Southern Blot Analysis Research Articles

Molecular Characterization and Genomic Function of Grapevine Geminivirus A.

A new grapevine geminivirus A (GGVA) isolate (named as GGVA-17YM1) and its associated defective genome (GGVA-D) were identified from a grapevine sample collected in Yuanmou, Yunnan Province, using sRNA high throughput sequencing and traditional Sanger sequencing. To explore the pathogenicity of GGVA and GGVA-D, infectious clones of GGVA-17YM1 and GGVA-D-17YM1 were constructed. Infection assays indicated that Nicotiana benthamiana plants inoculated with GGVA alone or a combination of GGVA and GGVA-D exhibited upward curled apical leaves and dwarfism. Southern blotting and quantitative real-time polymerase chain reaction analysis revealed that GGVA-D increased the accumulation level of GGVA DNA. Transient expression using a PVX-derived recombinant vector indicated that C2 and C4 encoded by GGVA are involved in symptom induction in N. benthamiana. Furthermore, the V2 protein inhibited local RNA silencing in co-infiltration assays in GFP transgenic N. benthamiana plants. Subsequently, full-length genome sequencing resulted in the identification of 11 different isolates of GGVA and 9 associated defective DNA molecules. Phylogenetic analysis based on whole genome sequences showed that all GGVA isolates, including our sequences, clustered into two distinct branches with no geographical grouping. Analyses of molecular variation indicated single nucleotide polymorphisms (SNPs) with more transitions (55.97%) than transversions (44.03%). Furthermore, the main variants for ORF C1, C3, or V1 were synonymous mutations, and non-synonymous mutations for ORF C2, C4, and V2. Genetic selection analysis indicated that negative selection acted on four ORFs (V1, C1, C2, and C3), while V2 and C4 were under positive selection. Our results contribute to the characterization of the genetic diversity of GGVA and provide insights into its pathogenicity.

Agrobacterium-mediated transformation of Persian walnut using BADH gene for salt and drought tolerance

ABSTRACT Transgenic walnuts (Juglans regia L.) were produced in the presence of the Agrobacterium strain LBA4404 and the plasmid pBI121. This plasmid contains a BADH gene driven by a CaMV 35S promoter. It further includes an NPTII gene as a selectable marker which, in turn, is driven by a NOS promoter. Somatic embryos of the walnut cultivar ‘Chandler’ were inoculated using an Agrobacterium suspension. Secondary embryos that developed on a selection medium were driven towards germination so as to develop micro-shoot cultures. The regeneration rate was 5.5%. PCRand southern blot analysis were used for confirming that the BADH transgene is successfully integrated into the plant genome. Transgenic and wild-type plants raised from somatic embryos were exposed to four osmotic stress levels (i.e. 0%, 2%, 4%, and 8% PEG) and four salinity levels (i.e. 0, 50, 100, and 200 mM NaCl). After 21 days, the transgenic plants grew almost vigorously in saline and drought-stress conditions, whereas the wild-type ones showed retarded levels of growth and did not survive after the cradle stage. These results show that the BADH gene can be expressed effectively in transgenic plantlets of walnut and can be used for breeding walnuts against drought and salinity.

Monitoring Gene Conversion in Budding Yeast by Southern Blot Analysis.

By using an inducible site-specific double-strand break (DSB) in budding yeast, it is possible to monitor-in real time-the repair of the break by homologous recombination. A method is described using an ectopic homologous donor sequence to repair an HO endonuclease-induced DSB. These gene conversion events can occur with or without crossing-over, the products of which are distinguished as different-sized restriction endonuclease fragments. The method of Southern blotting is described in detail.

Enhanced detoxification of exogenous toluene gas in transgenic Ardisia pusilla expressing AtNDPK2 gene

The Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene is known to regulate the cellular redox state, and to enhance tolerance to multiple stressors in plants. In this study, we transferred AtNDPK2 under the stress-inducible promoter SWPA2 into Ardisia pusilla to enhance the plants’ ability to detoxify toluene gas. Thirty transgenic A. pusilla lines were confirmed by PCR analysis with AtNDPK2 and NPTII gene-specific primers. In addition, four transgenic A. pusilla lines were further confirmed by Southern blot analysis to verify the gene copy number. Three transgenic lines showed a single-copy transgene insertion, and one transgenic line had two transgene insertions. To test the gene expression of AtNDPK2 in the transgenic A. pusilla lines exposed to and not exposed to toluene treatment, qRT-PCR analysis was performed. The gene expression of AtNDPK2 in transgenic A. pusilla plants exposed to toluene treatment was significantly higher than that of transgenic plants not exposed to toluene treatment. Finally, we measured toluene removal efficiency of the transgenic and non-transgenic A. pusilla lines exposed to toluene-contaminated air. There was a statistically significant difference between the transgenic and non-transgenic A. pusilla lines at all time points (p < 0.001). The highest toluene removal efficiency (797.33 ± 59.41 µg m−3 cm−2 leaf area) was recorded in the transgenic A. pusilla line NDPK2-12-4 after 3 h of exposure to toluene, while the non-transgenic line showed little toluene removal efficiency (206.2 ± 31.19 µg m−3 cm−2 leaf area). These results suggest that the capacity for detoxifying toluene gas is related to the AtNDPK2 gene in A. pusilla. Therefore, this study provides useful results to reduce toluene pollution in indoor air.

