| Turfgrass plays an important role in landscape design,ecological restoration,soil and water conservation.Bermudagrass is one of the warm season turfgrass and exhibits supreme tolerance to drought and heat stresses.However,under extreme weather conditions in summer,combined heat and drought stress seriously affects the growth of bermudagrass showing yellowish and reduced the ornamental quality.In this study,the physiological indexes of bermudagrass under drought,heat and the combination of drought and heat stresses were measured.Based on the previous transcriptome data of bermudagrass under the conditions of drought,heat individual stress and the combined stress,the differentially expressed genes Cd PAO1 and Cd HSP18.2 were selected for gene cloning and functional characterization.The main results are as follows:1.Effects of drought,heat individual stress(42oC)and the combined stress on the physiological indexes of bermudagrass.The physiological indexes were measured after 0,3 and 7 days of treatment under drought,heat individual stress and the combined stress conditions.The results showed that the relative water content of leaves decreased significantly and the electrolyte leakage increased significantly after 7 days of combined stress treatment.The content of soluble sugar increased significantly after 3 and 7days of the combination of drought and heat stress.The content of proline increased significantly in drought treatment for 3 days and the combination of drought and heat stress treatment for 3 and 7 days.There were no significant changes in SOD and POD activities.2.Analysis of bermudagrass HSPs gene family and functional dissection of Cd HSP18.2 gene.According to the transcriptome data of bermudagrass treated with drought,heat individual stress and the combined stress,we found that expression levels of Cd HSPs gene were significantly modulated,especially for Cd HSP70 s which were highly induced after stress treatments.Based on unigene sequence of bermudagrass,Cd HSP18.2 gene was successfully cloned and verified by sequencing.Cd HSP18.2 gene has 456 nucleotides encoding 151 amino acids.q RT-PCR data showed that expression of Cd HSP18.2 gene was highly induced by drought,heat individual stress and the combined stress.Combined heat and drought stress upregulated Cd HSP18.2 gene for 415-fold.Phylogenetic tree analysis showed that Cd HSP18.2 was highly homologous to HSP proteins in other Poaceae family.Ecotopic expression of Cd HSP18.2 gene in Arabidopsis stimulated root growth under osmotic stress by mannitol treatment.Seed germination and green cotyledon rate were significantly higher than these in wild type under mannitol simulated drought stress condition.These results indicated that Cd HSP18.2 might improve plant drought stress tolerance.Additionally,genetic transformation system of perennial ryegrass was modified and Cd HSP18.2was transformed into perennial ryegrass.3.Analysis of bermudagrass PAOs gene family and functional dissection of Cd PAO1 gene.Cd PAO1 gene was induced after drought,but inhibited by heat individual stress and the combined stress treatments.Cd PAO4 was down-regulated while Cd PAO4 was up-regulated by all treatments.q RT-PCR data showed that expression of Cd PAO1 gene was induced by drought,but inhibited by heat stress and the combined stress treatments.Gene sequence of Cd PAO1 was cloned and verified based on bermudagrass unigene sequence.Cd PAO1 has 1497 nucleotides encoding 498 amino acids.Phylogenetic tress analysis showed that Cd PAO1 is highly similar to PAO protein in Zea mays,Oryza sativa and Brachypodium distachyon.Ecotypic expression of Cd PAO1 impaired heat tolerance in Arabidopsis.Transgenic Cd PAO1 Arabidopsis was sensitive to heat stress and exhibited severe leaf chlorosis/necrosis severity index.In addition,the promoter region of Cd PAO1 was cloned.Cis-element analysis showed that Cd PAO1 promoter has ARE,GARE,MBS,LAMP motifs,indicating Cd PAO1 might be regulated by abiotic stress and hormones. |
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