Gene Cloning And Functional Analysis Of Odorant Binding Related Proteins In Chilo Suppressalis And Nilaparvata Lugens

In the long-term evolutionary process, insects evolved a highly sensitive olfactory system to detect volatile compounds in the environments, and accordingly trigger physiological or behavioral responses such as finding mates, food, and habitats. Insect olfaction could be divided into peripheral and central processes. In the peripheral process, odorant binding proteins (OBPs) and chemosensory proteins (CSPs) were mainly responsible for binding and transporting the hydrophobic volatiles to the odorant receptors (ORs) located on the dendrite membrane. Chilo suppressalis belongs to Lepidoptera, and Nilaparvata lugens belongs to Hemiptera. Both species are important pests of rice. The studies of their olfactory proteins were not only important for understanding the olfactory mechanism, but also could provide potential target genes for the development of efficient behavior control technology, and thus helpful for improving the sustainable control of rice pests. However, the related researches were rare and were restricted to the antennal transcriptome data analysis and electroantennanogram measurements. In this study, by genome or transcriptome data analysis and PCR experiments, the cDNA sequences of 18 OBP genes from C.sup and 9 CSP genes from N. lugens were identified. Among those,10 CsupOBPs and all the 9 NlugCSP were in full length. The tissue and temporal expression patterns of all these genes were determined by RT-PCR or qPCR. In addition, the function of CsupOBP8 was explored by the competitive fluorescence binding assay. The main results were as follows:1. Gene cloning and sequence analysis of CsupOBPsBy the genomic data analysis and PCR experiments,18 CsupOBP cDNA sequences were cloned, of which 10 were in full length. These 10 genes were named as CsupOBP3,8, 9,11,16,25,26,30,32 and 33. Sequence analysis showed that most of these genes had 6 conservative cysteins, however, CsupOBP3,16,23,26 had only 4 conservative cysteines, and thus were classified into the Minus-C OBP. Except for the CsupOBP9,23 and 24, all CsupOBPs showed higher identity (> 50%) with other Lepidopteran OBPs. In particular, CsupOBP11 and 32 shared the identity of 82% and 79% with other reported OBP, respecitively. Phylogenetic tree showed that an OBP gene was classified into the PBP branch, and thus was named CsupPBP4.2. Expression patterns of CsupOBPs at different development stage and in different tissuesRT-PCR technique was used to determine the expression patterns of 30 CsupOBP genes. Results showed that CsupOBP1,3,6,8,9,10,18 and 24 were specifically expressed in the head of larval stage, especially the CsupOBP1,8,10 and 24. In the pupal stage, CsupOBP30 was specifically expressed in male pupa; CsupOBP5,6,7, and 31 were expressed in higher levels in male pupae than that in the female pupae. During the adult stage, CsupPBP4, CsupOBP1,3,4,8,10,11 and 24 were specifically expressed in antennae; and CsupOBP20,30, and 33 were highly expressed in antennae. CsupOBP 18 was specifically expressed in female antennae. These antenna highly or specifically expressed CsupOBPs may play roles in the olfaction of C. sup.3. Prokaryotic expression and ligand-binding affinity analysis of CsupOBP8As the CsupOBP8 was highly expressed in the larval heads and adult antennae, it was postulated to play an important role in the olfaction in both larval and adult stages. CsupOBP8 was expressed in vitro and was functionally analyzed by fluorescence competitive binding assay. The gene was cloned into the pET-30a (+) expression vector, and then expressed into Escherichia coli with IPTG. The recombinant protein was purified through nickel ion affinity chromatography and enterokinase hydrolysis. The binding affinity of the recombinant CsupOBP8 was tested with 33 odorant compounds, including 30 plant volatiles and 3 C. sup sex pheromones. Results showed that CsupOBP8 displayed high binding affinities with some plant volatiles, such as P-ionone, nerolidol method, farnesol,2-hexanone (Ki=4.9-15.0μM) and with benzaldehyde, heptanol, 2,6-di-tert-butylphenol, cedrol and acetophenone (Ki=22.3-30.8μM). It suggests that CsupOBP8 may play an important role in the C. sup olfaction by binding and transporting these plant volatiles.4. Gene cloning and sequence analysis of NlugCSPsThe nine NlugCSP cDNA full-lengths from N. lugens were cloned by using RACE strategy, and were named NlugCSP 1-9, respectively. The deduced amino acid sequences of these NlugCSPs possessed all hallmarks of a classic CSP, including 4 conserved cysteine residues. The identities among the nine NlugCSPs ranged from 10%to 77%. The phylogenetic tree showed that NlugCSP5,7 and 8 were clustered into a same group.5. Expression patterns of CsupOBPs at different development stage and in different tissuesBy qPCR, the expression level of 9 NlugCSP was detected with respect to different developmental stages, genders and tissues. Generally, the expression levels of 9 NlugCSPs were changed obviously with the developmental stages. NlugCSP9 displayed a sharp decrease in expression from 4th instar to 5th instar. NlugCSP8 expressed higher in 5th instar nymph than nymph of other instars and adult significantly. As to the different tissues, NlugCSP 1 was mainly expressed in male abdomen; the expressions of NlugCSP2,4,5,7 were higher is female wings; the NIugCSP3,6,8,9 were highly expressed in male legs. Compared with other NlugCSPs, NlugCSP 7 was highly expressed in the chemosensory tissues, suggesting a role in the chemosensation of N. lug. In contrast, NlugCSP 1 was specifically expressed in male abdomens, suggesting that it may serve as a gene related to the male reproduction.