Invasive Haemophilus influenzae type b (Hib) disease in children in Italy, after 20 years of routine use of conjugate Hib vaccines

Haemophilus influenzae serotype b (Hib) was the leading cause of bacterial meningitis in children before the implementation of infant immunization with conjugate Hib vaccines. Despite the effectiveness of the vaccine, invasive Hib disease cases (i.e. isolation of Hib from a normally sterile site) are still reported in children.All invasive Hib disease cases in children ≤ 15 years reported through the National Surveillance System of Invasive Bacterial Disease, during 2012–2018 in Italy, were analyzed. Hib PCR-confirmed isolates were subjected to MLST and PFGE analysis. The number of copies of the capb locus, a virulence factor potentially contributing to true vaccine failures (TVFs), was determined by Southern blot analysis. Vaccine effectiveness (VE) was determined using a multiple Poisson regression model.31 cases of invasive Hib disease in children were reported. Fourteen children were vaccinated (TVFs), 14 were unvaccinated and 2 partially vaccinated (vaccination status was unknown for 1 case). The median age of children was 12 months (range 3 months-15 years). A decrease in vaccination coverage was observed in 2014–2016 (source Ministry of Health), and a rise in incidence was documented from 2016 until 2018, especially in children < 5 years. Vaccine effectiveness was estimated to be 83% (95% CI:45–95).24 isolates were available. The predominant ST was ST6 (70.8%). Cluster analysis of ST6 isolates by PFGE identified five variants. Six isolates (25%) contained multiple copies of the capb locus distributed among TVFs (30%) and unvaccinated children (16.7%).Our data show that both failures to vaccinate and TVFs are associated with invasive Hib disease in children in Italy, during the vaccination era. Most cases in children ≤ 2 years were vaccine-preventable, since they occurred in unvaccinated subjects (13/21 cases, 62%). No host predisposing factors for TVF were recognized. TVFs were not significantly associated with either specific genotypes or amplification status of the capb locus.

Genomic features of an Escherichia coli ST156 strain harboring chromosome-located mcr-1 and plasmid-mediated blaNDM-5.

The emergence of Escherichia coli strains coharboring the blaNDM and mcr genes has become a new challenge in clinical therapy because of their resistance to most antibiotics. This study reports the emergence of an E. coli strain GZEC065 isolated from blood sample of a patient in China with both chromosome-located mcr-1 and plasmid-mediated blaNDM-5 genes. Antimicrobial susceptibility testing showed that GZEC065 was resistant to most tested antimicrobials, including imipenem and polymyxin B. S1-nuclease-pulsed-field gel electrophoresis, Southern blotting analysis and conjugation assay was performed to determine the location and conjugative ability of mcr-1 and blaNDM-5 genes. Whole genome sequencing and analysis showed that GZEC065 belonged to sequence type ST156 and contained three different plasmids (46kb, 88kb and 142kb) with multiple resistance genes, such as mcr-1, blaNDM-5 and blaTEM-1. The blaNDM-5 gene was carried by the 46kb conjugative IncX3 plasmid, which has been reported frequently in China. The mcr-1 gene was located on the chromosome mediated by the Tn6330 mobile element and showed genetic complexity among different strains. The emergence of E. coli strains coharboring both chromosome-located mcr-1 and plasmid-mediated blaNDM-5 genes highlights the urgent need for alternative antibiotic treatment and continuous active surveillance.

Molecular Identification and Dual Functions of Two Different CXC Chemokines in Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare.

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1–10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.

Physical and biochemical differences in Agrobacterium rhizogenes-mediated transgenic hairy root lines of Echinacea purpurea

Echinacea (Echinacea purpurea L.), also known as purple coneflower, is one of the important medicinal plants commonly used for respiratory infections. The aim of this study was to compare different transgenic hairy root lines for growth and secondary metabolite biosynthesis and to select a superior hairy root line for the production of caffeic acid derivatives (CADs). Agrobacterium rhizogenes-mediated root lines showing hairy root phenotypic characteristics were analyzed by the Southern blot analysis and the copy number of the rolC gene determined. Significant differences were found among seven hairy root lines evaluated for the copy number of rolC gene, root growth, total phenolic, and CAD contents. It was concluded that these differences may be due to the position and copy number of rolC gene in the genome. Across the parameters compared, it was found that line 7 was superior to the other transgenic lines. The results show that the selection of superior transgenic lines is an important factor for the efficient production of secondary metabolites in hairy root cultures of Echinacea.

