Identification Of SnRK Gene Family In Tobacco And Studying On ABA Signaling Pathway Involved By NtSnRK2 Under Heavy Metal Stress

SnRK(sucrose non-fermenting-1-related protein kinase,SnRK)family genes are key stress resistance genes,especially SnRK2 family genes can participate in ABA-dependent pathway to mediate plant signal sensing and transmission under stress conditions.Heavy metal poisoning can seriously affect plant cell membrane structure,respiratory and photosynthetic enzymes system,carbon and nitrogen metabolism.It can inhibit the normal physiological metabolism of plants and affect the high yield and quality of crops.At present,there are few studies on the excavation of NtSnRK1/2/3 gene in tobacco,and the exploration of NtSnRK2 and its ABA-dependent pathway under heavy metal stress is also less.Therefore,based on the transcriptome results of tobacco K326 obtained earlier,the NtSnRK1/2/3 family genes were screened,identified and bioinformatics analyzed.Four NtSnRK2 genes of tobacco K326 were selected for expression pattern analysis under heavy metal cadmium stress.Analysis;Screening and identification of upstream gene(NtPP2C)and downstream gene(NtABF)of NtSnRK2 in ABA signal transduction pathway,sequence analysis and expression pattern analysis under heavy metal cadmium stress;determination of ABA content changes of K326 under heavy metal cadmium stress,screening and identification of ABA synthesis related gene(NtNCED)and decomposition related gene(NtCYP707A1),sequence analysis and heavy metal cadmium stress.Analysis of forced expression patterns.To explore the NtSnRK 1/2/3 family genes,and to explore the mechanism of NtSnRK 2 and its ABA signaling pathway in response to heavy metal stress in plants,so as to provide references for NtSnRK 1/2/3 gene mining and heavy metal resistance research in plants.The experimental results are as follows:1.Combining the results of K326 transcriptome sequencing and specific primer clones,18 NtSnRK 1/2 were screened,including 5 NtSnRK 1 and 13 NtSnRK2,named NtSnRK1.1-1.5 and NtSnRK2.1-2.13,respectively.CDS length of NtSnRK1/2 members ranged from 856bp to 1130bp,and the similarity with Arabidopsis homologous genes was more than 90%.The 18 NtSnRK 1/2 genes have C-terminal regulatory domain(AMPKA_C),SNF1 homologous conserved domain(UBA_NtSnRK 1),serine/threonine kinase catalytic domain(S_TKc),catalytic domain(PKc_like superfamily)and other domains.NtSnRK1/2 encodes amino acids ranging from 284aa to 440aa,with molecular weights ranging from 30356.06 Da to 49901.57 Da.They all encode acidic proteins,which do not contain signal peptides.The proteins are sorted and transported to mitochondria.Of the 10 conservative sequences,motif 1 and motif2 are the most conservative.Cluster analysis shows that there are 6,5 and 7 NtSnRK1/2 in Group 1,Group 2 and Group 3,respectively.In addition,alpha helix and irregular curl are important secondary structures of NtSnRK1/2,and their three-dimensional configurations are very similar.2.Six NtSnRK3 genes,named NtSnRK 3.1-3.6,were screened and identified.The homology with Arabidopsis thaliana genes was more than 85%.The length of CDS was 1077 bp to 1232 bp.The coding amino acids ranged from 335 aa to 409aa,and the molecular weight ranged from 38744.41 Da to 50922.02Da.They belong to transmembrane proteins and are sorted into cytoplasm by endoplasmic reticulum.ATP binding site,CBL interaction site,PP2C binding site,PKc_like superfamily,AMPKA_C_like superfamily,serine/threonine kinase catalytic domain(CIPK_C)are also found in their structural domains.Alpha helix and irregular curl are the main structures of its members.The three-dimensional structure is similar to that of Arabidopsis NtSnRK3 members.The results showed that motifl,motif2 and motif3 sequences were relatively conservative.Cluster analysis showed that NtNtSnRK3 was closely related to Arabidopsis homologous genes,but far from rice homologous genes.3.The seedlings of K326 were treated with cadmium at 45 micrometers.The NtSnRK2.1,NtSnRK2.2,NtSnRK2.3 and NtSnRK2.7 of NtSnRK2 family were selected for qRT-PCR.NtSnRK2.2 and NtSnRK2.7 were most strongly induced by heavy metals,followed by NtSnRK2.3 and NtSnRK2.2.The expression level of NtSnRK2.1 increased significantly at first,reached the highest level at 1 h,then decreased,and reached the lowest level at 24 h;NtSnRK2.2 was strongly induced by stress,and the expression level of NtSnRK2.2 increased to the highest level at 1 h,decreased at 24 h,and then increased to the same level as the control;NtSnRK2.3 was weakly induced,and the expression of NtSnRK2.3 gene was up-regulated at first and reached the highest level at 3 h.NtSnRK2.7 was also strongly induced by stress.The transcription level of the gene treated for 1 hour was 8 times higher than that of the control,with significant difference,and then decreased.In conclusion,under cadmium treatment,the four NtSnRK3 members of tobacco K326 were induced to increase first,then decrease,and finally tend to remain unchanged.4.One NtPP2C and one NtABF sequence were obtained by screening and cloning.CDS was 804 bp and 1212 bp,respectively,encoding 267 and 403 amino acids.The molecular weight was 34153.35 Da and 59207.31 Da,and the theoretical isoelectric point(pI)was 6.48 and 5.72.No signal peptide was found in the encoding proteins,and subcellular localization was found in the cytoplasm.In addition,irregular curl and alpha helix are the main secondary structures of NtPP2C and NtABF,and their three-dimensional structures are highly similar to Arabidopsis homologous genes.NtPP2C members contain active site,PP2Cc superfamily,NtABF members contain DNA binding site,dimer interface,coiled coil and bZIP superfamily.Under cadmium treatment,the transcription level of NtPP2C in K326 seedlings increased first and then decreased,and the relative expression of NtABF increased first and then decreased.5.The content of ABA in K326 seedlings was determined by enzyme-linked immunosorbent assay(ELISA).Between 0 h and 72 h of cadmium stress,the level of ABA increased continuously,and the rate of ABA increased first and then decreased.In addition,one NtNCED(ABA synthesis gene)and one NtCYP707A1(ABA decomposition gene)were obtained by screening and cloning.The similarities with Arabidopsis homologous genes were 97%and 96%,respectively.Sequence analysis showed that their CDS were 1800 bp and 1410 bp,respectively,encoding 599 and 469 amino acids,all of which were hydrophilic transmembrane proteins.Molecular weights were 65858.34 Da and 61862.22 Da respectively,and theoretical isoelectric points(pI)were 6.16 and 6.68 respectively.There are RPE65 superfamily and P450 superfamily respectively.Irregular curl and alpha helix are important spatial arrangements.Three-dimensional structure analysis showed that the spatial structure of the two proteins was highly similar to that of Arabidopsis homologous genes.Heavy metal stress analysis showed that cadmium could induce the expression of NtNCED and NtCYP707A1.The expression of NtNCED increased first and then decreased,while the expression of NtCYP707A1 decreased first and then increased.Therefore,under heavy metal stress,K326 can regulate the expression of ABA synthesis and decomposition genes to promote the accumulation of ABA in plants.Therefore,tobacco may respond to heavy metal stress through ABA signaling pathways involving NtSnRK2,NtPP2C and NtABF.