Cloning And Resistance Study Of Ubiquitin Ligase DtE3 Gene In Dracocephalum Tanguticum Maxim

Dracocephalum tanguticum Maxim.is a Labiata,Dracocephalum perennial herb,a traditional Tibetan medicinal plant distributed on the Qinghai-Tibet Plateau.Growing on the banks of dry river valleys,fields,grasslands,or margins of pine forests at 1,900-4,000 m.The whole herb can be used as medicine and has a long history of Tibetan medical medicine.In clinical has the exact drug effect,has the liver heat,hemostasis,healing sores,dry yellow water and other functions.In this study,the transcriptome database was compared and DTE3 gene was screened.PHB-DTE3 overexpression vector was constructed and transformed into Escherichia coli DH5α.The positive monoclonal colonies were sent for sequencing and the detection results were analyzed by bioinformatics of protein sequences.The internal reference genes of Dracocephalum tanguticum under drought stress were screened.Optimized the system of germinating antimicrobial seedlings of Dracocephalum tanguticum.The sterile leaves were infected by Agrobacterium tumefaciens leaf disc transformation method and hairy roots were induced.The hairy roots with vigorous growth were simulated with 400 m M mannitol to observe the morphological changes of hairy roots under drought stress,and the three drought resistance indexes of MDA,SOD and POD were measured.The specific research results were as follows:1.Seven reference genes commonly used in plants were screened from Dracocephalum tanguticum transcriptome database: ACT,TUB-β,TUB-α,UBC,MDH,GAPDH,EF.Finally,UBC and ACT were identified as internal reference genes under drought stress.2.The DTE3 gene was screened from the transcriptome database of Dracocephalum tanguticum,primers were designed to amplify the target bands,and PHB ultrasonographic expression vectors were constructed.The transvector was tested positive and the samples were sent for sequencing.Vector NTI 11.5.1software was used to assemble the sequencing results and bioinformatics analysis was performed.The BLast X results of NCBI online database showed that the amino acid sequence of DTE3 gene in Salvia miltiorrhiza(TEY49278.1)had the highest homology of 81.86%.The prediction of physical and chemical properties showed that the isoelectric point of DTE3 protein sequence was 5.37,the instability index(II)was 41.15,the fat index was 70.42,and the total mean hydrophilicity(GRAVY)was-0.478.The secondary structure of E3 protein consisted of 25.76% Alpha helix,13.35% Extended strand,3.75% Beta turn and57.14% Random coil.The Ring-HC domain was found to belong to Ring-type ubiquitin ligase.The hydrophilic region of DTE3 protein is larger than the hydrophobic region,and the protein is hydrophilic protein.The signal peptide and transmembrane region analysis showed that there was no transmembrane structure and signal peptide region indication,and the protein did not transport across the membrane.N-glycosylation and phosphorylation sites analysis revealed that there were 3 N-glycosylation sites and 51 phosphorylation sites,with serine phosphorylation as the main site.3.Three kinds of disinfectants(10% H2O2,0.1% HgCl2,and 2% NaClO)and five time gradients(6 min,9 min,12 min,15 min,18 min)were used to optimize the germination conditions of Dracocephalum tanguticum sterile vaccine.Finally,the optimal condition for Dracocephalum tanguticum sterile vaccine germination was determined to be 10% H2O2 for 12 min disinfection.The leaves of Agrobacterium tumefaciens were infected with Agrobacterium tumefaciens C58C1 and Agrobacterium tumefaciens Dt E3-PHB-C58C1 respectively by the method of Agrobacterium tumefaciens leaf disk transformation to induce hairy roots.4.The hairy roots of control group and experimental group Dracocephalum tanguticum were treated with 400 m M mannitol for 6 days.Morphological analysis of hairy roots showed that overexpression of Dt E3 gene could increase root length,reduce root diameter,and slow down root senescence.MDA analysis showed that the content of MDA in hairy roots with overexpression of Dt E3 gene was lower than that in the control group,and overexpression of Dt E3 gene could slow down the damage of plasma membrane in hairy roots.The analysis of superoxide dismutase(SOD)activity showed that SOD content in hairy roots with overexpression of Dt E3 gene was higher than that in the control hairy roots.The peroxidase(POD)activity analysis showed that the content of POD in hairy roots of overexpressed Dt E3 gene was higher than that in the control hairy roots,and the increase of POD content was much higher than that in the control group.