| The rapid development of Chineseeconomy in recent years has been caused more and more environmental pollution problems.In particular,the environmental problems caused by the extensive use of petroleum and its repair technology have become a research hotspot.At present,the phytoremediation technology for oil-contaminated soil has attracted wide attention due to its beautiful appearance and no secondary pollution.Our previous study found two small heat shock proteins,MjsHsp18.2 and MjsHsp17.7,that are resistant to petroleum-contaminated plants Mirabilis jalapa Lcan be highly expressed under oil pollution stress.The expression vectors fromthe two genes were constructed by RACE technology,fusion PCR,and 2A peptide technology(pYES2-MjsHsp18.2-Flag,pYES2-MjsHsp17.7-HA,and pYES2-MjsHsp-fusion.)and obtained Saccharomyces cerevisiae transformed strains.On this basis,this project carried out the mechanism of MjsHsp in the process of yeast endoplasmic reticulum stress,thenlaid the foundation for revealing the function of these gene in the stress response of plant oil stress.The results of the study are as follows:1.Bioinformatics prediction MjsHsp18.2 gene encodes 161 amino acids,the molecular weight of the protein is 18.2kDa.The MjsHsp17.7 gene encodes 158 amino acids and has a protein molecular weight of 17.7 kDa.The binding sites of MjsHsp18.2 and MjsHsp17.7 are different,so the function between them are different.These two types of small heat shock proteins are located in the cytoplasm/nucleus,theycan be combined with proteins,ions,organic rings and heterocycles to participate in the stimuli response of biological metabolism,protein folding and abiotic stress.2.The expression patterns of MjsHsp18.2 and MjsHsp17.7 genes in the roots of Mirabilis jalapa L seedling under petroleum stress were analyzed by real-time fluorescent quantitative PCR.It showed that the expression level of MjsHsp18.2 was the highest when the concentration was 30g/kg oil treatment for 12 h.MjsHsp17.7 expressed the highest expression at 20g/kg oil treatment for 24 h.Western blot results of transformed strains confirmed the successful transformation of Saccharomyces cerevisiae.And the sensitivity experiment verified that the transgenic strains were stronger than the original species INVSCI1..3.To explore the relationship between sHSPs,endoplasmic reticulum,UPR response,apoptosis and oxidative stress in adversity.The use of tunicamycin to simulate the stress environment,real-time quantitative determination of the relative expression of BiP and PDI1 in the original species and transformed strains of Saccharomyces cerevisiae,and the SOD activity changes of Saccharomyces cerevisiae treated with different time.The results showed that the expression levels of BiP and PDI1 were significantly increased in the transformed strains.The expression level of BiP in the transformed strain MjsHsp17.7 was significantly higher than that in the non-transformed strain.The expression of PDI1 was more evident in the MjsHsp18.2 strain.MjsHsp-fusion strain the amount of expression of both genes is between them.The expression of Yca1 treatment with tunicamycin in the Saccharomyces cerevisiae strain and the experimental results of the survival rate and apoptosis rate of Saccharomyces cerevisiae after tunicamycin showed that the Yca1 expression level of the transgenic strain showed a downward trend,and MjsHsp17.7 The transformed strain showed the strongest cell activity with the lowest seed viability.It was demonstrated from the molecular and cellular levels that the transgenic strain had the activity to reduce the apoptosis rate.In summary,the small heat shock protein of Mirabilis jalapa L that is overexpressed in Saccharomyces cerevisiae helps to reduce endoplasmic reticulum stress brought by tunicamycin and reduce apoptosis and improve cell resistance. |
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