Screening Of Novel Insecticidal Proteins For Cadherin Toxin Binding Region From Cnaphalocrocis Medinalis

Rice leaf roller is one of the important lepidoptera pests in rice,which is a kind of migratory insect with wide distribution and strong adaptability to the environment.Bacillus thuringiensis is a very important insect pathogen,which can produce a variety of insecticidal crystal toxins in the process of spore formation,among which Bt Cryl toxin has insecticidal activity against lepidoptera insect larvae.Cadherin is recognized as the primary receptor for Cry1A insecticidal proteins,and its affinity with Cryl A insecticidal proteins is much higher than that of other receptors.As an important receptor of Bt Cry protein,studies have confirmed that the main reason for insect resistance to Bt toxin may be the combination of toxin and receptor has changed.Therefore,whether it is to study the expression of insect cadherin receptor and its ability to bind to Bt Cry toxin,reveal the insecticidal mechanism and resistance mechanism of Bt Cry toxin;or to improve the insecticidal activity of Bt toxin,it is necessary to understand the molecular mechanism of binding of toxin to receptor.Based on mature genetic engineering and phage display technology,the receptor proteins of the cadherin toxin binding region of rice leaf roller were obtained,it is applied to screen to obtain novel insecticidal proteins with potential Bt toxic protein functional activity,and the activity analysis of the obtained materials.1.Construction,expression and activity identification of cadherin toxin binding region of rice leaf rollerBy comparing the cadherin-like gene sequences of various lepidopteran insects on NCBI,the gene sequence of the CR7-MPR region of the rice leaf roller was successfully obtained by PCR.Using genetic engineering techniques,designing primers and amplifying the toxin binding region gene of rice leaf roller(CmCad TBR).Then construct the prokaryotic expression vector CmCad TBR-pET-26b,transferred into Escherichia coli BL21 compenent cells and purified by His-Trap HP nickel column.The expression level of CmCad TBR was identified by SDS-PAGE and Western Blot,and the binding activity of CmCad TBR to Cry1 Ac toxin was analyzed by ELISA.The results showed that the prokaryotic expression system of CmCad TBR-pET-26b was successfully constructed,and successfully expressed in E.coli BL21.Its binding activity with CrylAc toxin was verified by Western Blot and ELISA,to provide a material basis for using purified receptor protein to screen human single-domain antibody insecticidal protein.2.Screening,identification and activity analysis of insecticidal proteins derived from human single domain antibodyUsing the purified TBR receptor protein as a screening molecule,screening of single-domain antibodies with higher binding activity to cadherin-like receptors from DAb library by phage display technology and obtaining their gene sequences.The results showed that after three rounds of affinity screening,three positive clones C11,E8 and F3 were finally identified by monoclonal ELISA and PCR sequencing.We identify the high binding activity of C11 and purified receptor protein by specific binding ELISA method.Using competitive inhibition ELISA to identify its ability to compete with receptors for CrylAc toxins,the inhibition rate is 29.86%.The results showed that the ajusted mortality of 107 cfu/mL C11 against C.medinalis was 11.1%by leaf soaking method.