Cloning, Chemical Synthesis And Directed Evolution Of Marker Genes And Application In Fruit Genetic Engineering

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.