Cloning And Functional Analysis Of Ornithine Aminotransferase Encoded Genes TaOAT And AtOAT In Wheat

Wheat is a very important crop worldwide.But wheat production is seriously affected by the gradually increased environmental stresses.It is quite necessary to improve wheat tolerances to drought and salt by exploring some resistant genes to these stresses.Proline(Pro)accumulation is a common response to both abiotic and biotic stress conditions.Ornithine aminotransferase(OAT),alternatively known as ornithine delta aminotransferase(?OAT),is a pyridoxal phosphate(PLP)-dependent enzyme involved in the conversion of ornithine into glutamyl-5-semi-aldehyde(GSA)and vice versa,which catalyses the transamination of ornithine(Orn)into GSA for proline biosynthesis.The OAT enzyme functions in stress-induced proline accumulation in cytoplasm,programmed cell death and non-host disease resistance in plants through an alternative pathway known as the ornithine pathway.Up till now,there has been no study on OAT in wheat despite the success of its isolation from rice,maize,and sorghum.This study focuses on identification and molecular characterization and functional characterization of OAT encoded genes in wheat and Arabidopsis.The achievements obtained in this study will be theoretically and practically valuable for breeders to develop new wheat varieties with tolerance to environmental stress conditions.The main results achieved in this dissertation are listed as follows.1)In total,three homoeologous OAT genes in wheat genome were cloned on group 5 homologous chromosomes by bioinformatics strategy,polymerase chain reaction(PCR)amplification and then confirmation by sequencing,named as TaOAT-5AL,TaOAT-5BL,and TaOAT-5DL.Sequence alignment between gDNA and its corresponding cDNA of TaOAT-5AL,TaOAT-5BL,and TaOAT-5DL showed that the three alleles all obtained ten exons and nine introns.Two types of transcripts for TaOAT-5AL were revealed in the course of molecular cloning,among which one transcript showed interrupted gene structure between exon 9 and intron 8.2)A phylogenetic tree was constructed and results indicated that OATs is highly conserved among prokaryotes bacteria eukaryotic animal and plants.Among eukaryotic plants,it formed two distinguished groups,one among the monocots and the other among the eudicots.The subcellular localization analysis indicated that OAT is functioned in mitochondria.Protein-protein interactions supported their role in proline biosynthesis and nitrogen reutilization through interactions with proteins,such as delta 1-pyrroline-5-carboxylate synthetase(P5CS)and pyrroline-5-carboxylate reductase(P5CR),and arginase(ARG).Promoter analysis exposed the presence of several stress responsive elements such as abscisic acid responsive element(ABRE),myeloblastosis(MYB)cis-elements,ROS-related motifs(G-box and W-box),ethylene-responsive element(ERE),heat shock element(HSE),APETALA2-like(AP-2-like)element and low temperature responsive(LTR)element,which suggested their involvement in stress regulation.Expression profiling by quantitative real-time PCR(qPCR)illustrated that TaOAT was highly induced in the wheat plants exposed to 50% PEG or 200 mM NaCl stress conditions.Its expression was highly upregulated in drought resistant cultivars as compared to drought sensitive cultivars.Presence of plant AP-2 like cis-acting element showed its potential role in floret development.Up regulated expression of TaOATs was observed in stamens at heading stage,which revealed its role in anther dehiscence.3)The gene TaOAT-5BL was overexpressed in wheat by Agrobacterium-mediated transformation for primarily functional characterization.The transgenic plants were identified by Bar QuickStix stripe and PCR.After self-pollination stable independent transgenic lines overexpressing the gene was obtained by PCR detection.On 150 mM salt containing medium,the mature embryos of transgenic plants germinated with a rate of 71-85% while the mature embryos of the wild type were germinated only with a rate of 27%.After the stressed for thirty days,transgenic plants and the wilt type showed survival rates of 35-40% and 12%,respectively.Under water withholding conditions started from three-leaf-stage for seventeen days after the drought stress,the transgenic lines gown normally while the wild type was severely affected in growth.Free proline showed that there is no difference in proline accumulation between transgenic plants and the wild type under normal condition while under drought stress condition more proline accumulation was found in the transgenic plants than the wild type plants.4)To further dissect the function of this enzyme AtOAT from Arabidopsis was expressed in two wheat verities Fielder(drought sensitive)and Ningchun4(drought resistant)by Agrobacterium-mediated transformation for its functional characterization under drought,salinity and heat stress conditions.Stable transgenic plants were obtained by haploid technique and identified by fluorescence in situ hybridization(FISH).Three independent transgenic lines were selected from Fielder and Ningchun4.Respectively,for functional characterization of the target gene.Semi-quantitative PCR revealed that the transgene was highly expressed in the homozygous transgenic wheat plants derived from Fielder and Ningchun4.On the 200 mM NaCl containing medium,the transgenic plants showed higher survival rate than the wild type Ningchun4.On the 150 mM NaCl containing medium,the transgenic plants derived from Fielder showed a higher survival rate than the wild type.Under water stress condition for 15 days,the transgenic plants from Fielder showed a much better growth status than the wild type.Under water stress condition for 18 days,the transgenic plants from Fielder showed a higher drought resistance than their wild type.Additionally,proline content was significantly enhanced in the transgenic lines derived from Fielder and Ningchun4 as compared to the two wild types under salt and drought stress condition.The POD activity was also enhanced in the transgenic plants compared to their wild types in response to salt and drought stress conditions.5)Quantitative real-time PCR analysis showed that the expression of AtOAT upregulated the proline biosynthesis associated genes including TaOAT,Ta P5 CS and TaP5 CR,and downregulated the proline catabolism related gene P5 CDHin the transgenic wheat plants upon stress condition.It was observed that the expression of the genes involved in ornithine pathway of Orn-OAT-P5 C and GSA-P5CR-Pro was upregulated along with the upregulation of those genes involved in glutamate pathway of Glu-P5CS-P5C/GSA-P5CR-Pro.We concluded that AtOAT is involved in proline biosynthesis by activating glutamate pathway and ornithine pathway during stress conditions.