Genome Walking Method Research Articles

PCR-based genome walking methods (review)

The review discusses a range of classical and modern methods used to determine the nucleotide sequence of unknown DNA regions flanking known ones. These methods are applied to decipher the regulatory regions of genes, identify integration sites of T-DNA or viruses, and so on, in cases where the use of whole-genome sequencing is not justified. To amplify a DNA segment, a binding site for a primer must be added to the end of the unknown sequence. This can be achieved either by ligating an adapter or by annealing a degenerate primer under gentle conditions, or by looping the DNA fragment so that the target region is surrounded by known sequences. The second important task is to eliminate the inevitable products of nonspecific binding of adapters or degenerate primers, which is often resolved through multiple rounds of nested PCR. Different methods vary significantly in terms of complexity, prevalence, and the availability of required reagents.

Primer extension refractory PCR: an efficient and reliable genome walking method.

Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3' parts of the secondary and tertiary primers. The 5' parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5' part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.

Transposon-aided capture (TRACA) of plasmids from the human gut.

The gut microbiota consists of a vast and diverse assemblage of microorganisms that play a pivotal role in maintaining host health. Nevertheless, a significant portion of the human gut microbiota remains uncultivated. Plasmids, a type of MGE, assume a critical function in the biological evolution and adaptation of bacteria to varying environments. To investigate the plasmids present within the gut microbiota community, we used the transposon-aided capture method (TRACA) to explore plasmids derived from the gut microbiota. In this study, fecal samples were collected from two healthy human volunteers and subsequently subjected to the TRACA method for plasmid isolation. Then, the complete sequence of the plasmids was obtained using the genome walking method, and sequence identity was also analyzed. A total of 15 plasmids were isolated. At last, 13 plasmids were successfully sequenced, of which 12 plasmids were highly identical to the plasmids in the National Center for Biotechnology Information (NCBI) database and were all small plasmids. Furthermore, a putative novel plasmid, named pMRPHD, was isolated, which had mobilized elements (oriT and oriV) and a potential type II restriction-modification (R-M) system encoded by DNA cytosine methyltransferase and type II restriction enzyme (Ban I), whose specific functions and applications warrant further exploration.

Fork PCR: a universal and efficient genome-walking tool.

The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Bridging PCR: An Efficient and Reliable Scheme Implemented for Genome-Walking.

The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5'-end of IWP's 5'-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice.

Fusion primer driven racket PCR: A novel tool for genome walking.

The limitations of the current genome-walking strategies include strong background and cumbersome experimental processes. Herein, we report a genome-walking method, fusion primer-driven racket PCR (FPR-PCR), for the reliable retrieval of unknown flanking DNA sequences. Four sequence-specific primers (SSP1, SSP2, SSP3, and SSP4) were sequentially selected from known DNA (5'→3') to perform FPR-PCR. SSP3 is the fragment that mediates intra-strand annealing (FISA). The FISA fragment is attached to the 5' end of SSP1, generating a fusion primer. FPR-PCR comprises two rounds of amplification reactions. The single-fusion primary FPR-PCR begins with the selective synthesis of the target first strand, then allows the primer to partially anneal to some place(s) on the unknown region of this strand, producing the target second strand. Afterward, a new first strand is synthesized using the second strand as the template. The 3' end of this new first strand undergoes intra-strand annealing to the FISA site, followed by the formation of a racket-like DNA by a loop-back extension. This racket-like DNA is exponentially amplified in the secondary FPR-PCR performed using SSP2 and SSP4. We validated this FPR-PCR method by identifying the unknown flanks of Lactobacillus brevis CD0817 glutamic acid decarboxylase genes and the rice hygromycin gene.

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA

Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5’ root homologous to the known sequence and a heterologous 3’ bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

Identification and functional characterization of the CVOMTs and EOMTs genes promoters from Ocimum basilicum L.

Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This shows these promoters can drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

Molecular Characterization of a Vector-Based Candidate Antigen Using the 3'-RACE and Genome Walking Methods and In Silico Analysis of the Correspondent Protein for Vaccine Design and Development.

The main objectives of developing vaccines to prevent malaria transmission are malaria control and preventing the reemergence of the disease in endemic regions. Molecular and in silico characterization of a candidate molecule is the first step in the vaccine design process. Determining the sequence and amplification of full-length cDNA copies from the mRNA transcripts is often challenging. The methods in this chapter provide a protocol for the rapid amplification of cDNA ends (RACE) and genome walking. Carboxypeptidase B2 enzyme from A. stephensi (CPBAs-2) was selected as the target molecule and the steps in its characterization and in silico analysis are explained in this chapter.

