Functional Analysis On The Olfactory Genes In Bombyx Mori

As a most prosperous kind of animals in the world,insect could adapt to various environment to sustain a strong ability to survive and reproduce.Many behaviors of insects,including foraging,courting,mating,reproducing,defense and migration,are initiated by olfactory system.So studying on the function of important olfactory genes could facilitate our understanding of insect survival strategies.The domesticated silkworm,Bombyx mori,is not only a well-known insect in sericulture,but it is also considered as a model for Lepidopteran insects.In addition,thousands of years of artificial rearing have resulted in specialized behavior.As a typical oligophagous insect,B.mori primarily consumes mulberry leaves and seldom feeds on the other species.Besides,compared with other Lepidopteran insects,B.mori has the simplest sex pheromone system in which the pheromone bombykol can trigger the full sexual behavior of the male moth.Considering both feeding and sexual behaviors are initiated by olfactory perception,studying on the function of olfactory genes in the silkworm will provide insights to insect olfaction and promote further understanding of olfactory detection mechanism.This research studies on the olfactory genes functioned in silkworm feeding and mating behavior by initially mutated the genes with transgenic CRISPR/Cas9 system in which CRISPR/Cas9-based genome editing technology and piggyBac-based germline transformation technology are combined,followed by analyzing and validating the function of these olfactory genes with chemical ecology methods.The main work contains the following aspects.1.Functional study of BmOrco.By using a binary transgenic CRISPR/Cas9 system,we mutated the olfactory receptor co-receptor gene Orco.By microinjecting the transgenic plasmids to the silkworm embryo followed by fluorescence screening,the Orco mutant was generated in G2 generation.By PCR amplification and self-cross screening,a stable,heritable Orco homozygous mutant line was successfully established in G5 generation.To examine the electrophysiological response of olfactory neurons in the antenna of Orco homozygous mutants to the two sex pheromone components,bombykol and bombykal,the single sensillum recording(SSR)technology was used.The results showed a significantly reduced electrophysiological response to both pheromone components in Orco mutants.To determine the food selection behavior of Orco mutants,the mulberry leaves or different concentrations of potent volatile component in the mulberry leaf,cis-jasmone,were used as attractive source.The distance the silkworm had moved at the end of 1 hour and their walking trail in different time point within 1 hour were recorded and analyzed.The results showed that the Orco mutants displayed defective selection for mulberry leaves or low concentrations of cis-jasmone along with an impaired repellant behavior for the highest concentration of cis-jasmone.To examine the mating behavior of Orco mutants,a competitive choice assay was used in which a natural pheromone released by a wild type female moth,bombykol,bombykal or a simulated 11:1 mixture of bombykol and bombykal were respectively applied.The results showed that the mating behavior was severely impaired in Orco mutants while the mating behavior totally disappeared in Orco homozygous mutants.Above all,these results validated that Orco is indispensible for detecting odorants in silkworm olfactory system,which controls the larval feeding behavior and adult mating behavior directly.2.Functional study of BmOR54 and BmOR56.Two olfactory receptors OR54 and OR56 with close relationship to silkworm feeding behavior were mutated respectively and with the aid of Golden-Gate assembly method,both two receptors were mutated simultaneously as well.As a result,three mutant silkworm line were established,BmOR54?BmOR56 ? BmOR54-OR56.To examine the feeding behavior of fifth instar silkworm in the three mutants obtained,the mulberry leaves and different concentrations of cis-jasmone were used as attractive source.The results showed that compared with wild type silkworms,the feeding behavior of OR56 mutant was increased,while both feeding behavior of OR54 mutant and OR54-OR56 mutant were impaired.The expression pattern of OR54 and OR56 in three mutants showed that the depletion of OR56 downregulated OR54,and decreasing of OR54 downregulated OR56.This implied that OR56 has a function to promote feeding while OR54 has a role to inhibit feeding.All results above showed that the two mutual antagonistic receptors,OR54 and OR56 co-regulate the feeding behavior of B.mori.3.Functional study of BmPBPs.With transgenic CRISPR/Cas9 system,we obtained three mutant silkworm lines including BmPBP1,BmPBP1-PBP2 and BmPBP1-PBP2-PBP3.The SSR technology was used to examine the electrophysiological response of olfactory neurons in the mutant antenna to the two sex pheromone components,bombykol and bombykal.The results showed that the olfactory neuron of PBP1 mutant presented a stronger response to bombykol than wild type,while for a comparison,the response of olfactory neuron to bombykol in PBP1-PBP2 mutant disappeared,suggesting that when PBP1 was knocked out,PBP2 would compensate by binding with bombykol.In mating behavior assay,when natural pheromone released by a wild type female moth,bombykol or 11:1 mixture of bombykol and bomykal,the mating behavior of PBP1-PBP2 mutant increased,while exposed to the same stimulation,the mating behavior of PBP1-PBP2-PBP3 mutant decreased,suggesting that PBP3 could compensation the absence of PBP1 and PBP2.With far-western blotting and mass spectrum methods,the proteins interacted with PBP1 in both wild type and PBP1 mutant were investigated.By comparision,one protein named transaldolase was figured out.Considering the function of transaldolase is to catalyze the exchange of alcohol group and aldehyde group,we inferred that the trace PBP1 remained in PBP1 mutant could interact with transaldolase,making the bombykol molecular transformed to bombykal,after which the transformed bombykol would be bound and transported by PBP2,making a compensation to PBP1.Based on this speculation,a model for explanation of compensation effect was put forward.