【作者】 熊爱生；

【导师】 章镇； 姚泉洪；

【作者基本信息】 南京农业大学 ， 果树学， 2007， 博士

【摘要】 标记基因是一种用来筛选和鉴定转化细胞、组织和转基因植株的DNA片段,包括起富集转化细胞作用的选择基因和易于检测表达产物的报告基因。抗生素类和抗除草剂类是当前转基因作物中采用的选择标记基因。目前常用的报告基因主要包括β-葡萄糖苷酸酶基因(gus)、β-半乳糖苷酶基因(LacZ)、荧光素酶基因(Luc)、绿色荧光蛋白基因(gfp)和冠瘿碱合成酶基因等。植物转基因中最广泛应用的报告基因是gus。转基因作物进入商业化生产阶段,其外源基因的逃逸是不可避免的。因此使用安全的标记基因和报告基因是一个很好的选择。体外定向分子进化是发现和改造生物活性分子的重要方法,提供了一种高效获得多样性的方法。DNA改组是重要的体外分子进化技术,结合高通量筛选在农业、环境污染治理、化学工业、基因治疗、疫苗、蛋白药物等方面有着广泛的应用。近年来,许多体外分子进化的新策略和新方法层出不穷。DNA改组技术的发展和成熟是在上世纪九十年代末期。特别是进入21世纪以来,体外分子进化技术在新技术和新理论的推动下,又得到了长足的发展。推理设计是基于基因和蛋白质信息进行基因和蛋白质序列的改造,作为基因和蛋白质体外快速改造有其重要的一面。化学法合成DNA是生物学基础研究和生物工程应用的一个非常重要的手段,化学法合成基因提供了一种改造基因,阐明基因功能,分析蛋白质-核酸相互作用的强有力手段,通过化学法合成和改造后能够实现高表达,也是消除多个点突变的好方法。近年来化学法合成DNA的发展使得大规模合成较长DNA序列成为可能。随着化学合成寡核苷酸链广泛的用于各类引物、基因全合成、DNA芯片等领域,实现了合成寡核苷酸链的小型化,自动化及高通量化。诞生了一些界面友好的设计基因软件。基因全合成自从出现后一直就有着广泛的应用,近年来应用的范围也越来越广泛。在重叠延伸PCR合成基因方法的基础上,建立了一种基于两步PCR的简单高效经济合成长DNA序列方法:PTDS。该方法主要涉及两步:首先通过12个长度60 nt且20 nt重叠的寡核苷酸,使用高保真的DNA聚合酶pfu合成4个长度为500 bp左右的的DNA片段;其次通过第一步PCR产物作为模板,使用最外侧的引物和高保真DNA聚合酶pyrobest进行第二步的PCR扩增,从而合成完整的目的抗虫基因vip3aI。另外进一步通过改良的PCR精确合成方法(PAS)合成了一编码普鲁兰酶的pulA基因和一编码耐高温β-葡萄糖苷酸酶的Tm-gus基因。大肠杆菌的β-葡萄糖苷酸酶基因,是一个使用广泛的报告基因。由于其具有检测灵敏,酶性质稳定,完善的酶学分析方法和可见的表型等特点,已经成为研究体外分子进化技术的一个好的模式。本文建立了一个高通量β-葡萄糖苷酸酶基因的体外分子进化体系,并通过该高通量体系获得了一个较野生型显著耐受高温的突变体:GUS-TR。该突变体的大肠杆菌转化子在膜上能够耐受100℃的高温达30分钟,而对照不能耐受70℃的高温。通过6-His表达和镍离子螯合层析得到纯化的突变体和野生型蛋白GUS-TR和GUS-WT。酶活性测定结果显示,经过70℃处理10分钟,表达的野生型的GUS-WT活性明显下降,而表达的耐高温的GUS-TR,经过80℃加热10分钟处理活性依然保留88%左右,经过80度加热30分钟,活性依然保留在50%以上。gus-tr基因序列测定分析共有15个核苷酸位点发生改变,导致了12个氨基酸位点发生突变,分别是:I149T,N181S,D436E,V446A,A451V,Q493R,T509A,M532T,N550S,G559S,N566S和M591I。以gus-tr基因为模板进一步使用定点突变结合耐高温性状分析,发现存在于定点突变个体GUS-TR中的六个突变位点(Q493R,T509A,M532T,N550S,G559S和N566S)对于耐高温性的获得是重要的,且存在关联效应。其中Q493R和T509A位点是两个新发现的耐高温β-葡萄糖苷酸酶关键位点。通过定点突变技术获得含有上述六个关键位点的突变个体GUS-TR3337。通过对突变个体GUS-TR3337和野生型大肠杆菌β-葡萄糖苷酸酶(GUS-WT)的蛋白质纯化后分析,野生型蛋白质在70℃处理10分钟后酶活性完全丧失。而突变体GUS-TR3337在80℃处理10分钟后酶活性保留在75%以上。进一步通过蛋白质模型分析研究了耐高温突变个体和野生型之间的关系,从分子机制上探讨了β-葡萄糖苷酸酶之结构和功能的关系。为了直观地证明获得的耐高温β-葡萄糖苷酸酶突变体在植物转基因中作为报告基因的可行性,将获得的突变基因gus-tr3337和来自大肠杆菌的野生型gus基因分别转入拟南芥植株中进行植物中耐高温性能研究。通过农杆菌蘸花法分别获得多个转基因株系,编号分别为YG8557(耐高温GUS基因gus-tr3337)和YG8555(野生型GUS基因gus-wt)。通过对转基因YG8557拟南芥植株和YG8555拟南芥植株进行了高温处理后X-Gluc染色的实验发现,野生型GUS基因(gus-wt)转基因拟南芥未经高温处理显色良好,呈现蓝色。而经过60℃处理5分钟时略微显蓝色,且非常微弱,随着处理温度的升高和处理时间的延长,转基因植株均不能显蓝色。而耐高温改组GUS基因(gus-tr3337)转基因拟南芥植株未经高温处理显色良好,呈现蓝色。经过60℃处理20分钟、30分钟;70℃处理10分钟、20分钟、30分钟;80℃处理10分钟、20分钟很好的显蓝色。甚至80℃处理30分钟仍能够较明显的显示蓝色,说明经过体外定向分子进化技术改组突变的耐高温β-葡萄糖苷酸酶(GUS-TR3337)在植物中依然能够保持耐高温性能。通过RACE技术从苹果中克隆了一个编码5-烯醇丙酮莽草酸-3-磷酸合成酶的基因(mdepsps),多重序列比对分析发现苹果来源的EPSPS与蒺藜苜蓿的EPSPS关系最近。将mdepsps基因构建入原核表达载体pYPX251中,发现含有该表达质粒的大肠杆菌菌株不能够在含有30 mM的草甘膦的M9培养基正常生长。通过体外定向分子进化技术对mdepsps进行了改造,获得了在M9培养基上添加50 mM的草甘膦仍能正常生长的阳性克隆15个。本文从果树基因工程研究中广泛使用的报告基因(β-葡萄糖苷酸酶)和有潜在应用前景的选择标记基因(5-烯醇丙酮莽草酸-3-磷酸合成酶)两方面入手,分别通过基因分离、设计、化学合成、体外定向分子进化等手段进行研究。通过化学合成和体外定向分子进化获得的耐高温的β-葡萄糖苷酸酶可以作为新型报告基因在果树基因工程中应用,尤其是一些具有较高内源GUS的果树物种。从苹果中克隆并通过改组提高功能的编码5-烯醇丙酮莽草酸-3-磷酸合成酶的基因具有:来源于苹果,安全可靠,功能良好等特点,且具有自主知识产权,有较强的理论和实际意义。上述研究丰富了果树基因工程中的报告基因种类,对果树遗传转化有一定的意义,同时也进一步丰富了果树基因工程中的基因来源,尤其是安全性的来源果树本身的基因,为果树基因工程应用奠定基础。更多还原

【Abstract】 Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional on the presence of external substrates.Green fluorescent protein(GFP),β-galactosidase(GAL),luciferase(LUC),β-glucuronidase(GUS),and oxalate oxidase (OxO) have been important in transgenic plant research or crop development,and have been assessed for efficiency,biosafety,scientific application and commercialization. Despite approximately fifty marker genes existing for plants,only a few marker genes are used for most plant research and crop development.Many of these genes have specific limitations or have not been sufficiently tested to merit their widespread use.Theβ-glucuronidase gene(gusA) isolated from E.coli is still to date the most widely used reporter gene in