Functional Characterization Of Wheat TaSnRK2.9 Gene In Response To Drought And Salt Stresses

Abiotic stresses such as drought,high salinity and extreme temperatures severely affect plants growth,development,and finally reduce crop productivity and quality and threaten the sustainable development of agriculture.To cope with such abiotic stresses,plants have evolved a series of complex physiological mechanisms such as ROS detoxification,ion balance,and osmosis regulation.Previous researches showed that many genes,such as transcription factors and protein kinase genes,participated in response to these abiotic stresses.Sucrose non-fermenting 1-related protein kinase 2s?SnRK2s?are plant-specific protein kinases,which are involed in response to abiotic stress and ABA singal pathway.While research on SnRK2 in wheat,an important crop plant,is quite limited.In this study,we identified and cloned a new number of SnRK2 gene family from commen wheat,and designated TaSnRK2.9.The main results are as follows:1)The qRT-PCR was used to examine the expression patterns of TaSnRK2.9 under different abiotic stress and signal molecule treatments,as well as in different organs during development.The results indicated that PEG and NaCl treatmentments could upregulate TaSnRK2.9 expression.The expression of TaSnRK2.9 could reach as much as 3.7-folds under PEG treatment compared to that of the controls.While the expression of TaSnRK2.9could be induced by signal molecules including H₂O₂,ABA,MeJA,and Ethrel.These results suggested that TaSnRK2.9 may be involved in plants'response to abiotic stresses and participated in many singal pathways.Organ specific expression analysis showed that TaSnRK2.9 expressed in young root,stamen,pistil,and lemma at relatively higher levels.2)The subcellular localization assay in onion epidermal cells showed that TaSnRK2.9-GFP was localized in the whole cell,which is consistent with results of other SnRK2s.3)Transgenic tobacco plants expressing TaSnRK2.9 were obtained via Agrobacterium-mediated genetic transformation.The function of TaSnRK2.9 in response to abiotic stresses was analyzed and results showed that expressing TaSnRK2.9 in transgenic tobaccos enhanced plants tolerance to drought and salt,improved their survival rate under stress conditons.4)Seed germination experiment results showed that expressing TaSnRK2.9 increased the germination rate of transgenic plants.Seeds of transgenic plants on the mannitol or NaCl containing plates germinated earlier,and had higher germination rate compared to that of controls.5)Root elongation experiment results showed that under mannitol or NaCl treatment,transgenic lines expressing TaSnRK2.9 had significant longer root than the controls,which is consisitant with the result of TaSnRK2.9 organ expression pattern in that TaSnRK2.9expressed at higher level in young root,suggesting that TaSnRK2.9 plays a key role in plants root development.6)To investigate the role of TaSnRK2.9 in transgenic plants,we examined the physiological indices under stresses treatments.Results showed that,transgenic lines had a lower ROS content,higher atioxidant enzyme activity and antioxidants content,higher RWC,lower MDA content,lower IL,higher proline and soluble sugar content,and increased endogenous ABA content.7)To investigate the molecular mechanism of TaSnRK2.9 in response to abiotic stresses,we analyzed the expression of some stress-and ABA-related genes.Results showed that the expressions of ROS scavenging enzyme genes such as NtSOD?NtCAT?NtPOX2?NtAPX?NtGSHI increased under osmostic stress in transgenic plants.While the ABA related genes such as NtNCED1 and NtRD29A also showed higher expressions in transgenic plants.These results indicated that TaSnRK2.9 was involved in ROS-and ABA-mediated signal pathways.8)Under osmotic stresses,the stress-related genes such as NtERD10C?NtERD10D?NtLEA5?NtLTP1?NtSPSA?NtADC1?NtSAMDC?and NtP5CS1 showed higher expression level in transgenic plants than the controls,indicating that TaSnRK2.9 functions through activating the stress response mechanism.9)Yeast two-hybrid and BiFC expremients verified the protein interaction between TaSnRK2.9 and NtABF2 in tobaccos,demonstrating that TaSnRK2.9 participates in the ABA mediated signal pathway.10)Promoter sequence of TaSnRK2.9 was predicted to contain a DRE core element,therefore,in order to study the regulation of TaSnRK2.9 in our future work,we identified and cloned a new member of superfamily of AP2/EREBP transcription factor gene,named TaERF56D.We carried out expression pattern analysis of TaERF56D.Transgenic tobacco plants expressing TaERF56D were obtained and were subjected to the functional analyses of TaERF56D under abiotic stresses.Results showed that TaERF56D was upregulated by multiple abiotic stress treatments,and expressing TaERF56D tobacco plants enhanced tolerance to drought and salt stresses.11)To further investigate the mechanism of TaERF56D in wheat,we transferred TaERF56D into wheat through particle bombardment transformation method,and positive transgenic wheat plants were obtainted.Future studies will focus on the TaERF56D signal pathway and the interaction with TaSnRK2.9 in wheat.