Cloning And Functional Analysis Of Plant Transcription Factor Genes OsHOX12, OsHOX14and ABS2

The plant specific HD-Zip transcription factors include a conserved60or61amino acidsequence, known as the homeodomain (HD) motif, and a Zip motif that is immediatelyadjacent to the C-terminus of the HD motif. The HD-Zip transcription factor family membersplay vital roles in plant development, morphogenesis and plant's responses to biotic andabiotic stresses. In barley, a recessive mutation in Vrs1changes two-rowed barley tosix-rowed barley. The Vrs1(HvHox1) gene encodes a HD-Zip transcription factor. The spatialand temporal specificity of Vrs1gene expression suggests that VRS1is a transcription factorinvolved in the development (suppression) of lateral spikelets in two-rowed barley. Sequenceanalysis showed that rice genes OsHOX12and OsHOX14were homologous of Vrs1. In thisstudy, we isolated these2novel HD-Zip I genes from rice, OsHOX12and OsHOX14, andcarried out bioinformatics analysis, functional analysis, expression pattern analysis,subcellular localization analysis and DNA-binding activity analysis to investigated thefunction of these2new transcription factor.The petal identity is determined by class A (APETALA1(AP1), APETALA2(AP2)), class B(APETALA3(AP3), PISTILLATA (PI)) and class E (SEPALLATA(SEP)) genes. Some other factors, such asPETAL LOSS (PTL) and RABBIT EARS (RBE), have been reported to be involved in the initiation andgrowth of petal primordium. Research on the mechanism of petal development can contribute to the basictheory research as well as later application, but till now the molecular mechanisms about the petaldevelopment remain poorly understood. In our previous work, we isolated an Arabidopsis B3transcriptional factor member, ABS2. Using an activation-tagging method, we isolated a RAVtranscriptional factor member, abs2-1D (abnormal shoot2-1D). In this study we found thatthe phenotype of abs2-1D was caused by NGAL1(ABS2). We cloned the ABS2gene, andcarried out bioinformatics analysi, functional analysis, tissure expression analysis and ABS2sub-cellular localization, to provide data for exploring the functional mechanism of ABS2inthe petal development.The main results are as follows:(1)The full length cDNA of OsHOX12and OsHOX14were identified from rice by RT-PCR approach, sequence and phylogenetic analysis showed that OsHOX12and OsHOX14were homologous of Vrs1, belonging to the HD-Zip subfamily I and containing typical HDdomain. OsHOX12,OsHOX14and Vrs1were in the same clade. OsHOX14is more closelywith Vrs1than OsHOX12.(2) To investigate the functions of these HD-Zip proteins, we generated OsHOX12andOsHOX14over-expression transgenic plants. Transgenic plants were identified at RNA landDNA levels by Northern and Southern blotting. The result showed that the growth of thetransgenic plants was obviously suppressed compared to the non-transgenic ones.Over-expression of OsHOX12causes apical meristem up and low fertility. Over-expression ofOsHOX14causes the panicle couldn't come out from the sheath, and sterility. Furtherhistological section showed there were differences in the structures of sheath betweenover-expression plants and the WT.(3) oshox12T-DNA insertion mutant plants exhibited no visible phenotypic alterationscompared with the WT; oshox14mutant causes early mature.(4) The promoter regions of the OsHOX12and OsHOX14gene were fused to GUSreporter gene in pCAMBIA1391Z vector, respectively and the constructs were transformedinto rice callus and Arabidopsis by Agrobacterium-mediated transformation and floral dippingmethod. Histochemical analysis of the GUS activities indicated that pOsHOX12::GUS washighly expressed in anther, glume and palea in mature plants; pOsHOX14::GUS was mainlyexpressed in reproductive organ, such as anther and pistil.(5) Tissue expression profiles revealed by semi quantitative RT-PCR showed thatOsHOX12and OsHOX14were highly expressed in10DAF and15DAF of young panicle.OsHOX12and OsHOX14were found to be expressed in roots and stems.(6) The subcellular localization results showed that the OsHOX12and OsHOX14proteins were both localized in the nucleus.(7) Yeast one hybrid and protoplast transformation results indicated that both OsHOX12and OsHOX14could bind AH1(CAAT(A/T)ATTG) DNA sequence.(8) Phylogenetic analysis showed that ABS2belongs to the RAV subfamily of B3transcription factor, and was closely related to NGA genes,which are involved in flower organdevelopment..(9) Overexpression of ABS2showed the same phenotypes as the abs2-1D, and alsocaused petal loss phenotype,(10) There were no obvious phenotypic differences between abs2T-DNA insertionmutant and the WT. (11) Tissue expression profiles revealed by semi quantitative RT-PCR showed that ABS2was highly expressed in the flower, and it also expressed in roots and siliques, but no signialwas found in other tissues examined.(12) The sub-cellular localization results showed that the ABS2proteins was localized inthe nucleus,(13) The promoter region of the ABS2gene was fused to GUS reporter gene in pCB308vector and the construct was transformed into Arabidopsis, T1transgenic plants wereobtained.