Establishment of a chloroplast transformation system in Tisochrysis lutea

Tisochrysis lutea is a haptophyte microalga commonly used as a commercial feed for juvenile fish and shellfish larvae. Genetic modification is of considerable importance for developing the potential economic value of T. lutea. However, the genetic transformation system of T. lutea has not yet been established, which limits functional genomic studies and strain improvement of this important microalgal species. In the current study, a chloroplast transformation vector harboring the phosphinothricin resistance gene (bar) as a selectable marker was established, and two short peptide-encoding genes (ant1 and ant2) driven by the endogenous psbA and rbcL promoters were cloned in this vector. The recombinant plasmid was transformed using a biolistic method into the trnI/trnA locus of the chloroplast genome via homologous recombination. After continuous selection on phosphinothricin, the integration of foreign genes and the expression of specific products in the transformants were detected using polymerase chain reaction (PCR), Southern blotting, and western blot analysis. This is the first report of establishment of a stable transformation system in T. lutea, which is a prerequisite for functional genomics and applied research on this species.

Poly (ADP-ribose) polymerase inhibitor 3-methoxybenzamide enhances in vitro plant growth, microtuberization, and transformation efficiency of blue potato (Solanum tuberosum L. subsp. andigenum)

Poly(ADP-ribose) polymerases (PARP) are involved in the regulation of plant growth and development, especially under stress conditions. These enzymes post-translationally modify target proteins by adding ADP-ribose polymers. This study presents the effect of PARP chemical inhibitor 3-methoxybenzamide (3 MB) on in vitro plant growth, microtuber formation, and Agrobacterium-mediated transformation of blue potato (Solanum tuberosum L. subsp. andigenum). The addition of 3 MB (0.2 to 0.6 mM) significantly increased the growth and microtuber formation of in vitro propagated plants. The highest gain in plant fresh weight, root mass, and microtuberization was observed in clonal propagation medium with 0.4 mM 3 MB. Transformation frequency of blue potato internodes increased to 43%, 2.7-fold over the non-treated control (16%), when 0.2 mM 3 MB was included in the culture medium during the whole process. The addition of 3 MB during Agrobacterium transformation did not affect transgene copy number in regenerated plants. Southern blot analyses and histochemical staining for s-glucuronidase activity confirmed the presence and expression, respectively, of the uidA transgene in plant tissues. From these results, it can be concluded that the inhibition of PARP activity can increase biomass production and transformation rates for genetic improvement of blue potato.

Enhanced tolerance to Phytophthora root and stem rot by over-expression of the plant antimicrobial peptide CaAMP1 gene in soybean

BackgroundAntimicrobial peptides play important roles in both plant and animal defense systems. Moreover, over-expression of CaAMP1 (Capsicum annuum antimicrobial protein 1), an antimicrobial protein gene isolated from C. annuum leaves infected with Xanthomonas campestris pv. vesicatoria, confers broad-spectrum resistance to hemibiotrophic bacterial and necrotrophic fungal pathogens in Arabidopsis. Phytophthora root and stem rot (PRR), caused by the fungus Phytophthora sojae, is one of the most devastating diseases affecting soybean (Glycine max) production worldwide.ResultsIn this study, CaAMP1 was transformed into soybean by Agrobacterium-mediated genetic transformation. Integration of the foreign gene in the genome of transgenic soybean plants and its expression at the translation level were verified by Southern and western blot analyses, respectively. CaAMP1 over-expression (CaAMP1-OX) lines inoculated with P. sojae race 1 exhibited enhanced and stable PRR tolerance through T2–T4 generations compared with the wild-type Williams 82 plants. Gene expression analyses in the transgenic plants revealed that the expression of salicylic acid-dependent, jasmonic acid-dependent, and plant disease resistance genes (R-genes) were significantly up-regulated after P. sojae inoculation.ConclusionsThese results indicate that CaAMP1 over-expression can significantly enhance PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways. This provides an alternative approach for developing soybean varieties with improved tolerance against soil-borne pathogenic PRR.

Extraction of high-quality genomic DNA and identification of different DNA barcoding markers for chickpea (Cicer arietinum L.)

