亚洲玉米螟免疫相关模式识别受体基因β-1,3-GRP的cDNA克隆及表达分析
发布时间：2018-12-17 03:22
【摘要】：为研究亚洲玉米螟Ostrinia furnacalis (Guenee)幼虫体内一种与免疫相关的模式识别受体β-1,3-葡聚糖识别蛋白(ββGRPs)表达调控的分子机理,本文利用RACE技术从亚洲玉米螟幼虫体内克隆得到β-1,3-eRr,的cDNA序列,对β-1,3-GRP基因序列进行了生物信息学分析,摸索了原核表达β-1, 3-GRP蛋白的条件。对亚洲玉米螟5龄幼虫进行细菌注射,通过荧光定量实时PCR检测注射前后β-1,3-GRP基因在不同组织中mRNA水平上的表达变化情况。主要研究结果如下：(1)采用RT-PCR及RACE技术从亚洲玉米螟幼虫中克隆β-1,3-GRP基因全长cDNA序列。该基因全长1570 bp核苷酸(GenBank登录号：KF425324),包含一个1452 bp的开放阅读框(ORF),一个14 bp的5’非编码区(5’UTR)和一个104 bp的带有加尾信号的3’非编码区(3'UTR) 。开放阅读框从第15个核苷酸开始,终止于第1469个核苷酸,起始密码子ATG,终始密码子TGA,由其推导的氨基酸序列以甲硫氨酸为起始氨基酸,长为484个氨基酸。Of-β-1, 3-GRP蛋白的计算分子量约为53.37 kDa,估测等电点pI为6.46。生物信息学分析表明：β-β-1, 3-GRP蛋白无跨膜结构域,N端含有信号肽,切割位点为25与26位氨基酸之间,有2个糖基化位点,分别位于139位和141位,含有44个磷酸化位点,均匀分布于整个多肽链中。BlastP分析结果表明：Of-β-1, 3-GRP的氨基酸序列与棉铃虫Helicoverpa armigera β-1,3-GRP3,家蚕Bombyx mori β-1, 3-GRP2,烟草天蛾Manduca sexta GNBP,烟草天蛾M. sexta β-1, 3-GRP3,玉带凤蝶Papilio polytes革兰氏阴性菌结合蛋白3、柑橘凤蝶Papilio xuthus革兰氏阴性菌结合蛋白3高度同源。(2)采用pET-28b原核表达系统对β-1,3-GRP蛋白进行了原核表达,结果显示,Of-βGRP重组蛋白在IPTG的梯度诱导之后在沉淀中均有大量表达,重组表达蛋白质分子的大小与预测蛋白分子量大小一致,约为53kDao。pET-28b-β-1, 3-GRP在28℃温度下,可以明显看到1.0 mmol/L 和 1.2 mmol/L IPTG诱导下碎菌上清中有明显的蛋白表达。(3)通过Real time PCR检测注射后不同时间(0h, 2h, 4h, 6h, 8h, 10h, 12h, 24 h, 36 h) fl-1, 3-GRP基因在亚洲玉米螟幼虫不同组织(血细胞、中肠、脂肪体、体壁)中的表达变化。结果表明：血细胞中,注射后6h,生理盐水组、枯草芽孢杆菌组和大肠杆菌组β-,,3-GRP表达水平均开始上升(p0.05)。12h后,大肠杆菌处理组和枯草芽孢杆菌组防1,3-GRP表达水平都达到峰值,随着时间延长,表达量开始急剧下降。在中肠和脂肪体中,三个处理细β-1, 3-GRP表达平均呈先增后降的趋势,但在中肠中,大肠杆菌处理组表达水平变化趋势较枯草芽孢杆菌处理组明显,在脂肪体中二者差异不明显。在中肠组织中,β-1,3-GRP作为一种响应革兰氏阴性菌袁面p-1,3-葡聚糖的受体,对大肠杆菌的响应要较革兰氏阳性菌枯草芽孢杆菌快。在体壁组织中,与对照组相比,生理盐水缈1,3-GRP基因表达水平变化不大,枯草芽孢杆菌组β-1,3-GRP基因表达水平4h时达到峰值,之后缓慢下降,而大肠杆菌组β-1,3-GRP基因表达水平2h时达到峰值,之后缓慢下降。总体来说,不同组织β-1,3-GRP基因对革兰氏阳性菌和革兰氏阴性菌的反应速度以及注射后表达量的变化存在差别,但是两种细菌都能够引起β-1,3-GRP基因在转录水平上的表达量显著变化。
[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，
本文编号：2383566
[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，
本文编号：2383566
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