【作者】 章玉萍；

【导师】 樊美珍； 黄勇平； 苗雪霞；

【作者基本信息】 安徽农业大学 ， 森林保护学， 2009， 博士

【摘要】 G蛋白偶联信号传导系统是生物体内一类重要的细胞信号传导途径,其在高等动物、简单真核生物、昆虫及植物中普遍存在。国际上,以果蝇为模式昆虫,对G蛋白的结构与功能作了较多的研究,证实G蛋白参与了昆虫的嗅觉、视觉和内分泌激素等多种信号的传导过程。为了研究G蛋白在昆虫中的生理功能及其作用机理,本文选择家蚕(Bombyx mori)作为实验材料,利用生物信息学、RT-PCR技术及RACE方法进行异源三聚体G蛋白亚基的克隆,并将G蛋白亚基克隆入表达载体pET系列,构建了GST融合蛋白和His融合蛋白。并利用GST融合蛋白沉降技术研究了家蚕G蛋白β和γ亚基之间是否互作。本研究取得如下主要结果:1.家蚕G蛋白α亚基基因的克隆及时空表达分析运用生物信息学方法在已有的家蚕基因组数据库中找到了一段与G蛋白αlphα亚基(Gα)同源性很高的序列。通过设计特异性引物、PCR和RACE技术,成功地克隆了2个家蚕Gα基因的全长cDNA序列。分别命名为BmGα73B和BmGα12。BmGα73B基因全长1509bp,开放阅读框(ORF)1158bp,编码385个氨基酸。利用Blast、DNAstar等软件分析发现该基因编码的蛋白质与其他物种已知的Gα具有一定的同源性,与果蝇(Drosophila melanogaster)中的Gfα同源性最高,达到了47%。BmGα12基因全长1374bp,开放阅读框(ORF)1128bp,编码375个氨基酸。与果蝇的Concertina亚基氨基酸序列同源性达69%,与哺乳动物的Gα12/13型亚基同源性达56%。两个亚基都具有G蛋白α亚基的保守域结构。利用RT-PCR技术检测BmGα73B基因在家蚕不同组织器官和不同时期的转录表达活性,结果表明它在家蚕各组织器官中都有表达。从组织水平上看,BmGα73B在中肠中表达量最高;另外在马氏管、头部和神经索等组织中也有适量表达。在家蚕的不同时期中,转录水平峰值出现在幼虫期,在蛹早期也有适量的表达,而在预蛹期、蛹后期和成虫期几乎没有表达。以上结果说明BmGα73B可能参与了家蚕生长前期的中肠发育过程。用RNAi干扰的方法检测BmGα73B在家蚕中肠发育中的作用,没有检测到明显的缺陷型家蚕。2.家蚕G蛋白β和γ亚基基因的克隆与序列分析从GenBank上下载β亚基的序列,设计引物进行RT-PCR扩增,经测序克隆的β亚基序列和网上公布的序列完全一致。并进行了序列分析。另外从家蚕中首次克隆到两个G蛋白γ亚基基因,根据氨基酸序列同源性比较结果,初步推测克隆的家蚕γ亚基与果蝇的γ1和γ30A相似,分别命名为BmGγ1和BmγG30A。BmGγ1亚基编码70个氨基酸,与果蝇脑中表达的Gγ1亚基氨基酸同源性为70%;BmGγ30A亚基编码72个氨基酸,与果蝇的Gγ30A亚基氨基酸同源性为81%。两种γ亚基都具有CCL(G protein gammasubunit-like motifs,GGL)保守区,C末端具有4个氨基酸CAVI保守基序。3.家蚕G蛋白亚基基因的原核表达(1)家蚕G蛋白α亚基的原核表达。根据编码G蛋白α亚基蛋白的基因序列,设计两对特异引物,通过PCR技术从pMD18-T-Gα质粒中扩增出Gα蛋白基因,连接到原核表达载体pET-41b(+),构建了C端带有6Gst-tag的融合表达载体pET-41b(+)-Gα73B和pET-41b(+)-Gα12。利用化学转化法将重组表达载体转化E.coli BL21(DE3),PCR检测阳性转化子。经IPTG诱导和SDS-PAGE分析,成功获得了能够表达目的蛋白的工程菌株。摇瓶培养确定工程菌BL21(DE3)-pET-41b-Gα73B在IPTG浓度为0.1mmol/L、37℃下诱导3h可达到最大表达量;工程菌BL21(DE3)-pET-41b(+)-Gα12在IPTG浓度为0.5 mmol/L、37℃下诱导4h可达到最大表达量。表达方式分析发现两个重组蛋白在大肠杆菌中都以包涵体的形式存在。Western blot分析诱导的重组蛋白能与抗GST单抗特异性反应。(2)家蚕G蛋白β亚基基因的原核表达。编码G蛋白β亚基蛋白的基因序列,构建成带有C端6Gst-tag和6His-tag的融合表达载体pET-41b-Gβ1,pET-41b-Gβ2和pET-22b-Gβ2,在大肠杆菌BL21(DE3)中都能诱导表达。表达方式分析发现该重组蛋白在大肠杆菌中都以包涵体的形式存在。通过优化可溶性蛋白表达条件,工程菌BL21(DE3)-pET-22b-Gβ2在IPTG浓度为0.001 mmol/L,20℃表达条件下,诱导6h可成功获得少量的可溶性的His-Gβ2融合蛋白。(3)家蚕G蛋白γ亚基基因的原核表达。根据BmGγ1和BmGγ30A的蛋白序列,构建重组表达质粒pET-41b-Gγ1和pET-41b-Gγ30A并在大肠杆菌BL21(DE3)中诱导表达。表达条件研究表明,IPTG浓度对蛋白表达量没什么影响,两个融合蛋白均在诱导4h达到最大表达量。表达方式分析发现两个重组蛋白在大肠杆菌中都以可溶性表达和包涵体表达两种形式存在。利用谷胱甘肽琼脂糖球珠亲和层析技术纯化了目的蛋白,并用Westren blot技术对融合表达的蛋白进行了鉴定。4.家蚕G蛋白亚基之间的相互作用研究用GST融合蛋白沉降技术研究克隆的家蚕G蛋白β和γ亚基之间是否存在互作。在生物体内,G蛋白β和γ亚基只有结合成二聚体才具有信号传导的功能。结果显示家蚕G蛋白β和γ30A亚基可以结合在一起。本试验没有发现β和γ1亚基之间的相互作用,可能这两个亚基之间不能结合或结合力很弱,因此推测家蚕中可能存在其它亚型的β和γ亚基,还有待进一步试验证明。5.家蚕G蛋白调节因子基因的初步研究本研究还克隆了家蚕G蛋白调节因子(RGS)基因的部分cDNA序列,获得了3个RGS的部分序列。通过序列比较分析,将其命名为Bmo-RGS7、Bmo-SNX14和Bmo-Axin,其中Bmo-RGS7具有RGS、GGL、DEP保守区,Bmo-SNX14具有RGS基本保守区。关于家蚕RGS的详细结构和功能还有待进一步研究。通过本研究,从家蚕中克隆了5个G蛋白亚基基因和3个G蛋白信号调节因子基因。通过转录水平的研究证实,克隆的G蛋白α亚基属于广泛组织分布型,可能参与细胞多种信号途径的传导。GST融合蛋白沉降技术研究表明,G蛋白β和γ30A亚基可以结合在一起。上述结果为进一步研究家蚕G蛋白的功能及其在昆虫信号传导途径中的作用奠定了基础,同时,这些研究对深入开展昆虫与寄主植物互作机理,开辟害虫防治新途径具有极为重要的理论和实践意义。更多还原

