| Wheat(Triticum aestivum L.)is an important food crop.Salt stress is a major abiotic stress that dramatically threatens crop yield and food supply throughout the world.Therefore,it is of great value to study the mechanism of wheat salt resistance at the physiological and molecular levels.In this study,the salt tolerance of five wheat cultivars was screened,and the result showed that Qingmai 7(Q7)was a salt-tolerant wheat cultivar and Kenong 199(K199)was a salt-sensitive wheat cultivar.Then the transcriptome sequencing of the roots and leaves was conducted in the two cultivars,with or without salt treatment.Finally,TaGF14b,a salt-related wheat gene was screened and cloned,and its function was preliminarily explored in wheat.The results are as follows:1.The salt-tolerant wheat cultivar Q7 was screened out and its physiological characteristics of salt tolerance was verifiedQ7 and K199 were treated with 0(control)and 150 m M Na Cl,respectively.Compared to control,Na~+and MDA content increased,the biomass,K~+content,net photosynthetic rate,chlorophyll content decreased at 150 m M Na Cl in Q7 and K199.The above indexes were significantly decreased or increased in K199 compared to Q7,indicating that Q7 is more salt tolerant than K199.2.Analysis of the transcriptome data of Q7 and K199 with or without salt treatmentTranscriptome sequencing was conducted for the roots and leaves of Q7 and K199,with or without salt treatment.Transcriptome data were analyzed according to gene function annotation,GO functional classification,and KEGG metabolic pathway analysis.Five metabolic pathways including ROS(reactive oxygen species)scavenging,hormone regulation,ion transport,transcription factors expression and photosynthesis were investigated to explore the gene expression differences in Q7 and K199 under salinity.Compared to K199,some key genes related to these five pathways in Q7,such as those related to GSTs,GPX,POD,PP2C,PYR,SAUR,SOS,HAK,NHX,MYB,WRKY,NAC,b HLH and AP2/ERF etc.,were significantly up-regulated and expressed at higher levels after salt treatment.Combined with the analysis of relevant physiological data,it can be concluded that Q7 has stronger ability to cope with salt stress than K199 in ROS scavenging,hormone regulation,ion transport,transcription factors expression and photosynthesis.3.Cloning and bioinformatics analysis of TaGF14bAccording to the high expression of TaGF14b in the roots and leaves of Q7 after salt treatment,and the complex and diverse connection between 14-3-3 protein and salt tolerance pathway,wheat TaGF14b gene was selected as one of the genes to explore the molecular mechanism of salt tolerance in Q7.The CDS sequence of the TaGF14b gene was cloned.The full CDS of TaGF14b was 789 bp,which encoded 263 amino acids.Bioinformatics prediction analysis of TaGF14b gene and its amino acid sequences was performed.Ex PASy website analysis showed that TaGF14b protein is hydrophilic.Signal P-5.0 analysis showed that TaGF14b has no signal peptide.TMHMM analysis revealed that there is no membrane helical structure in TaGF14b protein.TaGF14b had the highest homology to the 14-3-3-like protein GF-14B of emmer wheat and an unnamed protein of durum wheat,both of which were putative 14-3-3protein family members,indicating that TaGF14b may be a member of 14-3-3 protein family.4.Identification of wheat TaGF14b gene overexpression linesIn order to identify the function of TaGF14b in wheat salt tolerance,an overexpression vector p CAMBIA3300-UBI-TaGF14b was constructed and delivered into wheat by Agrobacterium mediated transformation.The wheat seedlings obtained after Agrobacterium transformation were identified by PCR,it showed that among the 47 regenerated wheat seedlings,38 plants were identified to be TaGF14b transgenic plants.Subsequently,real-time quantitative PCR was used to test the TaGF14b expression for all these transgenic plants.Transgenic plants with high expression of TaGF14b were selected for further salt-tolerance identification experiment. |
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