genetically modified plants.Its popularity is attributed to high stability in plant tissues and lack of toxicity even when expressed at high levels.The histochemical GUS staining protocol is a simple,rapid,highly-reliable and cost-effective method for analysis of transgenic plants.In addition,no specialized equipment is needed for histochemical assay of GUS activity.GUS in genetically modified plants and their products can also be regarded as safe for the environment and consumers.Directed evolution in vitro, especially DNA shuffling,is a powerful method used in academic study and industrial applications to create modified and functionally improved proteins.Directed evolution has brought significant advances in many fields,such as biocatalysts,plant improvement,and vaccine and pharmaceutical development.Rational Evolutionary Design utilizes structural and sequence alignment information to create new genes and proteins.Rational Evolutionary Design has recently emerged as an attractive approach for studying function of proteins.Chemical synthesis of DNA sequences provides a powerful tool to modifying genes for high level expression in heterologous systems and for characterization of gene structure,expression and function.Modified genes and consequently protein/enzymes can bridge and facilitate genomics and proteomics research.High-fidelity and cost-effective chemical synthesis of DNA has been central to recent progresses in biotechnology and basic biomedical research.Chemical synthesis of DNA sequences provides a powerful tool to modify genes and to study gene function,structure and expression.Here,we report a simple,high fidelity and cost-effective PCR-based two-step DNA synthesis(PTDS) method for synthesis of long segments of DNA.The method involves two steps:(ⅰ) Synthesis of individual fragments of the DNA of interest:Ten to twelve 60-mer oligonucleotides with 20-bp overlap in each are mixed and a PCR reaction is carried out with high fidelity DNA polymerase Pfu to produce DNA fragments that are about 500-bp in length.(ⅱ) Synthesis of the entire sequence of the DNA of interest:Five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest,with the two outermost oligonucleotides as primers.Compared to the previously published methods, the PTDS method is rapid(5 to 7 days) and suitable for synthesizing long segments of DNA (5 to 6-kb) with high G+C contents,repetitive sequences,or complex secondary structures. Thus,the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures.Using the newly developed PTDS method,we have successfully obtained several genes of interest with their size ranging from 1.0-5.4 kb.Here we also describe a simple and rapid PCR-based method for accurate assembly and synthesis(PAS) of long DNA sequences.The PAS protocol involves five steps:(1) Design of the DNA sequence to be synthesized and design of 60-bp overlapping oligonucleotides to cover the entire DNA sequence;(2) Purification of the oligonucleotides by polyacrylamide gel electrophoresis(PAGE);(3) First PCR,to synthesize DNA segments of 400- to 500-bp in length using 10 inner(template) and 2 outer(primer) oligonucleotides;(4) Second PCR,to assemble the products of the first PCR into the full length DNA sequence; (5) Cloning and verification of the synthetic DNA by sequencing and,if needed,error correction using an overlap extension PCR technique.This method,which takes about 1 week,is suitable for synthesizing diverse types of long DNA molecules.We have successfully synthesized DNA fragments from 0.5-kb to 12.0-kb,with high GC contents, repetitive sequences,or complex secondary structures.Using the PAS protocol,we chemical synthesized the pulA gene coding for pullulanase and Tm-gus gene coding for