Identification And Function Analysis Of Olfactory-Related Genes In Frankliniella Intonsa(Trybom)

Insects mainly use the olfactory system to identify odors in the environment to complete life activities such as host location,mating,laying eggs,and evading enemy,in which olfactory proteins play an important role in this process.Due to its small size,fast reproduction,strong adaptability and severe occurrence,flower thrips have posed a great threat to crop production in China.Based on flower thrips transcriptome data,three olfactory-related genes CSP1,CSP2 and OR were cloned and identified,and their expressions in the different developing stages and different tissues of flower thrips were clarified.Using RNAi combined with the electroantennagram(EAG)and behavior bioassay,the function of CSP1 protein of flower thrips was preliminary clarified.The research results can provide scientific basis for further revealing the olfactory mechanism developing new control technology for flower thrips.The main results are as follows:1.Using RT-PCR technology to clone and identify two chemosensory protein genes CSP1,CSP2,and one olfactory receptor gene OR,the Gene Bank accession numbers are: MT199111,MT211602 and MT211603.The three gene open reading frames are: 405 bp,390 bp and 1374 bp,respectively,encoding 135,130 and 458 amino acids.CSP1 and CSP2 have typical chemosensory protein structural characteristics: 4conserved cysteine,and a signal peptide of about 20 amino acidsat the N-terminal;the phylogenetic tree analysis showed that the 3 olfactory proteins were closely related to the olfactory-related proteins of western flower thrips.2.The prokaryotic expression vectors of p ET30a-CSP1,pET28a-CSP2,and p ET30a-OR were constructed to successfully induce the expression of recombinant proteins CSP1,CSP2 and OR.Among them,p ET30a-CSP1 was expressed in the supernatant,and p ET28a-CSP2 and p ET30a-OR were expressed in inclusion bodies.High-purity target proteins were obtained by Ni-sepharose affinity purification under denaturing conditions followed by renaturation.3.Using qRT-PCR to analyze the expression profile of CSP1,CSP2 and OR genes in the different development stages of flower thrips,namely: 1 instar nymphs,2 instar nymphs,pupae,2 d female adults,5 d female adults and 10 d female adults.The results showed that,the expression of the three genes was the highest in the 2 instar nymph and the lowest in the pupae.In addition,CSP1,CSP2 and OR were expressed in different tissues of female adults including: antennae,head,thorax,abdomen and legs.The CSP1 gene was most expressed in the legs,followed by the head and thorax;the CSP2 gene was most expressed in the antennae;the OR gene was most expressed in the head,followed by the thorax and legs.Immunocolloidal gold was used to mark CSP1 in the antennae of different developmental stages of flower thrips.The results showed that the CSP1 protein was highest expressed in the nymph stage,followed by the adults stage,with extremely low expression in the pupal stage.4.Using "Y"-type olfactometer to study the effect of CSP1 on the behavior selection of flower thrips.Compared with the control group(dsGFP injection),flower thrips injected with dsCSP1 had a significantly lower preference to p-Anisaldehyde and Ethyl nicotinate,and the number of thrips who did not respond to odor sources increased significantly.Further EAG experiments showed that the EAG response in flower thrips injected with dsCSP1 with p-Anisaldehyde stimulation was significantly lower than that of the control group injected with dsGFP.