High-efficiency Thermal Asymmetric Interlaced PCR Research Articles

A new l-serine binding orphan SerBP affects indole synthesis in Pantoea ananatis.

Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind tol-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds tol-serine ABC transports. As a precursor of indole synthesis, the transmembrane transportedl-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, andfliR), 4 curli fimbriae genes (csgD, csgA, csgF, andcsgG) and 3 type 1 fimbriae genes (fimA, fimD, andfimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, andcsgG) and other functional genes (fimC, glxK, yehE, 07045, and11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis.

A novel amidase gene (bami) was cloned from Brevibacterium epidermidis ZJB-07021 by combination of degenerate PCR and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The deduced amino acid sequence showed low identity (≤55%) with other reported amidases. The bami gene was overexpressed in Escherichia coli, and the resultant inclusion bodies were refolded and purified to homogeneity with a recovery of 22.6%. Bami exhibited a broad substrate spectrum towards aliphatic, aromatic and heterocyclic amides, and showed the highest acyl transfer activity towards butyramide with specific activity of 1331.0±24.0U mg(-1). Kinetic analysis demonstrated that purified Bami exhibited high catalytic efficiency (414.9mM(-1) s(-1)) for acyl transfer of butyramide, with turnover number (K cat) of 3569.0s(-1). Key parameters including pH, substrate/co-substrate concentration, reaction temperature and catalyst loading were investigated and the Bami showed maximum acyl transfer activity at 50°C, pH 7.5. Enzymatic catalysis of 200mM butyramide with 15μgmL(-1) purified Bami was completed in 15min with a BHA yield of 88.1% under optimized conditions. The results demonstrated the great potential of Bami for the production of a variety of hydroxamic acids.

Cloning, overexpression and characterization of a xylanase gene from a novel Streptomyces rameus L2001 in Pichia pastoris

A novel GC-rich xylanase gene named xynA from Streptomyces rameus L2001 was cloned by high-efficiency thermal asymmetric interlaced PCR. The open reading frame of the cloned gene contained 672bp and encoded 223 amino acid residues with a calculated molecular mass of 24.5kDa. The recombinant xylanase (XynA) was scaled up in a 2.5L fermenter using BSM and BMMY medium; the highest enzyme activity, 13626U/mL, was obtained from BSM. This is the first report of high-cell-density production of a xylanase from S. rameus in Pichia pastoris. The xylanase showed a single band at about 25.0kDa on SDS-PAGE after being treated with endo-β-N-acetylglucosaminidase H. Studies of enzymatic properties showed that the optimal temperature and pH of XynA were 55°C and 4.5, respectively. XynA was very stable in a broad pH range (3.0–11.0), and the residual activity was >70% after incubation at 50°C for 30min. Thin-layer chromatography analysis showed that the recombinant xylanase was able to digest xylan, forming xylotriose and xylobiose as the main products; hence XynA from S. rameus L2001 could potentially be useful for preparation of xylooligosaccharides.

Mining and characterization of two amidase signature family amidases from Brevibacterium epidermidis ZJB-07021 by an efficient genome mining approach

Amidases have received increasing attention for their significant potential in the production of valuable carboxylic acids. In this study, two amidases belonging to amidase signature family (BeAmi2 and BeAmi4) were identified and mined from genomic DNA of Brevibacterium epidermidis ZJB-07021 by an efficient strategy combining comparative analysis of genomes and identification of unknown region by high-efficiency thermal asymmetric interlaced PCR (HiTAIL-PCR). The deduced amino acid sequences of BeAmi2 and BeAmi4 showed low identity (< 40%) with other reported amidases. The two amidases displayed optimum activity toward a wide spectrum of substrates at a mild alkaline pH and 45 °C. Both of them were remarkably inactivated by serine-directed inhibitor and sulfhydryl-reducing agent. Kinetic analysis revealed that nicotinamide was the preferable substrate for both amidases and the chlorine substitutions on the pyridine ring had a negative effect on activity. The bioprocesses for hydrolysis of 100 mM nicotinamide, isonicotinamide, 2-chloronicotinamide and 5-chloronicotinamide with purified BeAmi2 (6 U mL−1) were complete in 60 min with full conversion except 2-chloronicotinamide. These results indicated BeAmi2 was an effective catalyst for hydrolysis of several nicotinamide derivatives.

Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis.

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Mutation in the coding sequence of atp6 are associated with male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

ATPase subunit 6 (atp6) is an important mitochondrial functional gene involved in energy supply. In this study, mitochondrial DNA was isolated from a cytoplasmic male sterility line P3A and its maintainer line P3B. The full-length sequence of atp6 was obtained by high-efficiency thermal asymmetric interlaced PCR. Coding sequences (CDSs) were determined using cDNA. P3A and P3B sequences had an overall mean similarity of 73.6 % with other atp6 genes deposited in GenBank. Sequences in atp6 CDSs were different between P3A and P3B, being 1209 and 1239 bp and encoding 402 and 412 amino acids in P3A and P3B, respectively. A 33-bp deletion (including an 18-bp deletion and a separate 15-bp deletion) and a 3-bp insertion in the CDS of atp6 in P3A were characterized. To confirm that the 33-bp deletion and 3-bp insertion were specific to the cytoplasm of male sterile lines, 104 varieties were analyzed. The deletion and insertion were specific to male sterile cytoplasm. A specific molecular marker was developed to distinguish the male sterility cytoplasm from male fertile cytoplasm.

The neutral metallopeptidase NMP1 of Trichoderma guizhouense is required for mycotrophy and self-defence.

Trichoderma guizhouense NJAU 4742 (Harzianum clade) can suppress the causative agent of banana wild disease Fusarium oxysporum f. sp. cubense 4 (Foc4). To identify genes involved in this trait, we used T-DNA insertional mutagenesis and isolated one mutant that was unable to overgrow Foc4 and had reduced antifungal ability. Using the high-efficiency thermal asymmetric interlaced-PCR, the T-DNA was located in the terminator of a neutral metalloprotease gene (encoding a MEROPS family M35 protease), which was named nmp1. The antifungal activity of the mutant was recovered by retransformation with wild-type nmp1 gene. The purified NMP1 (overexpressed in Pichia pastoris) did not inhibit the growth and germination of other fungi in vitro. Its addition, however, partly recovered the antifungal activity of the mutant strain against some fungi. The expression of nmp1 is induced by the presence of fungi and by dead fungal biomass, but the time-course of transcript accumulation following the physical contact depends on mode of interaction: it increases in cases of long-lasting parasitism and decreases if the prey fungus is dead shortly after or even before the contact (predation). We thus conclude that NMP1 protein of T. guizhouense has major importance for mycotrophic interactions and defence against other fungi.

Production of dammarane-type sapogenins in rice by expressing the dammarenediol-II synthase gene from Panax ginseng C.A. Mey

Ginsenosides are the main active ingredients in Chinese medicinal ginseng; 2,3-oxidosqualene is a precursor metabolite to ginsenosides that is present in rice. Because rice lacks a key rate-limiting enzyme (dammarenediol-II synthase, DS), rice cannot synthesize dammarane-type ginsenosides. In this study, the ginseng (Panax ginseng CA Mey.) DS gene (GenBank: AB265170.1) was transformed into rice using agrobacterium, and 64 rice transgenic plants were produced. The Transfer-DNA (T-DNA) insertion sites in homozygous lines of the T2 generation were determined by using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) and differed in all tested lines. One to two copies of the T-DNA were present in each transformant, and real-time PCR and Western blotting showed that the transformed DS gene could be transcribed and highly expressed. High performance liquid chromatography (HPLC) analysis showed that the dammarane-type sapogenin 20(S)-protopanaxadiol (PPD) content was 0.35–0.59mg/g dw and the dammarane-type sapogenin 20(S)-protopanaxatriol (PPT) content was 0.23–0.43mg/g dw in the transgenic rice. LC/MS analysis confirmed production of PPD and PPT. These results indicate that a new “ginseng rice” germplasm containing dammarane-type sapogenins has been successfully developed by transforming the ginseng DS gene into rice.

