Preliminary Study On The Response Of AmRanBP1 Protein From Amygdalus Mira Koehne To High Temperature And Salt Stress

Amygdalus mira Koehne is a unique peach germplasm resource in Tibet,China.It has strong drought resistance,barren resistance,disease resistance,radiation resistance,cold resistance and easy renewal.It has many uses such as ornamental,medicinal and edible,and has high ecological value,economic value and medicinal value.At the same time,it is also second-class protected plant in China.In this study,the AmRanBP1 gene was cloned,and the spatio-temporal expression pattern of AmRanBP1 gene and the expression pattern under stress were detected by q RT-PCR.Bioinformatics analysis and subcellular localization of AmRanBP1protein;AmRanBP1 protein was induced and purified in vitro by prokaryotic expression technology and monoclonal antibodies were prepared.Construction of plant overexpression vector for Arabidopsis thaliana heterologous expression analysis;The proteins interacting with AmRanBP1 were screened by using the yeast two-hybrid library of Amygdalusmira Koehne,and the results are as follows:1.The AmRanBP1 gene was successfully amplified by using the c DNA of walnut leaves as a template.Abiotic stresses such as NaCl,Na HCO₃,drought,H₂O₂,heavy metals,and mannitol were applied to one-month-old seedlings of Amygdalusmira Koehne.It was found that AmRanBP1 responded to NaCl,mannitol,Cu SO₄,and Mn SO₄stresses.2.Bioinformatics analysis of the protein AmRanBP1 encoded by AmRanBP1 showed that AmRanBP1 was closely related to Ran BP1 of Prunus persica and Prunus dulcis,which was a functional protein on non-biofilm.It was an acidic hydrophilic protein.The transient expression vector p BS-GFP-AmRanBP1 was constructed,and the gene was found to be located in the cytoplasm by gene gun transient transformation.3.p ET-21a（+）-AmRanBP1 was successfully obtained,and its induction conditions were optimized.It was found that AmRanBP1 was induced by 2m M IPTG at 40°C for 4 hours, and the expression level was the highest and soluble protein.The purified protein obtained after desalination was used for subsequent antibody preparation.The growth of p ET-21a（+） and p ET-21a（+）-AmRanBP1 transformants in E.coli was compared,and it was found that the advantages were significant under high temperature,NaCl and Cu SO₄stress.4.The T₃homozygous of p BI121MCS-GFP-AmRanBP1 transgenic Arabidopsis thaliana were successfully obtained.Overexpression of AmRanBP1 improved the tolerance of Arabidopsis thaliana seeds to high temperature and NaCl stress.The transgenic Arabidopsis thaliana seedlings had a better growth trend after high temperature（30°C）and NaCl stress.Analysis of soluble protein content and chlorophyll content data showed that AmRanBP1 transgenic Arabidopsis thaliana could reduce the damage caused by high temperature and NaCl to plants.The expression level of AmRanBP1 in overexpression under stress was detected by q RT-PCR.The results showed that the expression level of AmRanBP1 gene was higher under high temperature and salt stress.At the same time,Western-blot hybridization with AmRanBP1 monoclonal antibody showed that the hybridization signal of transgenic was significantly stronger than that of wild type and mutant after high temperature and salt stress.Western-blot hybridization was consistent with q RT-PCR expression trend,indicating that transgenic Arabidopsis thaliana can respond to high temperature（30°C）and NaCl stress.5.The p GBKT7-AmRanBP1 bait protein was non-toxic and had no self-activation activity. The interacting proteins were screened by yeast two-hybrid.The three interacting proteins were 60S ribosomal protein L27a-3,major allergen Pru ar 1 and uncharacterized protein.