| Plants often encounter in a variety of adverse conditions between their growth and development in natural conditions.Drought is one of the major abiotic stresses that adversely affect plant growth and crop yield worldwide.Lots of genes are thought to be involved in responses to abiotic stress.Plants have various response and defense systems at the molecular,cellular,and physiological levels in order to survive.Therefore,the research on abiotic stress related genes has important theoretical and practical significance.The transcription factors are involved in responses against biotic and abiotic stress,and they play an essential role in regulation of plant adaptation to environmental changes.As upstream regulatory factors,transcription factors play an important role in the study of plant resistance,which can activate a variety of stress-related and functional gene expression.Transcription factors also regulate vast downstream genes and play an important role in regulation of development,metabolism and response to environment in plants.Our country is the main origin of mulberry and owns the most germplasm resources of mulberry in the world.Mulberry is an excellent ecological and economic tree.The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses.Efforts to investigate the functional genes and their molecular adaptation mechanisms under stresses plays an important role in strengthening stress tolerance and improving production of mulberry,and are of fundamental importance to the preservation of these genetic resources.At present the main transcription factors,which are more researched in plant resistance,include MYB,WRKY,b ZIP,AP2/ERF and NAC,but the main transcription factors information on drought-resistant genes of mulberry is scare,which is of great significance to improve the drought resistance of mulberry trees by changing the gene resistance of mulberry trees and can reduce the adverse effects of mulberry trees on the yield and growth caused by drought and natural geographical conditions.Therefore,in this paper,we focused on the identification and analysis of trihelix,b ZIP,MYB,ERF transcription factors related to drought resistance in mulberry,the main results were summarized briefly as follows:1.Identification of drought-resistant related trihelix,b ZIP,MYB,ERF transcription factor family in mulberryThe bioinformatics methods were used to identify and analyze the drought resistance-related trihelix,b ZIP,MYB,and ERF families of four transcription factors in mulberry according toother species.The sequence characteristics of these four gene families were systematically analyzed and predicted from the genomic level.The results are as follows:In this study,twenty nine members of the trihelix family,which contain highly conserved and characteristic trihelix domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into 5 subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the trihelix family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the trihelix family were consistent in two species,which indicated that the differentiation of the trihelix gene family occur earlier than that of these species.The conserved motifs in the trihelix family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Thirty two members of the BZIP family,which contain highly conserved and characteristic b ZIP domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A?B?C?D?E?F?G?H?I and other subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the b ZIP family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the b ZIP family were consistent in two species,which indicated that the differentiation of the b ZIP gene family occur earlier than that of these species.The conserved motifs in the b ZIP family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Ninety nine members of the MYB family,which contain more two highly conserved and characteristic Myb_DNA-binding domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A?B?C?D?E?F?G?H?I and other subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the b ZIP family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the MYB family were consistent in two species,which indicated that the differentiation of the b ZIP gene family occur earlier than that of these species.The conserved motifs in the MYB family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Eighty five members of the ERF family,which contain highly conserved and characteristic trihelix domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A?B?C?D?E?F?G?H?I?J?K?L and other subfamilies based on the evolutionary relationship and domain characteristics,and J subtribe gathered the most.Clustering analyses of the ERF family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the ERF family were consistent in two species,which indicated that the differentiation of the ERF gene family occur earlier than that of these species.The conserved motifs in the ERF family of Mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Our results preliminarily identified the evolution,functional characterization,which will provide a basis to further reveal the molecular evolution and biological function of the trihelix,b ZIP,MYB?and ERF transcription factors in mulberry.2.Identification of transcription factors in drought stress RNA-seq of mulberryRNA-seq was carried out for transcriptome analysis of mulberry 71-1 between two samples in regular and drought stress condition.A total of 10 differentially expressed genes were screened out based on the identification of differential expression genes of trihelix,MYB,b ZIP and ERF family in mulberry,of which 2 was down-regulated and 8 up-regulated.The b ZIP transcription factor family has 1 up-regulated differential gene.The trihelix transcription factor family has a down-regulated differential gene.There were 3 up-regulated genes and 1 down-regulated gene in the MYB transcription factor family.ERF transcription factor family 4,are up-regulated genes.GO enrichment of differentially expressed genes shows that The implementation of the function of differentially expressed genes of four TFs(Transcription factors)family mainly focuses on the classification of biological processes.The differentially expressed protein in molecular functional classification performs transcription factor activity,sequence-specific DNA binding,uucleic acid binding,DNA binding and so on.Differential expressed protein performs nuclear function in classification of cellular components.3.Cloning and sequence analysis of Mntrihelix from mulberry and its expression patternMntrihelixsequence was cloned with containing a 567 bp open reading frame(ORF),encoding 188 amino acid residues.SMART analysis showed Mntrihelix contained one typical SANT domains.Phylogenetic analysis showed that Mntrihelix belonged to the GT1 subfamily of the trihelix family and was most closely related to morusnotablis SMARTblast analysis indicated that Mntrihelix was close to trihelix of Arabidopsis thaliana and Glycine max from the reference species,and of Gossypium arboretum,Theobroma cacao and Morus notabilisfrom the non-redundant protein database.Further expression profile analysis using the quantitative real time-polymerase chain reaction technique(q RT-PCR)showed that most of the Mntrihelix gene from mulberry was induced by drough,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.4.Cloning and sequence analysis of Mnb ZIPfrom mulberry and its expression patternMnb ZIPsequence was cloned with containing a 675 bp open reading frame(ORF),encoding 224 amino acid residues.SMART analysis showed Mnb ZIP contained one typical SANT domains.Phylogenetic analysis showed that Mnb ZIP belonged to the b ZIP_plant_GBF1 subfamily of the b ZIP family and was most closely related to morusnotablis.SMARTblast analysis indicated that Mnb ZIP was close to Arabidopsis thaliana and Glycine max from the reference species,and Ziziphusjujuba,Malus domestica and Morusnotabilis from the non-redundant protein database.Further expression profile analysis using q RT-PCR showed that most of the Mnb ZIP gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.5.Cloning and sequence analysis of Mn MYBfrom mulberry and its expression patternMn MYBsequence was cloned with containing an822 bp open reading frame(ORF),encoding 293 amino acid residues.SMART analysis showed Mn MYB contained two typical SMART SANT domains.Phylogenetic analysis showed that Mn MYB belonged myb_SHAQKYF subfamily of the MYB family and was most closely related to morusnotablis.SMARTblast analysis indicated that Mn MYB was close to Arabidopsis thaliana and Glycine max from the reference species,and Populustrichocarpa,Ziziphusjujuba and Morusnotabilis from the non-redundant protein database.Further expression profile analysis using q RT-PCR showed that most of the Mn MYB gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.6.Cloning and sequence analysis of Mn ERFfrom mulberry and its expression patternMn ERFsequence was cloned with containing a 321 bp open reading frame(ORF),encoding 106 amino acid residues.SMART analysis showed Mn ERF contained one conservative AP2 domains.Phylogenetic analysis showed that Mn ERF belonged ERF subfamily of the AP2/ERF superfamily.Phylogenetic analysis indicated that Mn ERF was close to morusnotablis and Broussonetiapapyrifera from the reference species and of Gossypium arboretum,Theobroma cacao and Morusnotabilis from the non-redundant protein database by SMARTblast.Further expression profile analysis using q RT-PCR showed that most of the Mn ERF gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses. |
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