The genetic studies of individual plants, especially self-pollinated species like chickpea need to be evaluated at the DNA level with the help of molecular markers for identifying genetic variations among the plants. High-quality DNA extraction is a prerequisite for genetic studies. Extraction of intact genomic DNA with high – molecular mass is essential for the study of many molecular biology applications like Polymerase Chain Reaction, endonuclease restriction digestion, southern blot analysis, and also for the construction of a genomic library. Several plant DNA extraction methods are available, even though the DNA isolation methods that give good yield employing both quantity and quality is quite difficult especially for self-pollinated crops like a chickpea. This work was focused on developing a standard protocol for the extraction of genomic DNA and identifying different barcoding markers. The result revealed that the CTAB extraction method with slight modification in protocol had been optimized for DNA isolation. The purified DNA, which was isolated through the CTAB method, had excellent spectral qualities and is efficiently digested by a restriction endonuclease, and is found to be more suitable for long-fragment PCR amplification. DNA barcoding is considered as a promising tool because it provides a practical and standard identification of plants. The isolated DNA sample was processed with a classical DNA barcoding approach by amplifying and sequencing with a universal primer. According to the result, among the different barcoding markers studied, the RbcL and Mat K were found to given the best result for molecular species identification in chickpea.

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination.

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.

Embryogenic callus induction and Agrobacterium-mediated genetic transformation of 'Shine Muscat' grape.

We established a method for embryogenic callus induction and highly efficient Agrobacterium-mediated genetic transformation of a table grape cultivar 'Shine Muscat' (Vitis labruscana). Embryogenic calli were induced using flower bud filaments from a dormant cane. Agrobacterium strain LBA4404 harboring the binary plasmid pBin19-sgfp, which contains the sgfp and nptII genes, was used to infect embryogenic calli. Infected calli were selected on 1/2 MS medium containing 5% maltose and 2% agar supplemented with 15 mg l-1 kanamycin. Efficiency of transformation of regenerated plants reached nearly 100% as determined by PCR and Southern blot analyses. The developed method will open a new avenue for genome editing of 'Shine Muscat' and contribute to the advancement of grape breeding.

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3′-junction sequence was isolated, whereas the 5′-junction remained unclear. In contrast, for T-DNA2, the 5′-junction sequence was isolated, whereas the sequence isolated from the 3′-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

Cotton leaf curl Kokhran virus in association with Chili leaf curl betasatellite infecting mungbean (Vigna radiata.) and black gram (Vigna mungo.) in Pakistan

In 2016, begomovirus-like symptoms were observed in mungbean (Vigna radiata) and black gram (Vigna mungo) in Pakistan. Symptomatic and asymptomatic leaf samples were collected and total genomic DNA was extracted. Full length begomovirus and betasatellite genomes were PCR amplified and cloned in plasmid pTZ/57RT cloning vector. The cloned molecules were subjected to Sanger sequencing. Based on Sequence Demarcation Tool, the begomovirus clone SR-3 identified in mungbean showed 94.50% highest identity with cotton leaf curl Kokhran virus-Kokhran (CLCuKoV-Ko), while the clone SR-5 isolated from black gram showed 99.7% highest identity with CLCuKoV-Ko. In phylogenetic tree analysis the identified isolates made a lower branch tree with CLCuKoV-Ko. The betasatellite clone SR-79 isolated from black gram showed 97% similarity with chili leaf curl betasatellite (ChLCB) and the betasatellite clone SR-93 isolated from mungbean showed 97.7% similarity with ChLCB. Southern blot analysis also confirmed the presence of CLCuKoV-Ko. This is the first report of CLCuKoV-Ko associated with ChLCB infecting both mungbean and black gram in Pakistan. This presents the new combination of disease complex and increase in host range of this virus species, which shows a real threat to other crops especially legume crops in Pakistan.

Overexpression of cphA gene from Nostoc flagelliforme improves the drought tolerance of Arabidopsis thaliana

Nostoc flagelliforme is a terrestrial cyanobacterium distributed in arid or semiarid steppes in western and northwestern China. Cyanophycin is synthesized by cyanophycin synthetase (CphA), which serves as a storage polymer for nitrogen, carbon, and energy in N. flagelliforme. In the present study, cphA (GenBank Accession No.: KX469292), an open reading frame (ORF) of 2706 bp encoding a protein of 901 amino acid residues, was cloned. We found that cyanophycin synthesis was promoted by the expression of cphA in E. coli. The function of the cphA gene in Arabidopsis thaliana under drought stress was investigated, and stable integration and expression of transgenes in transgenic lines were confirmed by PCR, Western blot, Southern blot, and real-time PCR analyses. The transcript abundance of cphA was markedly increased in transgenic A. thaliana compared with that in the untransformed plants. A subcellular localization assay confirmed that cphA protein was targeted to the cytoplasm. The survival rate was also significantly higher in cphA overexpression lines than that in untransformed plants under drought stress, which verified by drought tolerance assay of the transgenic plants. These findings highlight the potential importance of cphA in the drought tolerance of N. flagelliforme.

Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control.

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected Mr of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 108 and 8.27 × 107 cells produce 229 and 114 μM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.