Molecular Characterization And Functional Analysis Of Metallothioneins In Ostrinia Furnacalis

The Asian corn borer,Ostrinia furnacalis,is an important pest of corn and other crops in China.It has a wide distribution and is harmful to all corn producing areas.Metallothionein（MT）is a low molecular weight protein with metal binding ability and high inductive properties.Understanding the function of the Of MT is of great significance to clarify its detoxification mechanism under heavy metal stress.In this study,taking Ostrinia furnacalis as the research object,two Of MTs were cloned from Ostrinia furnacalis,and their nucleotide and protein sequences were bioinformatically analyzed.q RT-PCR technique was used to analyze the m RNA expression changes of two Of MTs genes under different concentrations of three heavy metals;prokaryotic expression vectors of Of MT1 and Of MT2 were constructed,and the tolerance of recombinant bacteria to three heavy metals was tested.The content of heavy metal thionin in Asian corn borer larvae treated with different heavy metals and different concentrations was determined by ELISA.The specific research results are as follows::1.The Of MT1 sequence contains a 144 bp ORF,encoding 48 amino acids.The Of MT2 sequence contains a 138 bp ORF,encoding 46 amino acids.The cysteine content in Of MT1 and Of MT2 was 21%and 26%,respectively.2.Under different concentrations of Cd,Cu,Zn stress,the expression level of Of MT1was lower than that of the control,and heavy metals inhibited the expression of Of MT1gene.Under different concentrations of Cd²⁺stress,the expression of Of MT2 showed a trend of increasing first and then decreasing;under different concentrations of Cu stress,the expression of Of MT2 showed a trend of first increasing and then decreasing,and the expression reached the highest at 100.0 mg/kg;Under different concentrations of Zn stress,the expression level of Of MT2 was significant only at 250.0 mg/kg,and there was no significant difference from the control at other concentrations3.The recombinant genes p ET42a-Of MT1 and p ET42a-Of MT1 had a specific band around 32 k D,and the size of the target protein was about 32 k D.The DE3 strains expressing metallothionein were spread on the medium containing Cd,Cu and Zn,respectively.The results confirmed that the tolerance of E.coli expressing p ET42a-Of MT1and p ET42a-Of MT1 to heavy metals Cd and Zn was significantly enhanced.There was no significant difference under the heavy metal Zn.4.The difference between the Cd and the control was significant of Of MT content,and there was no significant difference between the different concentrations of Cd.The Of MT content was the highest at 50 mg/kg,with a concentration of 13.291 ng/ml;There was no significant difference.Among them,the content of Of MT was the highest when the concentration of Cu was 250 mg/kg,and the concentration was 19.132 ng/ml;the difference between Zn treatment and the control was significant;the content of Of MT was the highest when the concentration of Zn was 500 mg/kg,and the concentration was16.261 ng/ml.