Isolation, Characterization And Genetic Transformation Of The Sb Gaba-ts Genes In Sorghum [Sorghum Bicolor,(L.) Moench]

Deterioration of the ecological environment seriously affects crop growth and yield. Sorghum bicolor, the fifth most important cereal grown worldwide, has high tolerance to abiotic and biotic stresses, thus has been regarded as a source of beneficial genes for agricultural improvement. Gamma-aminobutyric acid（GABA） is a non-protein amino and has been associated with signaling transduction, stress response, and carbon and nitrogen source storage in plants. GABA is metabolized via a pathway named GABA shunt, a branch of the TCA cycle and links to development and stress responses of plants. However, the biological function of GABA shunt in sorghum have so far not been reported.In this study, two GABA-T isoforms from sorghum were identified with experimental efforts in subcellular localization, enzyme activity and expression pattern of different tissues and development stages. Moreover, a time course measurement of salt treatment to sorghum seedlings was processed to explore the metabolic and transcriptional changes under stress. The results are as follows.1. Two putative SbGABA-T isoforms（GenBank accession number: XM₀02445162 and XM₀02447046） from sorghum BTx623 were characterized. Amino sequence alignment analysis indicated that the SbGABA-Ts possessed 79% homology to each other and 71%⁸8% to the identified GABA-Ts of other plants. Conserved domains analysis suggested that the two SbGABA-Ts belong to acetyl ornithine aminotransferase family. The results of phylogenetic tree analysis showed that the two SbGABA-Ts were attributed to monocots lineage, closely to Rice GABA-Ts.2. Transient expression of SbGABA-Ts which N-terminal signal peptide sequence fusing with GFP in tobacco leaf epidermal cells revealed that both of them located in the mitochondria, which were identical to the predicted results in silico analysis.3. Prokaryotic expression of the N-terminal truncated recombinant SbGABA-Ts proteins showed that they can both utilize pyruvate and glyoxylate as amino acceptors, but not α-ketoglutaric acid.4. The expression pattern of the two isoforms suggested that SbGABA-Ts exhibited a similar expression pattern, but varied with different tissue types and different development stages, in which the highest in roots and increased gradually with the tissue development. Time course study of salt stress on sorghum seedlings demonstrated that a rapid GABA accumulation was triggered by Na Cl treatment, along with the elevation of the SbGABA-Ts transcriptional level to some degree. Oscillation and interaction of GABA content and SbGABA-Ts mRNA abundant during the salt treatment suggested that a complex cross-talk between different regulatory levels maybe involved in plant response to saline stress.5. To resolve the brown issue during tissue culture and regeneration, more than 70 sorghum genotypes have been screened by comparison the callus induction of immature embryo from different genotype, and select the suitable genotype for further transformation. Through the optimization of transformation conditions, a sorghum agrobacterium-mediated transformation system was established and the SbGABA-Ts over-expression transgenic plants were sucessfully obtained.The present study will not only lay the foundation for further study the molecular mechanism of the GABA shunt in regulating the stress networks in sorghum, but also provide candidate gene resources for improving crop with tolerance to adverse environmental conditions.