【Abstract】 The G Protein-coupled signaling system,ubiquitous in simple eukaryotes,plants,insects, and the other animals,is one of the most important cellular signaling pathways.Using Drosophila melanogaster as a model,many studies have been done on the structure and function of G-Proteins,confirming that they are involved in diverse signaling pathways such as visual,olfactory and hormonal signaling.In this paper,genes of G Protein subunits were cloned from Bombyx mori using bioinformatics,RT-PCR and RACE methods.GST fusion proteins and His fusion proteins of the G protein subunits were constructed and cloned into the expression vector,pET,and the interactions between clonedβandγsubunits were determined by GST pull-down exoperiments.The main results were as follows:1.G-proteinα-subunit gene cloning and expression analysis of B.moriTwo novel G protein alpha subunits termed BmGα73B and BmGα12 were isolated from Bombyx mori using bioinformatics,RT-PCR and RACE techniques.The full-length BmGα73B gene was 1509 bp in length with an 1158 bp ORF coding for 385 amino acid residues.The deduced amino acid sequence of the Bmα73B subunit was 47%identical to the D.melanogaster Gfαsubunit.The full-length BmGα12 gene was 1374 bp in length with an 1128 bp ORF coding for 375 amino acid residues.The deduced amino acid sequence of theα12 subunit was 69%and 56%identical to the D.melanogaster concertina gene and animalα12/α13 subunits,respectively.RT-PCR was conducted to investigate the BmGα73B expression pattern in different tissues through the metamorphosis and demonstrated that BmGα73B was widely expressed in B.mori tissues.The staged expression patterns indicated that BmGα73B was mainly expressed in larvae;it also existed in early pupae,but expression was lower or absent in later pupae and adults.The results indicated that BmGα73B may play crucial roles in the early development stage of the B.mori midgut,which could be a clue for the further investigation of G proteins in silkworm development.RNAi interference was used to detect the role of BmGα73B in midgut development,but no obvious defect was found.2.G-proteinβandγsubunits gene cloning and sequences analysisOne pair of primers was designed in accordance with the G proteinβsubunit gene sequence published in GenBank.The gene was amplified from B.mori cDNA by PCR,cloned into the pMD18-T vector,and then sequenced and analyzed.Two genes of G proteinγsubunits termed BmGγ1and BmGγ30A were cloned for the first time from B.mori using bioinformatics,RT-PCR and RACE techniques.Gγ1 and Gγ30A genes,coded for 71 and 72 amino acids,respectively,and the deduced amino acid sequence of the Gγ1 and Gγ30A subunit was 70%and 81%identical to D.melanogaster Gγ1 and Gγ30A subunits,respectively. Clonedγsubunits both had GGL domains(G Protein gamma subunit-like motifs) and a carboxyl-terminal CAVI motif,in which a conserved cysteine was modified by an isoprenoid group.Based on the amino acid alignment,it was hypothesized that Gγ1 and Gγ30A subunits cloned from B.mori are similar to Gγ1 and Gγ30A subtypes in D.melanogaster3.The prokaryotic expression of G protein subunits from B.mori(1) The prokaryotic expression of G proteinαsubunits.Two pairs of primers were designed in accordance with sequences of the proteins encoded by two G proteinαsubunits. Genes was amplified from the pMD18-T-Gαplasmid by PCR and then inserted into the prokaryotic expression vector pET-41b with a C-terminal 6Gst-tag.The recombinant fusion expression vectors pET-41b-Gα73B and pET-41b-Gα12 were transformed into BL21(DE3) cells via chemical