Degradation Of Imidazolidinic Acid By Recombinant Protein Of TriHy1 Gene From Trichoderma Militaris

Imidazoic acid is one of the important pesticides controlled by weeds in China.It is a typical long-lasting herbicide.The disadvantage of this herbicide is that it will remain in the soil for a long time,usually up to 2?3 years,and up to 4 years or more,difficult to degrade.Whether using imidazolidinic acid in crop rotation or continuous cropping,it is easy to cause phytotoxicity of crops,reduce production and even produce.Trichoderma(Trichoderma)is currently the most effective biological control fungus.In recent years,its control effect and stability have been significantly improved,and it also has a control effect on biological pesticides.Therefore,screening for Trichoderma with chemical pesticides and evaluating the degradation efficiency of genetically modified proteins that degrade pesticides is very important for improving biological control and reducing environmental pollution.This experiment utilizes the T.militaris(T.brevicompactum)strains that efficiently degrade imidazolidinic acid residues in the soybean field environment that has been screened in the laboratory.Through transcriptome sequencing,Unigene performs seven functional database annotations(NR,NT,GO,KOG,KEGG,SwissPprot,and InterPro).There are 7 differential genes,and the target gene which has an effect on the degradation of imidazolium is analyzed,and the TriHyl gene is successfully cloned,encoding 256 amino acids,which is a hydrophobin.A conserved region domain with Hydrolase superfamily was predicted by BlastP.Fluorescence quantitative RT-qPCR analysis showed that the TriHyl gene was down-regulated.The eukaryotic recombinant expression vector pPIC9K-TriHyl of the gene TriHyl was successfully constructed and transformed into Pichia pastoris GS115 to obtain Pichia pastoris recombinant strain GS115-TriHyl,and the secreted Pichia pastoris recombinantly expressed by methanol was successfully induced.The protein G-rTriHyl was detected by Tricine-SDS-PAGE and the secreted protein was observed at 29.65 KDa,which was consistent with the theoretical value.It was proved that TriHyl protein can be expressed in Pichia pastoris.The degradation efficiency of the recombinant protein G-rTriHyl on imidazolidinic acid was analyzed in liquid phase,and the degradation rate was as high as 51.22%.It was proved that the recombinant protein G-rTriHyl has a great degradation effect on imidazolium.Through the cloning,bioinformatics analysis and construction of recombinant protein of TriHyl gene,it provides basic data and theoretical basis for the study of the degradation of imidazolidinic acid.This practice is practical and important for maintaining ecological balance and increasing the economic value of soybean fields.