Production of oleanane-type sapogenin in transgenic rice via expression of β-amyrin synthase gene from Panax japonicus C. A. Mey.

BackgroundPanax japonicus C. A. Mey. is a rare traditional Chinese herbal medicine that uses ginsenosides as its main active ingredient. Rice does not produce ginsenosides because it lacks a key rate-limiting enzyme (β-amyrin synthase, βAS); however, it produces a secondary metabolite, 2,3-oxidosqualene, which is a precursor for ginsenoside biosynthesis.ResultsIn the present study, the P. japonicus βAS gene was transformed into the rice cultivar ‘Taijing 9’ using an Agrobacterium-mediated approach, resulting in 68 rice transgenic plants of the T0 generation. Transfer-DNA (T-DNA) insertion sites in homozygous lines of the T2 generation were determined by using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) and were found to vary among the tested lines. Approximately 1–2 copies of the βAS gene were detected in transgenic rice plants. Real-time PCR and Western blotting analyses showed that the transformed βAS gene could be overexpressed and β-amyrin synthase could be expressed in rice. HPLC analysis showed that the concentration of oleanane-type sapogenin oleanolic acid in transgenic rice was 8.3–11.5 mg/100 g dw.ConclusionsThe current study is the first report on the transformation of P. japonicus βAS gene into rice. We have successfully produced a new rice germplasm, “ginseng rice”, which produces oleanane-type sapogenin.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

Cloning, expression, characterization and mutational analysis of the tfdA gene from Cupriavidus campinensis BJ71.

2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. Previous studies focused on the tfdA gene in Ralstonia eutropha JMP134 isolated in Australia. In this study, a new tfdA gene was cloned from Cupriavidus campinensis BJ71, an effective degrading bacteria from China, based on the iCOnsensus-DEgenerate Hybrid Oligonucleotide Primers (iCODEHOPs) protocol, combined with high-efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR). The open reading frame of 861bp encoded a putative 287 amino acid protein with a theoretical molecular mass of 32.32kDa. The gene was overexpressed in Escherichia coli BL21 (DE3) and the purified TfdA showed optimal activity at pH 6.75 and 30°C. This enzyme was more thermostable and it could use 3-hydrocinnamic acid as substrate, with a similar enzyme activity compared with 2,4-D. TfdA and its variants were created as maltose-binding protein (MBP) tagged fusion proteins to examine the roles of putative substrate-binding residues. The MBP-N110A, MBP-V198A and MBP-R207K proteins showed decreased k cat and increased Km, and MBP-R278A was inactive, suggesting these residues may affect 2,4-D binding or catalysis.

Identification of pathogenicity-related genes in the rice pathogen Ustilaginoidea virens through random insertional mutagenesis

Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. To gain more insight into the cellular and molecular mechanisms in the pathogenesis of Ustilaginoidea virens, the causal agent of rice false smut disease, a T-DNA insertion mutant library of U. virens was established using ATMT. We optimized a range of conditions to improve the transformation efficiency. Transformants were most effectively obtained when the optimal co-cultivation time is 72h, with 50μM AS in medium and 100μl A. tumefaciens for co-cultivation, leading to the production of 160–185 hygromycin B resistant transformants per 1×105 conidia. Southern blot analysis indicated that 58.14% of transformants had a single T-DNA copy. Among 5600 transformants tested for virulence, 37 mutants with reproducible pathogenic defects were obtained. The flanking sequences of three avirulent tranformants (B20, B1015 and B1465) and two pathogenicity-reduced transformants (B726 and B785) were amplified by high-efficiency thermal asymmetric interlaced PCR. Sequence analyses revealed that single T-DNA insertion in mutant B20 targeted the coding region of a gene encoding a protein highly similar to SUN family protein, and in mutant B726 targeted upstream of a gene with unknown function. The two T-DNA insertion sites in mutant B785 were found in the coding region of a gene encoding C6 transcription factor, but failed in amplified flanking sequence of another T-DNA. Chromosomal rearrangement occurred in the genome of mutant B1016 and B1465 with single T-DNA insertion. Among avirulent mutants, B20 showed altered colony growth and pigmentation. The T-DNA insert in B20 was detected in the coding region of a gene named UvSUN2. Morphophysiological characterization analysis suggested that UvSUN2 might be a virulence factor, and possibly required for proper fungal growth, cell wall construction, and stress responses in U. virens. This study optimize and validate the transformation procedure, maximizing the number of single copy transformants and developing an efficient procedure for rescuing adjacent host sequences along with the inserted T-DNA. This is the first report of identification several candidate virulence factors and validated one in U. virens. Together with identification of avirulent mutants and their associated genes, results suggested that ATMT can effectively be used to identify genes associated with pathogenicity in U. virens, and provided novel insights into molecular mechanisms underlying virulence of this pathogen.

