Analysis On Cloning And Expression Pattern Of Obp3of Apis Cerana Fabricius With Different Mite Resisting Property

This assay is aiming at analyzing the difference of the whole sequence of obp3(odorant binding protein3) between varroa-resistant and non-varroa-resistantApis cerana,and searching for its temporal and spatial expression patterns bet-ween the two honeybee samples, to find the relationship between obp3and t-he trait of varroa-resistance inhoney bee.Finally,the polyclonal antibody against recombinant OBP3was prepared toprovide material forfurther research.1. Primers were designed according to cDNA sequences of obp3of Apis mellifera L. inNCBI.The cDNAs of Apis cerana were taken as templates to ca-rry out gene cloning toobtain the wholesequence of obp3. There existed thre-e nonsense mutations of a/g, g/a,and g/a and one sense mutation of c/t(L/F) at164bp,200bp,341bp and192locirespectively in obp3sequence from non-varroa-resistant bees and varroa-resistantbees.a/c(I/L),a/g(I/L),a/t(M/L),g/a(G/D),a/g(M/V),c/t(Q/S),a/c(Q/S),a/g(N/D),g/a(D/N),g/a(V/T),c/g(T/S),a/g(K/R),c/g(L/V),t/c(L/V) and a/c(R/S) were detected between Apis cerana Fabricius and Apis mel-lifera at59,161,201,217,231,234,235,318,321,348,364,367,372,374and452bp locirespectively.AGln-absence was detected in Apis mellifera comparingwith Apis ceranadue to abse-nce of three consecutive bases c、a、g51-53bp loci.It revealed sequencesdifferrencesof obp3inspecies.2. RT-PCR test showed that there was high expression in abdomen and headof obp3ofthe varroa-resistingbees. Meanwhile, therewas siginificant differenceof obp3expressionlevel between the two tissuses.The expression level of obp3increased in abdomen andhead with the aging of varroa-resistant Apis cerana.The results above revealed that obp3may affectvarroa resistanceinApis cerana.3.Results of bioinformatics analysis showed that OBP3has148amino acids,and themolecular formulaof OBP3is C766H1221N205O235S16, its molecular weight is17575.3,PI is4.94,estimated half life period is30h.OBP3is an unstable protein, its stability index, fatindex and hydrophilicindex are40.94,89.53and-0.311, respectively.There’s no coil nortransmembrane domain in OBP3. The maximum hydrophobic index of OBP3is3.244,while the minimum is-2.944. OBP3belongs to α-proteins. The signal peptide sequenceof OBP3is MKTIVILLFTLCIVSYMQMVRCD, and the splice site is located in the22-23th amino acids.OBP3may participate in secretory pathway.4. pET-OBP3expression vector was successfully constructed. After digestion and sequencing, result showed the recombinant pET-OBP3was confirmed to have a fragment of obp3by enzyme digestion and sequencing. The recombinantpET-OBP3plasmid was transformed into E.coli cell strain Rosetta (DE3) to pr-oduce fusion protein pET-OBP3, which was used as antihelion to immune the rabbits to get the polyclonal antibody against recombinant pET-OBP3.The poly-clonal antibody against recombinant pET-OBP3was specifically combined with the antihelion pET-OBP3revealed by western-blot,which suggesting the expression of obp3in E.coli.Polyclonal antibody against recombinant OBP3of Apis cerna Fabricius was successfully prepared.