Cloning, Characterization And Expression Of βGRP,a Pattern Recognition Receptor Related To Immunity Of Ostrinia Furnacalis Guenee Larvae

In order to investigate the regulation mechanism of the pattern recognition receptors β-1,3-glucan recognition protein (βGRPs) related to insects’innate immunity,βGRP gene was cloned from the Ostrinia furnacalis (Guenee) larvae with RACE. The cDNA sequence and deduced amino acid sequence were bioinformaticly analyzed, and then the fusion protein of β-1,3-GRP was expressed in the Escherichia coli BL21(DE3) strain and further purified with Ni2+chelated affinity chromatography. Bacterial infection was applied to elucidate the defense mechanism of the larvae, and the mRNA expression of β-1,3-GRP gene in various tissues of the larvae was analyzed by the quantitative real time PCR (qRT-PCR). The main results are as follows:(1) In this study, RT-PCR and Rapid amplification of cDNA ends (RACE) methods were used for the cloning of full length cDNA encodingβ-1,3-GRP from the 5th instar larvae of O. furnacalis. The full length cDNA of β-1,3-GRP was 1570 bp (GenBank accession number: KF425324), containing an ORF of 1452 bp, a 5’UTR of 14 bp and a 3’UTR with polyadenylation signal of 104 bp. The open reading frame starts from the 15th nucleotide and ends at the 1469th nucleotide. the initiation codon is ATG and the termination codon is TGA. Its deduced amino acid sequence starts with Met with the length of 484 amino acids. The β-1,3-GRP has a predicted molecular weight of 53.37 kDa and pi value of 6.46. Bioinformatics analysis showed that there was no possible transmembrane protein model but an signal peptide in the N terminal of the peptide, and the cutting site is between 25th amino acids and 26th amino acids. There are two glycosylation sites which located at 139th and 141th amino acid, and 44 phosphorylation sites which distributed throughout the polypeptide chain. BlastP analysis showed that the amino acid sequence of the cloned cDNA from O. furnacalis was highly similar to those of Helicoverpa armigera β-1, 3-GRP3, Bombyx mori β-1,3-GRP2, Manduca sexta GNBR M. sexta β-1,3-GRP3, Papiliopolytes gram-negative bacteria binding proteins3 and Papilio xuthus gram-negative bacteria binding proteins3.(2) The pET expression system was applied to express β-1,3-GRP protein, after gradient IPTG induction the β-1,3-GRP protein was expressed in E. coli BL21(DE3), and the size of the recombinant protein was same as the predicted, about 53 kDa. With 1.0 mmol/L and 1.2 mmol/L IPTG induction at 28 ℃ temperature, the pET-28b-β-1,3-GRP recombinant protein was significantly expressed in the supernatant of E. coli.(3) The qRT-PCR was used to detect the temporal and spatial expression pattern of β-1, 3-GRP gene expression after the injection of physiological saline, gram-negative E. coli and gram-positive Bacillus subtilis, respectively. At different times (0 h,2 h,4 h,6 h,8 h,10 h,12 h, 24 h,36 h) after bacteria injection, the mRNA expression of β-1,3-GRP gene in different tissues such as hemocytes, fat body, midgut tissues and integument in untreated group, saline group, E. coli-challenged group and B. subtilis-chailenged group were analyzed, respectively. The result showed that gene expression of β-1,3-GRP started to increase at 6 hours post injection (HPI) (p<0.05).), and then it reached a peak in E. co/z-challenged group and B.subtilis-challenged group group at 12 HPI. As time progress, the expression level began to decline sharply. In the midgut and fat body, β-1,3-GRP expression levels showed a tendency to drop after the first increase in saline group, E. co/z-challenged group and B. sMZtf/fc-challenged group. But in the midgut, the expression levels of E. co//-challenged group was more severely than that in the B. subtilis-challenged group, but there was no significant difference between them in the fat body (p>0.05). The results revealed that β-1,3-GRP, a receptor responseing to β-1,3-glucan, responded to E. coli faster than that of gram-positive bacteria B. subtilisas in the midgut. In the integument, β-1,3-GRP gene expression level had no significantly changes in the saline injection group compared with the untreated group. The β-1,3-GRP gene expression level reached a peak at 4 HPI, then slowly declined in B. subtilis-chaUcnged group. Meanwhile β-1,3-GRP gene expression level reached a peak at 2 HPI, and then slowly declined in E. co/i-challenged group. Both gram-positive and gram-negative bacteria challenge could lead to the expression changes of Of-β-1,3-GRP gene on the mRNA level, although there were differences on the mRNA level and at the speed of Of-βGRP responding to challenge when injected with gram-positive and gram-negative bacterials, respectively.