Cloning And Expression Analysis Of A G-protein α Subunit-Gαo And Screening For The Interactive Proteins In The Rice Water Weevil Lissorhoptrus Oryzophilus Kuschel

Rice water weevil Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae), native to North America, is the key insect pest of rice. In China, since its first detection in 1988 from Tangshan city of Hubei province, it had been reported in 15 provinces. Olfactory and gustatory of insects play important roles in the detection of food, hosts, or partners and therefore determine the spreading process of the alien weevil in a new environment. G protein-coupled signaling system is an important signaling pathway involved in insect olfactory, visual and endocrine hormones and other signal transductions. The research on the G protein of insect is helpful to understand not only the molecular mechanism of how insect feel outside signals, but also the functions of G protein on the behavioral and physiological regulation of signal transduction of insect. This will offer an theoretical basis to study signal transduction and to explore new approach pest control in the future.In this dissertation, I used reverse transcription (RT)-PCR and RACE method to clone the ORF of a G protein a subunit from L. oryzophilus, and analysis the sequence and evolution relationship. The expression profile of transcript of G protein a subunit was conducted by real-time quantitative PCR. Meanwhile, we expressed it in prokaryotic system and prepared for polyclonal antisera, with the anti-Lo Gαo antiserum we immunolocalized the G proteinαsubunit in the antennae of the weevil. We constructed a L. oryzophilus head's cDNA library, and screened for the related proteins of Lo Gαo by Yeast Two-Hybrid system. The results are presented as follows:1. A cDNA encoding a G proteinαsubunit was cloned from the head of the rice water weevil L. oryzophilus Kuschel (Coleoptera:Curculionidae) by the combination of reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA contains a 1065 bp open reading frame (ORF) that encodes a 354 amino acid protein. The predicted molecular weight (MW) and pI was 18.192 kDa and 5.08, respectively. Through multiple sequence alignment, phylogenetic and similarity analysis, we found that this G protein a subunit belongs to the Gαo family, the amino acid sequences of main functional domain and binding sites are essentially the same with the other Go protein. From the results we speculated that the functions of Lo Gαo might be similar with other Gαo proteins had been reported, and might be combined with similar receptors and regulated the same downstream effector.2. Prokaryotic expression, polyclonal antibody preparation and tissue distribution of the Gαo. Prokaryotic expression vector of pET30a/Lo Gαo was constructed and successfully expressed in BL21. The recombinant protein was purified and prepared the polyclonal antibody. With ELISA and Western blot, the antibody titer and specificity was studied. The titer of anti-Lo Gao antiserum was 2.048×106, and the result of Western blot showed that there was only one specific band of 40 kDa was detected from crude extracts of various organs of the weevil, there was no cross-reaction with other G protein subunits. The titer and specificity of the antibody were consistent with the requirements of next experiments, it provided a precondition for the immunolocalization of Lo Gao in the weevil3. Spatial expression profile of Lo Gao transcript was analyzed by Real-time quantitative PCR. With the relative quantitative method, we studied the expression profiles of the transcription level of Lo Gao and found that the Lo Gao mRNA expressed in various organs, without tissue-specific, but the expression in the antennae was significantly higher than in other parts of weevil. Antenna is an important part of insect sensory systems, performed the sense of smell, taste and other functions, highly expressed of the gene in the antennae, lead we speculated that the protein may be involved in the sense of olfactory/gustatory signal transduction.4. SEM observation of the antennae of the rice water weevil. Using scanning electron microscopy, we observed types and distribution of antennal sensilla of parthenogenesis and bisexual rice water weevil; observed the differences between two reproductive modes and male and female, and explored the functions of various types of sensilla, as a basis to study the immunolocalization of Lo Gao in the olfactory sensilla of the rice water weevil.5 Immunolocalization of Lo Gao in the antennal sensilla of the rice water weevil. With anti-Lo Gao polyclonal antibody prepared in this study, using immune colloid labeling method to study the localization of G protein ao subunit in the antennal of rice water weevil, and found that Lo Gao was mainly expressed in the dendrites of the trichoid sensilla. According to the general functions of antennal sensilla proposed by previous reports and our fore-mentioned observation, trichoid sensilla were such kinds of olfactory/gustatory sensilla. We speculate that the Lo Gαo protein may be involved in olfaction or gustatory signal transduction of the weevil.6. Interaction proteins were screened from the cDNA library of L. oryzophilus head with Lo Gao as bait protein by Yeast Two-Hybrid System. We built a yeast cDNA library of the head of rice water weevil, the titer of the library was 6.8×109 cell/ml (>2.0×107cell/ml), can be used for the yeast two-hybrid experiments. The rice water weevil head cDNA library was screened with Gao bait protein, get some interaction clones, e.g. myosin light chain kinase, apolipoprotein D, mysin VI, ferritin, sorbitol dehydrogenase, and zinc finger protein etc. The interaction of these proteins with Gao need further experimental proof.