Molecular Cloning Of G Protein β And γ Subunits Genes And Screening For Their Interactive Proteins In Sitobion Avenae

G protein-coupled signaling system, ubiquitous in animals, simple eukaryotes, plants and insects, is one of the most important cellular signaling pathways in organisms. More studies, Drosophila melanogaster as model, have been done on structure and function of G-protein, and confirmed that G proteins involved in diverse signaling pathways such as visual, olfactory and hormonal signals, etc. However, little is known about G proteins and their signaling mechanisms in other insects, especially in those that severely damage crops. Studies on G protein-coupled signaling pathways not only help to understand signal-perception mechanism in insects, but also elucidate inhibitory effect on certain defect link of signaling pathway, and then provide theoretic base for exploring new control method. In this paper, genes of G protein p and y subunits were cloned from Sitobion avenae, an important pest in wheat production, by RT-PCR and RACE methods. The interaction between cloned p and y subunits and screening for related proteins from Sitobion avenae cDNA library were determined by Yeast Two-Hybrid System. The main results are as follows:(1) Two genes of G protein p and y subunits were cloned for the first time from Sitobion avenae by RT-PCR and (3'/5')-RACE techniques. Both cDNA sequences were deposited in GenBank and the accession numbers are AY205294 and AY339875, respectively. The deduced amino acid sequence of p subunit was 90% and 80% identical to Anopheles gambiae (Drosophila melanogaster) and animals, respectively. The cloned y subunit was 60% identical to y1 subunit, preferentially expressed in the brain of Drosophila melanogaster, and had 38% identity with y7 subtype in animals. Cloned P subunit contained seven WD-40 (Tryptophan-Aspartate) repeats, which is common structural feature of all known P subunits of G proteins. Cloned y subunit had GGL domain (G protein gamma subunit-like motifs) and carboxyl-terminal CAVI motif, in which conserved cysteine was modified by an isoprenoid group. Based on the alignment of amino acid, it was supposed that p and y subunits cloned from Sitobion avenae are similar to pi and yl subtypes in Drosophila melanogaster, and P2 and y7 subtypes in animals.(2) Tissue- and stage-specific transcriptive levels were determined by relative quantitative RT-PCR (RQRT-PCR) and showed that both P and y subunits were transcipted in multiple tissues and at different stages, but the transcriptive level in head was higher than that in breast and abdomen of Sioibion avenae. This ubiquitous distribution of G protein P and y subunits in Sitobion avenae indicated that they may involve in diverse signaling pathways.(3) Interaction proteins were screened from Sitobion avenae cDNA library with cloned P subunit as bait protein by Yeast Two-Hybrid System. More than 120 positive clones were identified by PCR Colony Screening and 40 representative clones were sequenced. Through GenBank blast, the cDNA inserts were partially similar to such mRNAs as Bos taurus retina G protein-coupled receptor kinase 7 (GPRK7), Homo sapiens G protein-coupled receptor 126 (GPR126), Tenebrio molitor cathepsin-L-like midgut cysteine proteinase, 28S and 18S ribosomal RNA genes. No similar genes were found aboutother clones, indicated that they may be some unpublished sequences. Obtaining of these valuable clones provided substantial foundation for further study the function of G protein p subunit in signaling pathway of Sitobion avenae. The same experiments were done as cloned y subunit and seven positive clones were obtained and four were sequenced. However, no similar sequences to these clones were found in GenBank.(4) The interaction between cloned P and y subunits was determined by Two-Hybrid System and no positive clones were obtained. One of the possible reasons is that proteins with weak affinities may escape detection or there is no interaction between the two subunits in Sitobion avenae. Further study is needed to testify cloned p-y proteins interaction. From the facts mentioned above, it was presumed that there ma...