Analysis Of Salt-tolerant Transcriptome,and Cloning And Characterization Of Resistance Associated Genes IbBBX24 And IbCPK28 From Sweetpotato(Ipomoea Batatas(L.)Lam.)

Sweetpotato,Ipomoea batatas(L.)Lam.,is an important crop for food,feed,industrial materials and bio-energy resource.Its growth and productivity are seriously limited by abiotic and biotic stresses.In this study,the root transcriptomes of the salt-sensitive variety Lizixiang and the salt-tolerant line ND98 were compared to identify the genes and pathways involved in salt stress responses.The differentially expressed ESTs were analyzed,and IbBBX24 and IbCPK28 genes associated to abiotic and biotic stress tolerance were cloned from salt-tolerant line ND98.The functions of IbBBX24 and IbCPK28 genes were characterized by the Agrobacterium tumefaciens-mediated transformation system of sweetpotato,respectively.The main results are as follows:1.In this study,systematic characterization of salt tolerance and Fusarium wilt resistance were performed between Lizixiang and ND98.This result provides a novel and valuable material for improving the salt tolerance and Fusarium wilt resistance of sweetpotato.The root transcriptomes of the salt-sensitive variety Lizixiang and the salt-tolerant line ND98 were compared to identify the genes and pathways involved in salt stress responses.The differentially expressed genes(DEGs)in Lizixiang and ND98,respectively,were involved in salt responses.A lower DNA methylation level was detected in ND98 than in Lizixiang.In both genotypes,the DEGs,which function in phytohormone synthesis and signalling and ion homeostasis,may underlie the different degrees of salt tolerance.Significant up-regulations of the genes involved in the jasmonic acid(JA)biosynthesis and signalling pathways and ion transport,more accumulation of JA,a higher degree of stomatal closure and a lower level of Na+were found in ND98 compared to Lizixiang.This is the first report on transcriptome responses to salt tolerance in sweetpotato.These results reveal that the JA signalling pathway plays important roles in the response of sweet potato to salt stress.This study provides insights into the mechanisms and genes involved in the salt tolerance of sweetpotato.2.A B-Box-type zinc finger protein(IbBBX24)gene was selected from the DEGs of Lizixiang and ND98.The cDNA sequence of 1069 bp contained a 696 bp ORF encoding a 231 aa polypeptide with a molecular weight of 25.44 kDa and an isoelectric point(pI)of 5.08.The 1705 bp genomic DNA contains 3 exons and 2 introns.qRT-PCR analysis revealed that the expression of IbBBX24 gene was significantly induced by salt,drought,H2O2 and ABA stresses.Laser confocal observations revealed that the IbBBX24 protein was localized on nucleus in N.benthamiana epidermal cells.IbBBX24-overexpressing plants exhibited significantly enhanced fusarium wilt resistance as well as salt,drought and oxidative tolerance.Proline,ABA,K+content,superoxide dismutase(SOD)and photosynthesis activities were significantly increased,whereas malonaldehyde(MDA),Na+and H2O2 contents were significantly decreased in the overexpressing transgenic plants under salt,drought and oxidation stresses.After fungal pathogens infection,the significant increase of SOD,POD activities,total phenolics and lignin content and significant reduction of MDA content were found,and a rapid increase of H2O2 levels was observed.IbBBX24 may serve as a useful biotechnological tool for the improvement of abiotic and biotic tolerance in sweetpotato.Moreover,the yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)technologies were used to screen and identify the proteins that interact with IbBBX24(IbAP2,IbbZIP,IbJAZ10,IbPOD,IbSOD,IbPsy and so on).The downstream genes of IbBBX24 were screened by ChIP-seq technologies(Leucine-rich repeat protein kinase,C2H2 zinc finger protein,TGA10,bHLH,calcium-binding EF-hand family protein,CDPK,chitinase,ATP synthase subunit Beta and so on).In summary,this is the first report on disease resistance function of IbBBX24 gene.This research not only provides important gene material for breeding sweetpotato varieties with biotic and abiotic stresses tolerance,but also provides the theoretical basis for the study of gene regulatory network about biotic and abiotic stresses tolerance of sweetpotato.3.A calcium-dependent protein kinase 28(IbCPK28)gene was selected from the DEGs of Lizixiang and ND98.The 1680 bp open reading frame(ORF)encoding a 559 aa polypeptide with a molecular weight of 62.83 kDa and an isoelectric point(p1)of 9.14.The 4619 bp genomic DNA contains 12 exons and 11 introns.qRT-PCR analysis revealed that the expression of IbBBX24 gene was higher in ND98 than Lizixiang in stems(38.09 times)and leaves(3.3 times),and was significantly induced by salt,drought,H2O2 and ABA stresses.Analysis of cis-regulatory elements revealed that the 2480 bp promoter of IbCPK28 contains predicted development-and stress response-related elements.Laser confocal observations revealed that the IbCPK28 protein was localized on cell membrane in N.benthamiana epidermal cells.Well-developed mesophyll cell and xylem,accompanied by significantly increased chlorophyll and lignin contents were observed in IhCPK28-overexpressing(IbCPK28-OX)plants,while mesophyll cell and xylem were underdeveloped in Ib CPK28-RNA interference(IbCPK28-RNAi)plants.Transcriptome and qRT-PCR analyses showed that overexpression of IbCPK28 up-regulated the key genes involved in chlorophyll and lignin biosynthesis,and auxin(IAA)and gibberellin(GA)signaling pathways.IbCPK28-OX plants exhibited significantly enhanced fusarium wilt resistance as well as salt,drought and oxidative tolerance.The activities of peroxidase(POD)and photosynthesis and K+content were significantly increased,whereas hydrogen peroxide(H2O2)and Na+contents were decreased in the IbCPK28-OX plants under salt,drought and oxidative stresses.After Fusarium oxysporum infection,the significant increase of total phenolic,lignin and salicylic acid(SA)contents and POD activity were found,and a rapid increase of H2O2 levels was observed,peaked at 0.5 day and thereafter declined in the IbCPK28-OX plants.The results revealed that IbCPK28 functions in multiple signaling pathways,and has potential to be used in regulating plant development and improving plants' resistance to biotic and abiotic stresses.