thermostableβ-glucuronidase of Thermotoga maritime.Escherichia coliβ-glucuronidase(gusA) gene,a versatile and efficient reporter gene, has been the model for studying in vitro directed evolution because its stability,easy analysis of the enzyme properties and conveniently visible phenotype.We developed a high efficiency,throughput system for in vitro directed evolution using gusA reporter gene as the model.The system consisted mainly of three aspects:a prokaryotic expression vector pYPX251,an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy.The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E.coli.Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter.A library which contained 100,000 individuals was screened by incubating ten filter papers with X-Gluc.The polymerase chain reaction products of the gusA gene,the fragments of 50-100 bp,with high mutation rates were purified using dialysis bag from 10%PAGE after electrophoresis.The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded.A GUS variant, named GUS-TR,was obtained through this system,which is significantly more resistant to high temperature than the wild type enzyme.The GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100℃for 30 minutes.To achieve a thermostableβ-glucuronidase and identify key mutation sites,we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene,gus-tr,after four rounds of DNA shuffling and screening. This variant had mutations in fifteen nucleic acid sites,resulting in changes in twelve amino acids(AAs).Using gus-tr as the template,we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein.We found that six sites(Q493R,T509A,M532T,N550S,G559S and N566S) present in GUS-TR3337,were the key AAs needed to confer its high thermostability.Of these Q493R and T509A were not reported previously as important residues for thermostability ofβ-glucuronidase. Furthermore,all of these six mutations must be present concurrently to confer the high thermostability.We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations.Compared with the wild-type protein which lost its activity completely after 10 min at 70℃,the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80℃for 10 min.The GUS-TR3337 exhibited high activity even heated at 100℃for 30 min on nitrocellulose filter.The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.In order to study the thermostableβ-glucuronidase in plant,over 20 samples of transgenic Arabidopsis thaliana were obtained by floral dip method.Compared with the transgenic plant YG8555,hosting the wild-type gus-wt gene,which lost its mostly activity after 5 min at 60℃,the transgenic plant YG8557,hosting gus-tr3337 gene,retained its activity when heated at 60℃for 20 min,30min;at 70℃for 10min,20min,30in;80℃for 10 min,20 min,even heated at 80℃for 30 min.Based the conserved sequence of abscisic acid responsive elements-binding factor,a cDNA sequence coding for an AREB transcription factor was cloned from Malus robusta. Alignment of predicted amino acid sequences of comparison of MrAREB transcription factor in different plants,the MrAREB was closest with AREB2 of Populus trichocarpa. The gene mdepsps coding for a 5-enolpyruvylshikimate- 3-phosphate synthase was isolated from Malus domestica.Alignment of predicted amino acid sequences of comparison of mdepsps transcription factor in different organisms,the mdepsps was closest with EPSPS of Medicago truncatula.The gene,mdepsps,originates from popularity fruit apple,has the more bio-safety than the epsps gene from bacillus.To achieve a high activity of MdEPSPS, we applied in vitro directed evolution strategy through DNA shuffling,screening and obtained some mutant clones,which resistance 50 mM glyphosate on the M9 culture medium.更多还原