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Rapid enhancement of ?-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes.

ABSTRACT: Agrobacterium-mediated transient gene expression have become a method of choice over stable plant genetic transformation. Tocopherols are a family of vitamin E compounds, which are categorized along with tocotrienols occurring naturally in vegetable oils, nuts and leafy green vegetables. This is the first report involving AtTC and AtHPT transient expression in Nicotiana benthamiana and this system can be used efficiently for large scale production of vitamin E. Agroinfiltration studies were carried out in N.benthamiana for the expression of Arabidopsis thaliana (At) genes encoding homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) individually and in combination (HPT?+?TC). The transgene presence was analyzed by reverse transcription PCR, which showed the presence of both the vitamin E biosynthetic pathway genes. The gene expression analysis was carried out by (reverse transcription quantitative real-time polymerase chain reaction) RT-qPCR and ?-tocopherol content was quantified using high performance liquid chromatography (HPLC). The relative gene expression analysis by RT-qPCR confirmed an increased expression pattern where TC?+?HPT combination recorded the highest of 231 fold, followed by TC gene with 186 fold, whereas the HPT gene recorded 178 fold. The ?-tocopherol content in leaves expressing HPT, TC, and HPT?+?TC was increased by 4.2, 5.9 and 11.3 fold, respectively, as compared to the control. These results indicate that the transient expression of HPT and TC genes has enhanced the vitamin E levels and stable expression of both A. thaliana genes could be an efficient strategy to enhance vitamin E biosynthesis in agricultural crop breeding.

SUBMITTER: Sathish S

PROVIDER: S-EPMC6237709 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Rapid enhancement of α-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes.

Sathish Sundararajan S Preethy Kuppuraj Sree KS Venkatesh Rajendran R Sathishkumar Ramalingam R

3 Biotech 20181115 12

Agrobacterium-mediated transient gene expression have become a method of choice over stable plant genetic transformation. Tocopherols are a family of vitamin E compounds, which are categorized along with tocotrienols occurring naturally in vegetable oils, nuts and leafy green vegetables. This is the first report involving AtTC and AtHPT transient expression in Nicotiana benthamiana and this system can be used efficiently for large scale production of vitamin E. Agroin ...[more]

PMID: 30498659

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Project description:BackgroundAgroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis.ResultsUsing the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1-2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform.ConclusionsIn this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

Project description:Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.

Dataset Information

Rapid enhancement of ?-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes.

Publications

Rapid enhancement of α-tocopherol content in <i>Nicotiana benthamiana</i> by transient expression of <i>Arabidopsis thaliana</i> Tocopherol cyclase and Homogentisate phytyl transferase genes.

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