| GRAS [GAI(GIBBERELLIN-INSENSITIVE),RGA(Repressor of ga1-3),SCR(SCARECROW)] is an important family of plant-specific transcription factors.GRAS transcription factors are involved in various physiological processes and regulatory networks in plants.It plays a variety of roles in tissue formation,hormone(gibberellin)signal transduction,light signal transduction,biotic and abiotic stress responses.GRAS transcription factors are involved in plant responses to abiotic stress and improve plant stress tolerance.In this study,a GRAS transcription factor gene was cloned from Betula platyphylla,which belongs to the SHR1 subfamily,was named BpSHR1,and the expression and salt tolerance function of the gene were analyzed.The open reading frame of BpSHR1 gene is 1425 bp,encoding 474 amino acids.Through the sequence analysis of the conserved domain sequence of GRAS protein similarity with other6 species such as Xanthoceras sorbifolium,the results show that BpSHR1 has the conserved domain sequence characteristics of GRAS family.The amino acid sequence similarity of the Cterminal is relatively high.The expression pattern of this gene under salt stress was analyzed by real-time fluorescence quantitative PCR(q RT-PCR)technology,and it was found that the expression of BpSHR1 in different birch tissues at different stress time points was up-regulated to different degrees.Among them,the expression of BpSHR1 gene was the highest in birch leaves treated with salt for 6 hours,indicating that this gene responds to salt stress.In order to further study the salt tolerance regulation mechanism that BpSHR1 gene may be involved in,plant overexpression(p ROKⅡ-BpSHR1)and gene knockout(p Eg P237-2A-BpSHR1)vectors were constructed respectively.The above vector was stably transformed into birch using the leaf disk method,and 17 overexpressing transgenic birch lines were obtained by screening.The expression of BpSHR1 gene in different strains was analyzed by q RT-PCR technology,and the results showed that its expression ranged from 4 to 226 times of wild type,and the expression levels of OE5 and OE14 were relatively high.The q RT-PCR technology was used to detect the targeting efficiency of birch plants transiently transformed with knockout expression vectors of different targets,and the target with the highest targeting efficiency was selected for stable transformation of birch.Five mutant birch lines were obtained by molecular sequencing technology.In order to further explore the possible downstream target genes regulated by birch BpSHR1,an overexpression vector containing a Flag tag was constructed,and RNA-seq analysis was performed on the overexpressed and wild-type transiently transformed birch plants under salt stress.Through the quality assessment of transcriptome data and the analysis of differentially expressed genes,the results showed that under salt stress,a total of 685 differentially expressed genes were obtained,of which 264 were up-regulated and 421 were down-regulated.The GO classification statistics of differentially expressed genes showed that the number of differentially expressed genes in connection,catalytic activity,metabolic process and cell membrane components and their proportion in the whole gene were relatively high.The results of differential gene metabolic pathway analysis showed that the metabolic pathway was the main distribution pathway of differentially expressed genes.The results of significant enrichment analysis of differential genes showed that the enrichment significance of differentially expressed genes in plant pathogen response and MAPK signaling pathway was reliable and the number of differential genes was also large.In BpSHR1-overexpressing birch,there are multiple differential genes up-regulated under salt stress conditions,such as protease inhibitors,peroxidase,lipid transporter,b HLH transcription factor,WRKY transcription factor,MYB transcription factor,etc.It was suggested that these differential genes might be regulated by the BpSHR1 gene and might respond to salt stress. |
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