Analysis Of Stress-resistant Genes In Glycyrrhiza Inflata Based On Transcriptome Sequencing

Land salinization seriously affects the growth of crops,and it is of great significance to understand the resistance mechanism of plants and to select resistance-related genes for molecular breeding of resistance plants.The previous research in this laboratory found that the callus induced by the roots of Glycyrrhiza inflata had certain salt tolerance.Transcriptome sequencing was performed on the callus after salt treatment to obtain transcriptome data of Glycyrrhiza inflata.This study is based on the sequencing data of the transcriptome of Glycyrrhiza inflata,bioinformatics analysis of late embryogenesis abundant protein?LEA?,NAC transcription factor?NAC?and proline-rich protein?PRPs?genes,the gene expression patterns of the differentially expressed genes screened were analyzed,and the gene functions of GiLEA₃-1,GiNAC06,GiPRP3 were analyzed.The main findings are as follows:?1?Bioinformatics analysis of LEA,NAC and PRPs genes of Glycyrrhiza inflata20 LEA family genes and 1 up-regulated LEA differentially expressed gene,86 NAC transcription factor genes and 14 NAC differentially expressed genes,5 PRPs family genes and 3 PRPs differentially expressed genes were identified from the transcriptome of Glycyrrhiza inflata under salt stress.The results of sequence alignment analysis showed that most LEA,NAC,and PRPs genes of Glycyrrhiza inflata were clustered closely with stress-related genes of other species.Most of the LEA family gene-encoding proteins of Glycyrrhiza inflata function in the alkaline environment of the cytoplasm;The NAC differentially expressed genes of Glycyrrhiza inflata were mostly function in the acidic environment in the nucleus;The Glycyrrhiza inflata PRPs family of genes encodes proteins that function in multiple regions of the cell via transmembrane transport in an alkaline environment.?2?Transcriptome data verification16 differentially expressed genes were selected for realtime fluorescence quantitative PCR,the results showed that 11 genes were expressed in the same way as the transcriptome results.The consistency rate of the verified gene expression and the transcriptome data was 68.75%,indicating that the transcriptome sequencing data had a strong credibility and was of guiding significance for the subsequent screening of stress-related genes.?3?Expression analysis of LEA,NAC and PRPs differentially expressed genes under different abiotic stressesThe callus of Glycyrrhiza inflata were treated with 0.8%NaCl,15%PEG and 100?mol/L ABA stress respectively,and the relative expression levels of each gene were measured at different time points.The results showed that GiLEA₃-1 and GiPRPl might be induced by drought and salt stress.GiNAC03 and GiNAC08 may be induced by drought and ABA stress.GiNAC06 may respond to salt,drought and ABA stress.GiPRP3 may be induced under drought stress.?4?Obtaining transgenic tobacco plants containing GiLEA₃-1,GiNAC06,GiPRP3 respectivelyUsing pEASY as the backbone of the cloning vector,we constructed recombinant cloning vectors containing GiLEA₃-1,GiNAC06,and GiPRP3 respectively.Using pBI121 as a plant expression vector,XbaI and Sma1 were used as restriction sites to construct plant expression vectors containing GiLEA₃-1,GiNAC06,and GiPRP3 genes respectively.Through the Agrobacterium tumefaciens-mediated genetic transformation method,the target genes were introduced into wild-type tobacco,and the resistant tobacco strains obtained through antibiotic resistance screening were tested for the DNA level and RNA level of the target gene,transgenic tobacco-positive plants containing GiLEA₃-1,GiNAC06 and GiPRP3 respectively were obtainedThis study verified the reliability of the sequencing data of the transcriptome of Glycyrrhiza inflata under salt stress,and it has a guiding role in gene screening and gene function research.Through bioinformatics analysis and gene expression pattern analysis,it was found that the three genes GiLEA₃-1,GiNAC06,and GiPRP3 were all abiotic stress-inducible genes,and successfully obtained respectively containing the three gene transgenic tobacco plants,which was used to study the ability of transgenic plants to resist abiotic stress in the later period.And lay the foundation for the regulation mechanism of abiotic stress,and provide new candidate genes for abiotic stress tolerance for molecular breeding of Glycyrrhiza inflata and other crops.