Studies On The Relationship Between Olfactory Receptor Orco And Feeding Harbits Of Silkworm,Bombyx Mori

The silkworm stains have different feeding habits for artificial diets.But the mechanism of this diversity is unclear.There are many factors influencing the feding habits,and olfactory system play an important role in this process.In insect,the gene expression and amino acid polymorphism of olfactory receptor may affect the sensibility for the ligand,and form different phenotypes.We have known that olfactory receptor Orco is important for olfactory recognition.The olfactory of insect is deprivation when Orco gene is silenced by gene mutation or RNAi.So,did Orco affect the feeding habits in silkworm? Is there diversity in gene expression and nucleotide polymorphism of Orco between different silkworm strains? And how the relationship of this diversity and feeding habits? To figure out the problems,we used two silkworm strains with different feeding habits for artificial diets as the experimental material.In this study,Orco antigen protein was prepared by prokaryotic expression system,and generated the polyclonal antibody by immuning mice.Then we studied the localization of Orco by immunofluorescence,and discussed the relationship between olfactory receptor Orco and the feeding habites of silkworm in DNA,mRNA and protein level.Finally,we applied the researches achievements into genetic breeding of silkworm.The main results were as follows:(1)Cloning Orco gene of different feeding habits silkworm strains ZhongGuang04,RiGuang03 and Lu7,there was no difference in Orco amino acid sequence.So,Orco had no difference of nucleotide in the silkworms with different feeding habits for artificial diets.(2)The molecular weight of atypical olfactory receptor Orco protein is 53.4 kD and isoelectric point is 8.69.Orco is alkaline protein.Prokaryotic expression was experimented with IC3 and gained specific antigen protein.Then we obtained high quality BmOrco polyclonal antibody.(3)Immunofluorescence using anti-BmOrco,Orco was expressed in antenna and maxillary in silkworm by immunofluorescence.There were specific signal in second and third tentacles of antenna.Specific signal were also discovered in the telos of maxillary palp and the root of three sensilla trichodea in maxillary galea.Orco were colocalizated with HRP-FITC.(4)Using qPCR and Immunofluorescence,we fund that Orco expression of Lu7 with low feeding habits was significantly higher than ZhongGuang04 with high feeding habits for artificial diets in mRNA and protein level.The fluorescence intensity of Lu7 with low feeding habits was stronger than ZhongGuang04 with high feeding habits for artificial diets by immunofluorescence.Orco expression of Lu7 was higher than ZhongGuang04 in the head of larvae.So,Orco expression of silkworm with low feeding habits was higher than silkworm with high feeding habits for artificial diets.(5)Using qPCR totest the Orco expression,the result was that Orco expression of F1 was reduced than the parent with low feeding habits using qPCR.But there was no change compared with the silkworm with high feeding habits.(6)We tested the change of Orco expression in day 1 third instar silkworms with starvation for 12 h.The result showed that Orco expression was significantly reduced compared with control after starvation.