Molecular Cloning And Functional Analysis Of A NAC Gene OoNAC72 From Oxytropis Ochrocephala Bunge

Oxytropis ochrocephala Bunge,one of poisonous grass,distributed widely in the northwest of China,which causes a tremendous losses of Livestock husbandry and destroys the grassland ecological balence.O.ochrocephala are mainly distributed in the bad environments such as drought,less rain and cold.They can rapidly spread in these areas,which is closely related to its strong resistance.Therefore,the study about its resistence mechanism is a significant breakthrough on explaining the spreading mechanism of O.ochrocephala.At present,studies about O.ochrocephala are mainly concentrated in the investigations and statistics of distribution situation,allelopathy and toxicology research et al,and have not ever touched upon the resistance mechanism.Many studies have shown that transcription factors involving in transcription regulation under adversity stress are crucial to plant growth.Now,in this study,we used the NAC transcription factors as the entry point and took full advantages of the received transcriptome sequencing data,screened the key NAC transcription factors participanting in theadversity stress of O.ochrocephala.In order to make clear its regulative way and explore the molecular mechanism of the resistence of O.ochrocephala,we carried out bioinformatics analysis,the analysis of biological characteristics,expression spectrum analysis and functional verification.In this study,the final aim is to provide a new basis to explain the internal mechanism of the spread of O.ochrocephala.(1)We determined Unigene comp71824 annotating for NAC transcription factors as the research target according to the data analysis of the transcriptome sequencing of O.ochrocephala.Then,we designed the specific primers by using primer 5.0 software and successfully cloned this gene from O.ochrocephala seedling,named OoNAC72.The length of OoNAC72 gene sequence is 1376 bp,ORF is 996 bp,and starting sites of codon locates in 49 bp position,termination sites of codon locates in 1045 bp position.The OoNAC72 gene encodes 331 amino acids.NCBI BlastX analysis found that N-terminal of O.ochrocephala OoNAC72 gene contains NAM domain structure which is a long 122(98-220)amino acids belonging to the typical NAC transcription factors.(2)The bioinformatics analysis of the cloned O.ochrocephala OoNAC72 gene sequences found that the protein which formed by amino acids of the O.ochrocephala OoNAC72 did not have the membrane spaning domains,signal peptide,and strong hydrophilicity.Subcellular localization analysis speculates that OoNAC72 locates in the endo-nucleus within the nucleus.Protein encoded by OoNAC72 gene contained 7 glycosylation sites and 19 phosphorylation sites.Mutiple sequence aligment between OoNAC72 and other known NAC proteins showed a high homology in their conservative areas,and the NAC domain of OoNAC72 protein can be divided into five regions(A to E).Phylogenetic tree analysis indicated that the sequence of OoNAC72 protein amino acids was closely related to those of Medicago truncutula and Cicer arietinum,with highest homology.(3)We constructed the plant fusion protein expression vector pCAMBI1302-OoNAC72-eGFP,and analyzed the subcellular localization of OoNAC72 by Agrobacterium-mediated transient transformation.The results showed that pCAMBI1302-OoNAC72-eGFP was mainly distributed in the nucleus of tobacco epidermal cells and was consistent with the characteristics of the transcription factor.We also constructed the recombinant vector pGBKT7-OoNAC72 and analyzed the transcriptional activation of OoNAC72 transcription factor by yeast one hybrid system.The results showed that OoNAC72 gene had transcriptional activation activity.(4)The qRT-PCR analysis indicated that the expressions profiles of O.ochrocephala OoNAC72 gene was differently induced by stress of drought,cold,salt and exogenous hormone ABA,GA,ETH.Abiotic stress can significantly induce OoNAC72 gene expression,and the most obvious change is under the stress of ABA,but ETH and GA is not.The results showed that the expressions of OoNAC72 gene induced by adversity abiotic stresses may rely on ABA signaling transaction pathway.(5)We transformed OoNAC72 gene into Arabidopsis(Col-0)by Agrobacterium mediated transformation method.Then through the offspring plants screening by hygromycin culture medium and the DNA level testing of positive transgenic Arabidopsis.The results showed that OoNAC72 gene has been transformed into Arabidopsis genome.