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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, Preethy, Kuppuraj Sree, Venkatesh, Rajendran, Sathishkumar, Ramalingam
3 Biotech 2018 v.8 no.12 pp. 485
Arabidopsis thaliana, Nicotiana benthamiana, agroinfiltration, alpha-tocopherol, biochemical pathways, biosynthesis, crops, gene expression, genetic transformation, green leafy vegetables, high performance liquid chromatography, leaves, nuts, plant breeding, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, reverse transcription, tocopherol cyclase, tocotrienols, transgenes, vegetable oil
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 α-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 α-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.