Functional Expression Of Cadherin And Characterization The Cry1Ac Resistance Mediated By Its Knockout In Ostrinia Furnacalis

The Asian corn borer,Ostrinia furnacalis（Guenée）,belongs to the genus Ostrinia of the subfamily Pyralidaeof Lepidoptera.It is one of the most important pests attacking corn in Asian countries,and its outbreak seriously threaten the corn production and planting benefits in China.For a long time,the management of O.furnacalis was relied on chemical control.However,the over use of chemical insecticides always resulting in a series of problems,such as pesticide residues in crops and soil media,and agro-ecological environmental pollution.Now,more than 60 million hectares of transgenic Bt corn were planted in the world.The application of transgenic insect-resistant corn producing Bt effectively has suppressed populations of target pest,reduced pesticide usage.However,the effectiveness of Bt cron has been seriously threatened by the adaptive resistacne evolution of target pests to Bt.Although the commercial Bt corn has not yet been approved in China,some Bt corn events received biosafety certificates.It is necessary to perform prospective study on Bt resistance mechanisms of O.furnacalis.Cadherin has been identified as a functional receptor for Bt toxin in sevral insects,and diverse mutations occurred in this gene were related with Cry1A resistance.In this study,we firstly showed that the O.furnacalis cadherin（OfCad）is the functional receptor for Cry1Ac using eukaryotic exogenous expression system and cytotoxicity assays.Subsequently,the CRISPR/Cas9 gene editing technique was empolyed to construct an OfCad gene knockout strain（OfCad-KO）.The resistance levels of OfCad-KO to four kinds of Cry1 toxins were determined.Additionally,the inheritance of resistance to Cry1Ac and its linkage with manipulated resistant allele were studied.The results enriched the knowledge of the mode of action of Bt Cry toxin,and provide theoretical basis for breeding design of Bt corn,and Bt resistance early warning and management in O.furnacalis.1.Heterologous expression of OfCad in Sf9 cell lines and its in vitro interaction with Cry1Ac toxinThe wild-type OfCad gene was successfully transient expressed in insect Sf9 cells using the Bac-to-Bac baculovirus expression system.Western blot analysis showed that the primary band of the recombinant OfCad protein was about 210 k Da,which was consistent with the expected molecular weight.Immunofluorescence localization revealed that the OfCad was expressed on the surface of cell membrane,which was a typical cadherin expression pattern.Co-localization experiments between heterologous-expressed OfCad protein and Cry1Ac were also performed,and reveled that significant Cry1Ac signals associated with cells expressing OfCad.The cytotoxicity of Sf9 cells expressing recombinant OfCad was tested with sevral concentrations of Cry1Ac toxin.The results showed that the mortality of Sf9 cells expressing OfCad was increased following with the increasing Cry1Ac concentration.In contrast,no significant swelling（mortality<6%）for the cells without transfection or producing empty bacmid,respectively.2.Construction of the OfCad knockout strain and its susceptibility to Bt toxinsUsing CRISPR/Cas9 gene-editing technique,we designed two sg RNAs targeting exon4 and exon35 of OfCad,respectively,according to the whole genome sequences of the Asian corn borer（NJ-S,our unpublished data）.After microinjection,the surviving individuals were single-pair crossed with wild-type NJ-S adults.And then,the OfCad knockout strain,named as OfCad-KO,was successfully obtained through three rounds of sigle-pair/mass-crossing with molecular marker-assisted selection.Molecular analysis confirmed that the OfCad gene was missing about 15.4 kb sequence between exon 4 and exon 35 in OfCad-KO strain.The large fragments deletion of OfCad will cause the gene lose its normal physiological function.When compared with the NJ-S strain,the susceptibility of OfCad-KO showed 14-and 4.6-fold reduction to Cry1Ac and Cry1Aa,respectively.In contrast,Cry1Ab,as well as Cry1Fa,showed no significant different toxicity against these two strains.3.Genetic analysis of Cry1Ac resistance in the OfCad-KO strainBioassays with larvae from the mutagenesis OfCad-KO strain,the original NJ-S Strain and their F₁ progeny at a diagnostic concentration of Cry1Ac were carried out.The results showed that the h values of two F₁ families were 0 after Cry1Ac treatment,which indicated that the Cry1Ac resistance of OfCad-KO strain was autosomal and completely recessive.The F₁ generation was self-crossed to produce F₂,and then the F₂ progeny were treated by diagnostic concentration of Cry1Ac.The survival rate was 16.6%,which closed to the expected value controlled by a monogenic trait.By genotyping,the distribution of genotype of untreated individuals（ss:rs:rr=25:47:16）was basically consistent with the Mendel’s Laws of inheritance.While,all the detected 40 survivals were homozygous bearing the deletion mutation of OfCad.The results indicate that the obtained Cry1Ac resistance in OfCad-KO strain was tightly linked with the OfCad knockout.