Transcriptome Analysis Of Potentilla Sericea And Preliminary Study On PSMYB2 Function Under Cadmium Stress

Worldwide,the heavy metal cadmium(Cd)pollution in soil had become an increasingly urgent problem.As one of the most dangerous environmental pollutants,cadmium could have a strong toxic effect on plants even at low concentrations,which posed a serious threat to the growth of plants and affected the yield and quality of crops.In addition,cadmium(Cd)also accumulated in the human body through the food chain,which had an impact on human health.Potentilla sericea was highly resistant and could adapt to the cold and arid climate in the north.It had good water conservation,soil and water conservation and slope protection functions.It could be used as a ground cover instead of lawn in gardens to reduce maintenance cost.In this study,P.sericea was used as the test material to analyze the transcriptome of P.sericea under different concentrations of cadmium.Through transcriptome sequencing analysis,it was found that a large number of MYB transcription factors were up-regulated.In order to explore the role of P.sericea MYB transcription factor in its growth and development,based on transcriptome data,the family of P.sericea MYB transcription factor was analyzed,and the PsMYB2 gene was screened and cloned for bioinformatic analysis.The expression of PsMYB2 gene under cadmium stress was detected.The plant expression vector of PsMYB2 gene was constructed,and the gene was transferred into the wild-type Arabidopsis thaliana by the dipping method to obtain transgenic resistant plants.(1)Two root samples(Cd0 and Cd20)of P.sericea were subjected to high-throughput sequencing,and a total of 78.06 Gb Clean Data was obtained.The Clean Data was spliced by using Trinity,and a total of 94,009 transcripts and 53,225 Unigenes were obtained.The N50 values of the transcript and Unigenes were 2,082 bp and 1,946 bp,and the average length was 1539.45 bp and 1395.46 bp,respectively.Among them,43,991 Unigenes obtained annotation information.A total of 11,684 Differentially Expressed Genes(DEGs)were screened,of which 8083 Differentially Expressed Genes were significantly up-regulated and 3601 were significantly down-regulated(padj<0.05).Among them,11,684 Differentially Expressed Genes were annotated to 127 KEGG metabolic pathways,5,877 DEGs were annotated to 52 GO secondary entries,and 5222 DEGs were assigned to 25 specific COG categories.In addition,the fluorescence real-time quantitative detection results were also consistent with the transcriptome sequencing results,confirming the reliability of the transcriptome sequencing analysis results.(2)Using the method of biological information,63 MYB transcription factors were screened from the P.sericea transcriptome database,and comprehensively analyzed the sequence characteristics,physical and chemical properties,conserved domains,subcellular location and evolutionary relationship of the MYB transcription factor family.At the same time.the MYB gene related to the regulation of heavy metal cadmium was screened.The results showed that the 63 P.sericea transcription factors screened included 22 1R-MYB transcription factors.37 R2R3-MYB transcription factors.3 3R-MYB transcription factors,and 1 4R-MYB transcription factor.and R2R3-MYB transcription factors were the most.Based on the results of the analysis,R2R3-MYB transcription factors related to abiotic stress was selected from the P.sericea MYB transcription factor,which was used as the target gene for the next step and named PsMYB2.(3)Using P.sericea cDNA as a template,the target fragment was amplified,and PsMYB2 was successfully cloned.The OFR of PsMYB2 gene is 852 bp in length and encoded 283 amino acids.Analyzing the conserved domains of PsMYB2 protein,the gene had a SANT motif(MYB domain)at 23-68 amino acids and 78-117 amino acids,respectively.The analysis of the physical and chemical properties of the PsMYB2 transcription factor found that the relative molecular mass of the protein was 32.76 kDa,the theoretical isoelectric point(pI)was 6.25,and the instability coefficient was 68.07.It was an unstable protein.Containing 230 hydrophilic regions.54 hydrophobic regions,it was a hydrophilic protein.In the secondary structure of PsMYB2 protein,α-helix(Alpha helix)accounted for 28.17%,β-sheet(Beta turn)accounted for 3.87%.extended strand accounted for 5.99%,and random coil(Random coil)accounted for 61.97%.Under the treatment of 20 mg/L CdCl2·2.5H2O solution,the expression of PsMYB2 gene in roots and leaves showed a trend of first increasing and then decreasing with the extension of treatment time.At 12 h,the gene expression reached highest.The expression level in the stem showed a trend of first increasing,then decreasing and then increasing.(4)The plant overexpression vector pBL121-PsMYB2-GFP of PsMYB2 gene was constructed by restriction enzyme digestion and ligation method,and the EHA105 Agrobacterium strain containing the recombinant plasmid was obtained.Afterwards,the PsMYB2 gene was transferred into the wild type plants of Arabidopsis thaliana using the dipping method to obtain the T3 generation transgenic plants.The T3 generation transgenic plants were identified,and Arabidopsis thaliana transgenic lines with high expression levels of the PsMYB2 gene were screened.By extracting the protoplasts of transgenic Arabidopsis thaliana plants,observation under a fluorescence microscope showed that PsMYB2 was localized in the nucleus.Order the transgenic Arabidopsis thaliana seeds on medium plates containing 100,150 mmol/L NaCl and 150,200 mmol/L Mannitol and 10,20 mg/L CdCl2·2.5H2O respectively to analyze the stress resistance of transgenic Arabidopsis thaliana during seedling stage,and preliminary researched on the function of PsMYB2 transcription factor.