ABP gene from rice with gaining mutant materials could build a firm foundation for genetic improvement of rice.The auxin-binding protein cDNA of rice was amplified and cloned.Then this sequence was analyzed by bioinformatics methods,then prokaryotic expression vector and RNA interference vector involved this cDNA were constructed.The following description was the main experimental results:1.A full length of rice ABP cDNA was cloned,the sequencing result was logged into GenBank numbered as EU595028.This cDNA which contains GAGA transcriptional cis-acting elements,ribosome binding site(RBS),tailing signal(AATAAA), glucocorticoid acting site and so on is 920bp long except polyA,encoding 206 amino acids.Sequence analysis indicated that there is 79%nucleotide homology with auxin-binding protein gene of Zea mays,and homology of the amino acid sequence is 78%.Moreover,the deduced polypeptide chain had three conservative domains,one endocytoplasmic reticulum identification signal KDEL motif at the C terminus,and one glycosylation site(NSTF) as well as the auxin-binding protein of Zea mays, which indicated that the conservatism of auxin-binding protein gene is very high among different plants.There is a transmembrane part at the signal peptide(1-44 amino acids) by transmembrane prediction.Therefore,ABP from rice might be a secreting protein mainly existing in endocytoplasmic reticulum,and anchored by another protein during signal transduction at plasma membrane.The secondary and tertiary structure prediction indicated that main functional domains concentrate in theβ-barrel area which might be involved in its biological function.And the auxin-binding area also might locate in this area according to the aggregate analysis.2.Prokaryotic expression vectors containing the full length cDNA or opening reading frame were constructed respectively.GST fusion expression was carried on in E.coli BL21(DE3) pLysS system hoping to obtain purified ABP of rice for biochemical, immunology and protein interaction researches.However,no different bands between the samples and negative controls were observed through several rounds of time and IPTG concentration gradient screening.Hence,it is possible that the expression condition is complicated,prokaryotic expression system is not competent for it or the ABP of rice is toxiferous for growth of E.coll.3.For manipulating RNA interference in plants by induction elements outside,HSP18.2 heat-inducible promoter from Arabidopsis thaliana was cloned into eukaryotic expression vector pCAMBIA1301.It was improved that this promoter could start a foreign gene's expression in rice calli with GUS gene as a reporter gene.The locations of introns and exons of ABP gene were identified through alignment of ABP cDNA sequence and genomic sequence of rice.An intron splicing hairpin DNA fragment (one intron of rice ABP gene as the loop and the next exon as the inverted repeated arms) was obtained by recombinant PCR method.And then a heat-inducible RNAi vector named as pCAMBIA1301-HSP-ABP was constructed,followed by infection of rice calli to obtain inducible mutant materials specific to ABP gene.The dead calli after infection implied that growth shutdown or lethality might because of ABP shortage.Thus,a gene encoding repressor protein would be inserted into the vector pCAMBIA1301-HSP-ABP in next research.