transformation.Positive clones were screened by PCR.The target proteins were successfully expressed in Escherichia coli.The highest amount of Gst-Gα73B was produced at 37℃after induction with 0.1mmol/L IPTG for 3h and of Gst-Gα12 was at 37℃, 0.5 mmol/L IPTG induced for 4h.The solubility analysis showed that both recombinant proteins existed as inclusion bodies in E.coli cells.Western blot analysis indicated that the recombinant proteins were reactive to an anti-Gst-tag polyclonal antibody.(2) The prokaryotic expression of G proteinβsubunits.The prokaryotic expression vectors,pET-41b-Gβ1,pET-41b-Gβ2 and pET-22b-Gβ2,with a C-terminal 6Gst-tag and 6His-tag,were successfully constructed and induced with IPTG.Solubility analysis showed that all of the recombinant protein existed as an inclusion body in E.coli cells.By optimizing conditions for soluble protein expression,a small amount of soluble fusion protein was obtained with recombinant plasmid pET-22b-Gβ2 after induction with an IPTG concentration of 0.001mmol/L,for 6h at 20℃.(3) The prokaryotic expression of G proteinγsubunits.The prokaryotic expression plasmids,pET-41b-Gγ1 and pET-41b-Gγ30A,were successfully constructed and induced with IPTG.Optimization of expression conditions found that IPTG concentration has no affect on protein expression and the two fusion proteins reached the largest amounts after induction for 4h.Solubility analysis showed that both recombinant proteins were located in supernate and inclusion bodies.The fusion proteins were purified by affinity chromatography and identified by western blotting with anti-Gst-tag monoclonal antibody.4.The interaction betweenβandγsubunits of B.mori.The interaction betweenβandγsubunits was determined by the GST pull-down method and the results showed that the G proteinβsubunit can be combined with theγ30A subunit in B.mori.Further study is needed to confirm the function ofβandγ30A subunits.From the observations described above,it was presumed that there may be other types ofβandγsubunits in B.mori.5.A preliminary study on regulation of G-protein signaling in B.moriThree partial cDNA regulators of G-protein signaling(RGS) were cloned from B.mori using bioinformatics and RT-PCR methods and were termed Bmo-RGS7,Bmo-SNX14 and Bmo-Axin according to sequence analysis.The cloned Bmo-RGS7 gene had RGS,GGL,and DEP domains,whereas Bmo-SNX14 had only an RGS domain.In this paper,seven genes,including G-Protein subunits and RGSs were cloned and isolated for the first time from B.mori.The study of tissue distribution showed that G Proteinαsubunit is ubiquitous and may be involved in diverse signaling pathways.The result of GST pull-down screening indicated that the G proteinβsubunit could interact directly with theγ30A subunit.Thus far no publications are available concerning the studies presented here on G Proteins of B.mori.These achievements provide a substantial foundation for further studies of G Proteins and G-Protein signaling Pathways in B.mori.This research also has important theoretical and practical significance in probing deeply into insect-host plant,interactions,the mechanism of plant pest-resistance and the exploration of new control methods.更多还原