Cloning And Functional Analysis Of The Salt-Tolerant Gene FvSnRK2 Of Fraxinus Velutina Torr

Environmental stress affects the growth and development of plants,and salt stress is one of the main abiotic stress factors.Salt inhibits the growth of plants,restricts the development of agriculture,and has a serious impact on human production and life.In the long-term evolution process,plants gradually form a series of physiological mechanisms that respond to salt stress.Among them,ABA is one of the most important stress hormones.When plants are subjected to various stresses,such as salt,low temperature and drought,plant cells rapidly accumulate ABA and respond to stress.SnRK2?sucrose non-fermenting1-related proteinkinases 2?is a positive regulator of downstream signaling in the ABA signaling pathway and plays a very important role in the response of ABA to stress.Fraxinus velutina Torr is a tall tree of the Oleaceae and Fraxinus L.,with deep roots,developed lateral roots,strong adaptability,resistance to various stress?harmful gases,pathogen,salt alkali and waterlogging etc.?.It is one of the most salt-tolerant arbor species in northern China.Therefore,it can be used as a greening tree in saline-alkali areas and it is extremely important to study its salt-tolerant mechanism.The results of transcriptome sequencing indicated that FvSnRK2 was highly expressed under salt stress?200 mmol/L NaCl?and it is speculated that FvSnRK2 may play a key role in plant salt tolerance.FvSnRK2 was cloned and its function was verified in Arabidopsis thaliana to explore its salt tolerance mechanism.The results are as follows:1.FvSnRK2182 and FvSnRK5271 were obtained from Fraxinus velutina Torr using bioinformatics methods and molecular biology techniques.FvSnRK2182 is1773 bp and open reading frame is 1092 bp,which encodes number of 363?10 kinds of amino acids?amino acid residues?with a large proportion are Ile,Asp,Glu and Leu?;and the FvSnRK2182 initiation codon sequence is“GGTTGGATGGATCGACC GGC”.There is a typical Ser/Thr enzyme activity domain at the N-terminus:“DFGYSKSSVLHSQPKSTVGTPAYIAPE”.FvSnRK5271 is 1267 bp and open reading frame is 1029 bp,which encodes number of 343?10 kinds of amino acids?amino acid residues?with a large proportion are Glu and Leu?.The FvSnRK5271initiation codon sequence is“CAACAATGGATAGGTAC”.The N-terminus also has a typical Ser/Thr enzyme activity domain.Bioinformatics analysis showed that both FvSnRK2182 and FvSnRK5271 belonged to the SnRK2 subfamily and had alpha-helix,?-sheet and?-turn.FvSnRK2182 and FvSnRK5271 were hydrophilic proteins.2.Tobacco epidermal cells were bombarded with a construct carrying GFP and FvSnRK2182-GFP by transient transformation method.By two-photon laser scanning confocal microscopy,it was found that GFP and FvSnRK2182-GFP fusion proteins were transiently expressed under the control of CaMV 35S promoter,and FvSnRK2182 was found to be localized in cell membrane and nucleus.This is consistent with the result that FvSnRK2182 was introduced into Arabidopsis thaliana after Kan screening by inflorescence dip method.3.Introduce FvSnRK2182 into Arabidopsis thaliana by inflorescence dip method to obtain transgenic seeds of T₀ generation.After 3 generations of culture,T₃generations of homozygous Arabidopsis thaliana were screened and functional analysis showed that:1).Compared with WT?wild type Arabidopsis thaliana?,the number of lateral roots of overexpressing Arabidopsis thaliana significantly increased;further analysis found that the auxin content of the overexpressing Arabidopsis thaliana remained high during the germination and growth of the seedlings;2).Under salt stress conditions,compared with WT,the salt tolerance of the overexpressing Arabidopsis thaliana was significantly improved,mainly showed in seed germination and seedlings growth;3).Compared with WT,overexpressing Arabidopsis thaliana was more sensitive to ABA and the seed germination rate,germination potential,germination index,main root length and number of lateral roots were significantly reduced under ABA treatment;4).Under the conditions of NaCl and ABA co-treatment,the resistance of the overexpressing Arabidopsis thaliana was significantly increased compared with WT;5).Under 100 mmol/L NaCl,compared with WT,shoot and root dry weight,shoot chlorophyll content and K⁺content of overexpressing Arabidopsis thaliana seedlings were significantly improved,and the Na⁺content and Na⁺/K⁺were significantly reduced.4.Transcriptome sequencing analysis found that:1).Compared with WT,573genes of overexpressing Arabidopsis thaliana seedlings showed significant changes in expression,of which 246 genes were up-regulated and 327 genes were down-regulated;These 573 differentially expressed genes are annotated into 37 GO classifications,including 15 biological process classifications,12 cell component classifications,and 10 molecular functional classifications;KEGG pathway annotation results showed that 573 differentially expressed genes from overexpressing Arabidopsis thaliana were annotated to 20 pathways?including signal transduction,biosynthesis and various metabolisms?;2).After 100 mmol/L NaCl treatment for 48 h,compared with WT,there were only 112 differential gene expressions in overexpressing Arabidopsis thaliana,of which 65 genes were up-regulated and 47genes were down-regulated.These 112 differentially expressed genes were annotated into 38 GO classifications,including 16 biological process classifications,12 cell component classifications and 10 molecular functional classifications;KEGG pathway annotation results analysis revealed that 112 differentially expressed genes were annotate to 20 pathways including cellular processes,environmental information processing,genetic information processing and metabolism.This can explain the effect of NaCl stress on the growth of Arabidopsis thaliana at the genetic level;3)Randomly selected 17 genes with high differential expression were verified the accuracy of transcriptome results by real-time quantitative PCR?qPCR?;4)Combined with transcriptome sequencing results,gene expression of ion transport-related proteins in the calcium-dependent protein kinase pathway under 100 mmol/L NaCl treatment was measured and the resulst showed that expression of HKT1,SOS1 and AKT1 were down-regulated,expression of NHX1 was up-regulated in wild-type Arabidopsis thaliana seedlings,while in overexpressing Arabidopsis thaliana seedlings,the expression of HKT1 and SOS1 were significantly down-regulated,and the expression of NHX1 and AKT1 was significantly up-regulated.Under 100 mmol/L NaCl treatment,expression of downstream genes of SnRK2 in ABA signaling pathway was measured and the resulst showed expression of KAT1 and SLAC1 were significantly down-regulated,and expression of Rboh,ABF2,ABER4 and ABF3 were significantly up-regulated in overexpressing Arabidopsis thaliana seedlings?compared with WT?.In summary,this study was the first to clone FvSnRK2182 kinase gene and FvSnRK5271 kinase gene from Fraxinus velutina Torr;FvSnRK2182 was expressed in roots and leaves of Arabidopsis thaliana and was up-regulated by salt stress;Overexpressing FvSnRK2182 significantly promotes lateral root growth and improves plant salt tolerance.These results indicated that FvSnRK2182 participates in stress-related physiological processes,which lays a foundation for further study of the salt tolerance mechanism of Fraxinus velutina Torr.