Isolation and functional characterization of seed-specific FAD2-1 promoter from cotton (Gossypium hirsutum L)

Using the high-efficiency thermal asymmetric interlaced PCR technique, a 1,279 bp fragment of 5′-flanking region of the Δ12-desaturase enzyme gene FAD2-1 from Gossypium hirsutum (GhFAD2-1), was successfully isolated. Cis-acting elements search in the fragment revealed the presence of a numbers of tissue-specific motifs including E-box (CANNTG) and −300 element (TGHAAARK). Functional analysis in transgenic Arabidopsis plants showed the 1,279 bp fragment could drive β-glucuronidase (GUS) reporter gene to express exclusively in seeds. Moreover, the GUS activity was detected mainly in the mid- and late-developmental phases of the developing seeds of transgenic plants. Compared to the developing seeds of the plants having GUS with 35S promoter, seeds of the transgenic plants having GUS with GhFAD2-1 promoter, the later has lower GUS activity. The GhFAD2-1 promoter-driven GUS expression has obvious tissue and developmental stage specificity, which could be applicable to the modification of seed phenotypes of transgenic plants. The results also established the first step toward our understanding of the spatial and temporal regulation mechanisms of the GhFAD2-1, playing a major role in controlling conversion of oleic acid to linoleic acid in the cottonseed.

A simple and efficient method to isolate LTR sequences of plant retrotransposon.

Retrotransposons (RTNs) have important roles in the formation of plant genome size, structure, and evolution. Ubiquitous distributions, abundant copy numbers, high heterogeneities, and insertional polymorphisms of RTNs have made them as excellent sources for molecular markers development. However, the wide application of RTNs-based molecular markers is restricted by the scarcity of the LTR (long terminal repeat) sequences information. A new, simple, and efficient method to isolate LTR sequences of RTNs was presented based on the degenerate RNase H nested primers and PPT (polypurine tract) primer of RTNs in tree peony. This method combined the characteristics and advantages of high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), annealing control primer (ACP) system, and suppression PCR method. Nineteen LTR sequences were isolated using this new method in tree peony and the applicability of the LTR sequences based markers was validated by further SSAP analysis. The results showed that the new method is simple, of low-cost, and highly efficient, which is just conducted by three rounds of PCR and does not need any restriction enzymes and adapters, much less the hybridizations. This new method is rapid, economical, and cost- and time-saving, which could be easily used to isolate LTR sequences of RTNs.

Generation of AQP2-Cre transgenic mini-pigs specifically expressing Cre recombinase in kidney collecting duct cells

The important differences in physiological parameters and anatomical characteristics of the kidney between humans and mice make it difficult to replicate the precise progression of human renal cystic diseases in gene modification mouse models. In contrast to mice, pigs are a better animal model of human diseases, as they are more similar in terms of organ size, structure, and physiological parameters. Here, we report the generation and initial examination of an AQP2-Cre transgenic (Tg) Chinese miniature (mini)-pig line that expresses Cre recombinase exclusively in kidney collecting duct cells. An 8-kb fragment of the mini-pig aquaporin 2 (AQP2) 5'-flanking region was utilized to direct Cre expression in Tg mini-pigs. Two Tg mini-pigs were generated by pig somatic cell nuclear transfer and both carried the entire coding sequence of Cre recombinase. RT-PCR and western blotting analysis revealed that Cre recombinase was uniquely expressed in the kidney, while immunohistochemical studies located its expression in kidney collecting duct cells. Furthermore, six integration sites and 12-14 copies of the Cre gene were detected in various tissues by high-efficiency thermal asymmetric interlaced PCR and absolute quantitative real-time PCR, respectively. Combined with previous studies of Cre recombinase activity, we believe that this AQP2-Cre Tg mini-pig line will be a useful tool to generate kidney collecting duct cell-specific gene knockout mini-pig models, thereby allowing the investigation of gene functions in kidney development and the mechanisms of human renal cystic disease.

