【摘要】：為研究亞洲玉米螟Ostrinia furnacalis (Guenee)幼蟲體內一種與免疫相關的模式識別受體β-1,3-葡聚糖識別蛋白(ββGRPs)表達調控的分子機理,本文利用RACE技術從亞洲玉米螟幼蟲體內克隆得到β-1,3-eRr,的cDNA序列,對β-1,3-GRP基因序列進行了生物信息學分析,摸索了原核表達β-1, 3-GRP蛋白的條件。對亞洲玉米螟5齡幼蟲進行細菌注射,通過熒光定量實時PCR檢測注射前后β-1,3-GRP基因在不同組織中mRNA水平上的表達變化情況。主要研究結果如下：(1)采用RT-PCR及RACE技術從亞洲玉米螟幼蟲中克隆β-1,3-GRP基因全長cDNA序列。該基因全長1570 bp核苷酸(GenBank登錄號：KF425324),包含一個1452 bp的開放閱讀框(ORF),一個14 bp的5’非編碼區(qū)(5’UTR)和一個104 bp的帶有加尾信號的3’非編碼區(qū)(3'UTR) 。開放閱讀框從第15個核苷酸開始,終止于第1469個核苷酸,起始密碼子ATG,終始密碼子TGA,由其推導的氨基酸序列以甲硫氨酸為起始氨基酸,長為484個氨基酸。Of-β-1, 3-GRP蛋白的計算分子量約為53.37 kDa,估測等電點pI為6.46。生物信息學分析表明：β-β-1, 3-GRP蛋白無跨膜結構域,N端含有信號肽,切割位點為25與26位氨基酸之間,有2個糖基化位點,分別位于139位和141位,含有44個磷酸化位點,均勻分布于整個多肽鏈中。BlastP分析結果表明：Of-β-1, 3-GRP的氨基酸序列與棉鈴蟲Helicoverpa armigera β-1,3-GRP3,家蠶Bombyx mori β-1, 3-GRP2,煙草天蛾Manduca sexta GNBP,煙草天蛾M. sexta β-1, 3-GRP3,玉帶鳳蝶Papilio polytes革蘭氏陰性菌結合蛋白3、柑橘鳳蝶Papilio xuthus革蘭氏陰性菌結合蛋白3高度同源。(2)采用pET-28b原核表達系統(tǒng)對β-1,3-GRP蛋白進行了原核表達,結果顯示,Of-βGRP重組蛋白在IPTG的梯度誘導之后在沉淀中均有大量表達,重組表達蛋白質分子的大小與預測蛋白分子量大小一致,約為53kDao。pET-28b-β-1, 3-GRP在28℃溫度下,可以明顯看到1.0 mmol/L 和 1.2 mmol/L IPTG誘導下碎菌上清中有明顯的蛋白表達。(3)通過Real time PCR檢測注射后不同時間(0h, 2h, 4h, 6h, 8h, 10h, 12h, 24 h, 36 h) fl-1, 3-GRP基因在亞洲玉米螟幼蟲不同組織(血細胞、中腸、脂肪體、體壁)中的表達變化。結果表明：血細胞中,注射后6h,生理鹽水組、枯草芽孢桿菌組和大腸桿菌組β-,,3-GRP表達水平均開始上升(p0.05)。12h后,大腸桿菌處理組和枯草芽孢桿菌組防1,3-GRP表達水平都達到峰值,隨著時間延長,表達量開始急劇下降。在中腸和脂肪體中,三個處理細β-1, 3-GRP表達平均呈先增后降的趨勢,但在中腸中,大腸桿菌處理組表達水平變化趨勢較枯草芽孢桿菌處理組明顯,在脂肪體中二者差異不明顯。在中腸組織中,β-1,3-GRP作為一種響應革蘭氏陰性菌袁面p-1,3-葡聚糖的受體,對大腸桿菌的響應要較革蘭氏陽性菌枯草芽孢桿菌快。在體壁組織中,與對照組相比,生理鹽水緲1,3-GRP基因表達水平變化不大,枯草芽孢桿菌組β-1,3-GRP基因表達水平4h時達到峰值,之后緩慢下降,而大腸桿菌組β-1,3-GRP基因表達水平2h時達到峰值,之后緩慢下降�？傮w來說,不同組織β-1,3-GRP基因對革蘭氏陽性菌和革蘭氏陰性菌的反應速度以及注射后表達量的變化存在差別,但是兩種細菌都能夠引起β-1,3-GRP基因在轉錄水平上的表達量顯著變化。
[Abstract]:In order to study the molecular mechanism of the expression and control of an immune-related pattern recognition receptor 1-1, 3-Dextran in the larvae of Ostrinia furnita (Guenee), the cDNA sequence of 1-1, 3-eRr and 3-eRr was obtained by the RACE technique. The sequence of P-1, 3-GRP was bioinformatics, and the conditions of prokaryote-1, 3-GRP were found. The expression of IL-1, 3-GRP gene in different tissues of the 5-instar larvae of maize in Asia was detected by fluorescence quantitative real-time PCR. The main results of this study were as follows: (1) The full-length cDNA sequence of the 3-GRP gene was cloned from the larvae of maize by RT-PCR and RACE. The total length of the gene was 1570 bp (GenBank accession number: KF425324), a 1452 bp open reading frame (ORF), a 14 bp 5 'non-coding region (5' UTR) and a 104 bp 3 'non-coding region (3' UTR) with a tail signal. The open reading frame is terminated at 1469 nucleotides, the initiation codon ATG, and the final codon TGA from the 15th nucleosonic acid, and the amino acid sequence deduced therefrom is methionine as the starting amino acid and has a length of 484 amino acids. The calculated molecular weight of the Of-1-1, 3-GRP protein was about 53. 37 kDa, and the isoelectric point pI was estimated to be 6.46. Bioinformatics analysis showed that the 3-GRP protein has no cross-membrane domain, and the N-terminal contains signal peptide, the cleavage site is between 25 and 26 amino acids, and there are two glycosylation sites, which are located in 139 and 141 positions, respectively, and contain 44 phosphorylation sites and are uniformly distributed in the whole polypeptide chain. The results of BlastP analysis showed that the amino acid sequence of Of-1-1, 3-GRP and Helicoverpa armigera Helicoverpa armigera-1, 3-GRP3, bombyamori-1, 3-GRP2, Manduca sexta GNBP, M. sexta-1, 3-GRP3, Papilio poland gram-negative bacteria binding protein 3, The Papilio xuthus gram-negative bacteria binding protein 3 of the citrus fruit is highly homologous. (2) Prokaryotic expression of the P-1, 3-GRP protein was carried out by using the prokaryotic expression system of the pET-28b, and the results showed that the recombinant protein of the Of-GPRP was expressed in the precipitation after the gradient induction of IPTG, and the size of the recombinant expression protein molecule was consistent with the predicted protein molecular weight. The expression of 1. 0 mmol/ L and 1. 2 mmol/ L IPTG in the supernatant of the strain was obviously observed at the temperature of about 53kDato. pET-28b-1-1, 3-GRP at 28 鈩，

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