Transcriptome-Wide Identification and Expression Profiling of SPX Domain-Containing Members in Responses to Phosphorus Deprivation of Pinus massoniana

The SPX domain-encoding proteins are believed to play important roles in phosphorus (Pi) homeostasis and signal transduction in plants. However, the overall information and responses of SPXs to phosphorus deficiency in pines, remain undefined. In this study, we screened the transcriptome data of Pinus massoniana in response to phosphorus deprivation. Ten SPX domain-containing genes were identified. Based on the conserved domains, the P. massoniana SPX genes were divided into four different subfamilies: SPX, SPX-MFS, SPX-EXS, and SPX-RING. RNA-seq analysis revealed that PmSPX genes were differentially expressed in response to phosphorus deprivation. Furthermore, real-time quantitative PCR (RT-qPCR) showed that PmSPX1 and PmSPX4 showed different expression patterns in different tissues under phosphorus stress. The promoter sequence of 2284 bp upstream of PmSPX1 was obtained by the genome walking method. A cis-element analysis indicated that there were several phosphorus stress response-related elements (e.g., two P1BS elements, a PHO element, and a W-box) in the promoter of PmSPX1. In addition, the previously obtained PmSPX2 promoter sequence contained a W-box, and it was shown that PmWRKY75 could directly bind to the PmSPX2 promoter using yeast one-hybrid analysis in this study. These results presented here revealed the foundational functions of PmSPXs in maintaining plant phosphorus homeostasis.

Characterization of MYB35 regulated methyl jasmonate and wound responsive Geraniol 10-hydroxylase-1 gene from Bacopa monnieri.

BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter. Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having Vmax of 0.16µMsec-1μg-1 protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.

Molecular Cloning and Characterization of Five Glutathione S-Transferase Genes and Promoters from Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) and Their Response to Tannic Acid Stress.

Plants accumulate phenolic compounds such as tannic acid to resist insect herbivores. The survival of insects exposed to toxic secondary metabolites depends on the detoxification metabolism mediated by limited groups of glutathione S-transferases (GSTs). Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is an important foliar pest of poplar trees. GSTs play an important role in xenobiotic detoxification in M. troglodyta. Five GST genes were identified in M. troglodyta and were classified into five different cytosolic GST classes, delta, omega, sigma, theta, and zeta. Real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to determine the mRNA expression of the five cloned GSTs in the midguts and fat bodies of M. troglodyta. The mRNA expression of the five GSTs was significantly induced when M. troglodyta was exposed to tannic acid. To further understand the tannic acid regulatory cascade, the 5′-flanking promoter sequences of the five MtGSTs were isolated by genome walking methods, and the promoters were very active and induced by tannic acid. In summary, the induction of GST mRNA expression was due to the response of five MtGST promoters to tannic acid. Therefore, MtGST promoters play an important role in the regulation of GST transcription.

Identification, molecular characterization and expression of aminopeptidase N-1 (APN-1) from Anopheles stephensi in SF9 cell line as a candidate molecule for developing a vaccine that interrupt malaria transmission

BackgroundAccording to the World Health Organization reports, billions of people around the world are at risk for malaria disease and it is important to consider the preventive strategies for protecting the people that are living in high risk areas. One of the main reasons of disease survival is diversity of vectors and parasites in different malaria regions that have their specific features, behaviour and biology. Therefore, specific regional strategies are necessary for successful control of malaria. One of the tools that needs to be developed for elimination and prevention of reintroduction of malaria is a vaccine that interrupt malaria transmission (VIMTs). VIMT is a broad concept that should be adjusted to the biological characteristics of the disease in each region. One type of VIMT is a vector-based vaccine that affects the sexual stage of Plasmodium life cycle. According to recent studies, the aminopeptidase N-1 of Anopheles gambiae (AgAPN-1) is as a potent vector-based VIMT with considerable inhibition activity against the sexual stage of Plasmodium parasite.MethodsSystems for rapid amplification of cDNA ends (3ʹ-RACE) and genome walking methods were used for sequence determination of apn-1 gene from Anopheles stephensi and distinct bioinformatics software were used for structural analysis. AsAPN-1 was expressed in Spodoptera frugiperda (Sf9) insect cell line using the baculovirus expression system. Recombinant AsAPN-1 was purified under the hybrid condition and its biological activity was assayed.ResultsAsapn-1 gene and its coded protein from An. stephensi were characterized for the first time in this study. Subsequently, the structural features and immunological properties of its coded protein were evaluated by in silico approaches. Enzymatic activity of the recombinant AsAPN-1, which was expressed in Sf9 insect cell line, was equal to 6 unit/μl.ConclusionsResults of this study revealed that AsAPN-1 is very similar to its counterpart in An. gambiae. In silico evaluation and fundamental data which are necessary for its evaluation as a VIMT-based vaccine in the next steps were acquired in this study and those could be useful for research groups that study on malaria vaccine for countries that An. stephensi is the main malaria vector there.

Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

A Simple Genome Walking Strategy to Isolate Unknown Genomic Regions Using Long Primer and RAPD Primer.

BackgroundGenome walking is a DNA-cloning methodology that is used to isolate unknown genomic regions adjacent to known sequences. However, the existing genome-walking methods have their own limitations.ObjectivesOur aim was to provide a simple and efficient genome-walking technology.Material and MethodsIn this paper, we developed a novel PCR strategy (termed SLRA PCR) that uses a single long primer (SLP), a set of gene specific primers (GSP), and a random amplified polymorphic DNA (RAPD) primer for genome walking. SLRA PCR consists of two processes: the first amplification using SLP, and three successive rounds of nested PCR amplified by GSP and RAPD primer. The novelty of the approach lies in the use of long primers (SLP and GSP) and same annealing and extension temperature 68℃ in combination. This method offers higher amplification efficiency, superior versatility, and greater simplicity compared with conventional randomly primed PCR methods for genome walking.ResultsThe promoter regions and the first introns of the insulin-like androgenic gland hormone (IAG) gene and the hemocyanin gene of Macrobrachium nipponense were cloned using SLRA PCR, respectively.ConclusionsThis genome walking strategy can be applied to a wide range of genomes.

Construction of a Genomic Bacterial Artificial Chromosome (BAC) Library for the Prawn Macrobrachium rosenbergii and Initial Analysis of ZW Chromosome-Derived BAC Inserts.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4kb, corresponding to ∼ 4× coverage of the estimated 5.38Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

C-Jun regulates the promoter of small subunit hemocyanin gene of Litopenaeus vannamei

Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5′-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression.

Cloning and functional characterization of the Na+/H+ antiporter (NHX1) gene promoter from an extreme halophyte Salicornia brachiata

Salinity is one of the major abiotic stresses which affect plant growth and productivity by imposing dual stress, ionic and osmotic stress, on plants. Halophytes which are adapted to complete their life cycle in saline soil keep the transcript expression of stress-responsive genes constitutively higher in the optimum growth environments, which can be further increased by several folds under stress conditions. The transcript expression of SbNHX1 gene, cloned from a leafless succulent halophyte Salicornia brachiata, was up-regulated under salinity stress, but its transcriptional regulation has not been studied so far. In the present study, a 1727 bp putative promoter (upstream to translation start site) of the SbNHX1 gene was cloned using a genome walking method. The bioinformatics analysis identified important stress-responsive cis-regulatory motifs, GT1, MBS, LTR and ARE, in addition to two leaf-specific enhancer motifs. The GUS expression analysis of stable transgenic tobacco plants, transformed with a transcriptional fusion of GUS with the full SbNHX1 promoter (NP1) or any of its five deletion fragments (NP2 to NP6), showed that the deletion of two enhancer motifs resulted in the sudden decrease in GUS expression in leaves but not in the stem or root tissues. In contrast, under salinity stress, the higher induction of GUS expression observed in NP1 and NP2 was correlated by the presence of salt-inducible GT1- and MBS-motifs which is distributed only in NP1 and NP2 deletion promoter fragments. Finally, we concluded that the SbNHX1 promoter has a 624 bp (−1727 to −1103 bp) regulatory region which contains the two leaf-specific enhancer motifs and salinity stress-inducible GT-1 and MBS motifs. We suggest the SbNHX1 gene promoter and fragments as a candidate alternative promoter/s for crop engineering for better stress tolerance, which can be amended according to the desired level of expression needed.

Molecular cloning, bacterial expression and promoter analysis of pathogenesis-related protein 10 gene from Panax notoginseng

Based on transcriptome analysis and RT-PCR techniques, a PR10 gene was isolated from P. notoginseng root and named PnPR10. Bioinformatics and phylogenetic tree analyses revealed that the open reading frame (ORF) of PnPR10 was 465 bp in length, encoding 154 amino acids and containing one typical conserved domain of the pathogenesis-related protein Bet v I family, with high similarity to the gene from P. ginseng. Using the genome walking method, a 458 bp promoter upstream of the PnPR10 transcription start site was isolated from P. notoginseng root genomic DNA by hiTAIL-PCR. The PnPR10 promoter contained multiple cis-acting regulatory elements. The CGTCA-motif and TGACG-motif, which have been shown to play important roles in response to MeJA, were identified. The expression of PnPR10 can be induced by typical root rot disease pathogens (F. solani and C. destructans). The purified recombinant PnPR10 protein could clearly inhibit the growth of two pathogens. The results demonstrate that the PnPR10 gene is involved in the defense response against root rot disease pathogens. This experiment lays a theoretical foundation for revealing the mechanism of resistance in P.notoginseng.