High-Efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR) for Determination of a Highly Degenerated Prophage WO Genome in a Wolbachia Strain Infecting a Fig Wasp Species

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.

Analysis of Integrated Structure of the Exogenous Gene Insertion and the Establishment of Event-specific Detection Method in Transgenic Insect Resistant Cotton

In this study, the complete sequence of exogenous DNA insertion was obtained from transgenic cotton line 06N-119 by using 3'-terminal high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) two times, 5'-terminal hiTAIL-PCR four times and long distance PCR (LD-PCR) once. The insertion of the exogenous gene is 11578 bp in length and consists of the Nptâ…i gene expression cassette and the Bt gene expression cassette in series. There is a 75 bp fragment lost at insertion site but no gene recombination occurred. The event-specific qualitative PCR method was established based on the 3'-terminal flanking sequence, which was proved to be highly specific and sensitive. The detection limit was as low as 0.05% every 100 ng of template, that is equivalent 11 target sequence copies. The results of this study would be importance on the detection of exogenous gene in transgenic cotton and biosafety assessment.

Mutagenesis of barley malting quality QTLs with Ds transposons

Various functional genomic tools are being used to identify and characterize genes in plants. The Activator/Dissociation (Ac/Ds) transposon-based approach offers great potential, especially in barley, due to its limited success of genetic transformation and its large genome size. The bias of the Ac/Ds system towards genic regions and its tendency toward localized transpositions can greatly enhance the discovery and tagging of genes linked to Ds. Barley is a key ingredient in malting and brewing industry; therefore, gene discovery in relation to malting has an industrial perspective. Malting quality in barley is a complex and quantitatively inherited trait. Two major quantitative trait loci (QTLs) affecting malting quality traits have been located on chromosome 4H. In this study, Ds was reactivated from parent transposants (TNP) lines, TNP-29 and TNP-79, where Ds was mapped in the vicinity of important malting QTLs. Reactivation of Ds was carried out both by conventional breeding and in vitro approaches. A threefold increase in reactivation frequency through the in vitro approach enabled the development of a new genomic resource for the dissection of malting QTL and gene discovery in barley. Identification of unique flanking sequences, using high-efficiency thermal asymmetric interlaced PCR and inverse PCR from these populations, has further emphasized the new location of Ds in the barley genome and provided new transposon mutants especially in β-GAL1, β-amylase-like gene and ABC transporter for functional genomic studies.

Establishment of an improved high-efficiency thermal asymmetric interlaced PCR for identification of genomic integration sites mediated by phiC31 integrase

Streptomyces phage phiC31 integrase is widely used to mediate the integration of exogenous genes into host genomes for gene therapy and genomic modification, as it autonomously performs efficient, unidirectional, site-specific integration into pseudo attP sites of the host genome. Although pseudo attP sites are rarely found within exons, it is necessary to map their precise locations to avoid the risk of insertion mutagenesis. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is a technique that has been developed to recover genomic sequences that flank insertion tags. We have found, however, that this technique is poorly efficient, as it amplifies many non-specific targets and frequently does not generate sufficient product for downstream analysis. Therefore, we have modified the hiTAIL-PCR procedure and re-designed the random primers. As a result, both the amount and specificity of the reaction product were enhanced for each integration site. Restriction analysis of known sequences within the integrated vector, which co-amplified with the flanking genomic sequences, validated 90% of these bands for sequencing. In contrast, only 30% of the bands produced by previous hiTAIL-PCR could be validated. Compared with the original hiTAIL-PCR, our improved hiTAIL-PCR procedure identified phiC31 integration sites more accurately and efficiently.