Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor

Ko, Suk-Min; Sun, Hyeon-Jin; Oh, Myung Jin; Song, In-Ja; Kim, Minjae; Sin, Hyun-Sook; Goh, Chang‐Hyo; Kim, Yong-Woo; Lim, Pyung-Ok; Lee, Hyo-Yeon; Kim, Suk Weon

doi:10.1007/s13580-011-0007-x

Cited by 30 publications

(18 citation statements)

References 15 publications

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“…In comparison to previous Agrobacterium ‐mediated transformation protocols in L. minor (Cantó‐Pastor et al ., ; Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , , ), the increased efficiency of our protocol is likely resulted from adopting sonication coupled with vacuum infiltration infection method. In several previous studies, such as banana (Subramanyam et al ., ), chickpea (Indurker et al ., ) and cowpea (Bakshi et al ., ), it has been shown that the combination of vacuum infiltration and sonication can greatly improve the efficiency of Agrobacterium infection.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, duckweeds are capable of rapidly accumulating heavy metals, nutrients and pollutants, which can be used for bio‐remediation of wastewater (Cheng and Stomp, ; Khellaf and Zerdaoui, ; Megateli et al ., ; Stout et al ., ; Xu et al ., ). The extremely fast population doubling rate (3–5 days per asexual doubling under ideal conditions) also makes duckweeds a good candidate for the production of recombinant proteins including antigens, monoclonal antibodies and exogenous enzymes (Cox et al ., ; Firsov et al ., , ; Khvatkov et al ., ; Ko et al ., ; Naik et al ., ; Stomp, ; Sun et al ., ), as well as dietary supplements for humans and animals because of its high nutritional value (Appenroth et al ., ; Beukelaar et al ., ; Bhanthumnavin and Mcgarry, ). Despite the potential for both basic research and industrial applications that established duckweeds as one of the most promising alternative future crops (Appenroth et al ., ; Lam et al ., ), current research and development in duckweeds are constrained by the lack of efficient genetic manipulation tools that allow to knockout or knock‐in a gene of interest in the duckweed genome within a short time.…”

Section: Introductionmentioning

confidence: 99%

“…The development of genetic manipulation tools requires both an efficient transformation protocol and an effective genome editing system. Among the 37 duckweed species that belong to five genera: Spirodela , Landoltia , Lemna , Wolffiella and Wolffia (Landolt, ; Sree et al ., ), the Agrobacterium‐ mediated transformation system has been developed in at least seven of them, including Lemna gibba (Cantó‐Pastor et al ., ; Yamamoto et al ., ), Lemna minor (Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , ), Lemna turionifera (Yang et al ., ), Landoltia punctata (formerly Spirodela oligorrhiza ) (Rival et al ., ; Vunsh et al .,), Spirodela polyrhiza (Yang et al ., ), Wolffia arrhiza (Khvatkov et al ., , ) and Wolffia globosa (Heenatigala et al ., ). However, the efficiency of the transformation varied dramatically among species (from 0.14% to 82.5%).…”

Section: Introductionmentioning

confidence: 99%

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Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

Liu

Wang

et al. 2019

Plant Biotechnology Journal

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Summary The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

Liu

Wang

et al. 2019

Plant Biotechnology Journal

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“…Several duckweed species, e.g. Lemna minor, have been engineered to produce antigens, monoclonal antibodies and exogenous enzymes (Sun et al 2007;Ko et al 2011;Naik et al 2012). Additionally, duckweed can be used as a dietary supplement for humans and animals because of its high nutritional value (Bhanthumnavin & Mcgarry 1971).…”

Section: Introductionmentioning

confidence: 99%

Survey of duckweed diversity in Lake Chao and total fatty acid, triacylglycerol, profiles of representative strains

Tang

et al. 2015

Plant Biol J

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Lemnaceae (duckweeds) are widely distributed aquatic flowering plants. Their high growth rate, starch content and suitability for bioremediation make them potential feedstock for biofuels. However, few natural duckweed resources have been investigated in China, and there is no information about total fatty acid (TFA) and triacylglycerol (TAG) composition of duckweeds from China. Here, the genetic diversity of a natural duckweed population collected from Lake Chao, China, was investigated using multilocus sequence typing (MLST). The 54 strains were categorised into four species in four genera, representing 12 distinct sequence types. Strains representing Lemna aequinoctialis and Spirodela polyrhiza were predominant. Interestingly, a surprisingly high degree of genetic diversification within L. aequinoctialis was observed. The four duckweed species revealed a uniform fatty acid composition, with three fatty acids, palmitic acid, linoleic acid and linolenic acid, accounting for more than 80% of the TFA. The TFA in biomass varied among species, ranging from 1.05% (of dry weight, DW) for L. punctata and S. polyrhiza to 1.62% for Wolffia globosa. The four duckweed species contained similar TAG contents, 0.02% mg · DW(-1). The fatty acid profiles of TAG were different from those of TFA, and also varied among the four species. The survey investigated the genetic diversity of duckweeds from Lake Chao, and provides an initial insight into TFA and TAG of four duckweed species, indicating that intraspecific and interspecific variations exist in the content and composition of both TFA and TAG in comparison with other studies.

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“…Several commercially important proteins have already been successfully expressed in duckweed plants: the endoglucanase E1 from Acidothermus cellulolyticus (Sun et al, 2007 ), and monoclonal antibodies against CD20, CD30, and interferon α2b (Cox et al, 2006 ). Protective antigen of swine epidemic diarrhea (Ko et al, 2011 ), tuberculosis antigens ESAT6 and Ag85B (Peterson et al, 2015 ), and hemagglutinin of avian influenza virus H5N1 (Bertran et al, 2015 ; Huong et al, 2015 ) have also been successfully expressed in these plants.…”

Section: Introductionmentioning

confidence: 99%

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

et al. 2018

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The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

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Expression of the protective antigen for PEDV in transgenic duckweed, Lemna minor

Cited by 30 publications

References 15 publications

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

Survey of duckweed diversity in Lake Chao and total fatty acid, triacylglycerol, profiles of representative strains

Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

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