Title: Construction of FATTY ACID DESATURASE 2 (FAD2) gene knockouts using RNAi novel technique in the model plant Thlaspi arvense.
Abstract: FATTY ACID DESATURASE 2 and FATTYU ACID ELONGASE 1 (FAE1) are the two genes of interest present in the plant pennycress. FAD2 is in the pathway that synthesizes the polyunsaturated linoleic (18:2) and linoleic (18:3) fatty acids from the monounsaturated oleic acid (18:1). Polyunsaturated fatty acids are undesirable in biodiesel because they confer oxidative instability, but greater energy (J.C. Sedbrook, et al, 2014). Targeting gene function reduction of FAD2 in Thlaspi arvense using RNA interference method, lowered linoleic/linoleic acid content and increased oleic acid content in seed oil. We are hoping …show more content…
FAD2, a very important enzyme in the fatty acid biosynthesis pathways. Pennycress has relatively high Erucic (22:4) acid content, and disadvantages of Erucic acid is that it has a relatively poor cold flow properties. Cold flow is the tendency of a solid material to move slowly or deform permanently under the influence of mechanical stress (cold). The goal is to construct a knockout of FAD2 in the seeds, which we are hoping will reduce the amount of unsaturated fatty acids, making the oil content more appropriate for Biodiesel production. Dr. Sedbrook and his team are trying to domesticate Pennycress to make it economically …show more content…
Two sets of primers were used to obtain the DNA of both the open reading frames. A standard set of protocol was followed to obtain highly amplified open reading frame of FAD2, but with an increased annealing temperature of 55 ° C as the primer’s boiling point was high of 75 ° C. The reaction were labelled RNAIFAD2.1 and RNAIFAD2.2. The amplified PCR DNA was then run on 1% agarose gel to separate the amplified DNA. The gel was excised after the electrophoresis, and later subjected to gel purification to recollect the RNAIFAD2.1 and RNAIFAD2.2 DNA using the standard protocol provided with the GeneJet Gel Extraction Kit. The DNA after transcribed in the cells of the pennycress will adopt a miRNA shape due to its complementary sequence flanked by inverted repeats favoring a hairpin formation.
The purified gel extracted RNAIFAD2.1 and RNAIFAD2.2 were then subjected to a restricted digest using BAMH1 and XHO1 restricted enzymes along with our donor vector pENTR 2B. The reaction was incubated for 1 hour in 37° water bath. The reaction was then run on 1% agarose
Abstract: FATTY ACID DESATURASE 2 and FATTYU ACID ELONGASE 1 (FAE1) are the two genes of interest present in the plant pennycress. FAD2 is in the pathway that synthesizes the polyunsaturated linoleic (18:2) and linoleic (18:3) fatty acids from the monounsaturated oleic acid (18:1). Polyunsaturated fatty acids are undesirable in biodiesel because they confer oxidative instability, but greater energy (J.C. Sedbrook, et al, 2014). Targeting gene function reduction of FAD2 in Thlaspi arvense using RNA interference method, lowered linoleic/linoleic acid content and increased oleic acid content in seed oil. We are hoping …show more content…
FAD2, a very important enzyme in the fatty acid biosynthesis pathways. Pennycress has relatively high Erucic (22:4) acid content, and disadvantages of Erucic acid is that it has a relatively poor cold flow properties. Cold flow is the tendency of a solid material to move slowly or deform permanently under the influence of mechanical stress (cold). The goal is to construct a knockout of FAD2 in the seeds, which we are hoping will reduce the amount of unsaturated fatty acids, making the oil content more appropriate for Biodiesel production. Dr. Sedbrook and his team are trying to domesticate Pennycress to make it economically …show more content…
Two sets of primers were used to obtain the DNA of both the open reading frames. A standard set of protocol was followed to obtain highly amplified open reading frame of FAD2, but with an increased annealing temperature of 55 ° C as the primer’s boiling point was high of 75 ° C. The reaction were labelled RNAIFAD2.1 and RNAIFAD2.2. The amplified PCR DNA was then run on 1% agarose gel to separate the amplified DNA. The gel was excised after the electrophoresis, and later subjected to gel purification to recollect the RNAIFAD2.1 and RNAIFAD2.2 DNA using the standard protocol provided with the GeneJet Gel Extraction Kit. The DNA after transcribed in the cells of the pennycress will adopt a miRNA shape due to its complementary sequence flanked by inverted repeats favoring a hairpin formation.
The purified gel extracted RNAIFAD2.1 and RNAIFAD2.2 were then subjected to a restricted digest using BAMH1 and XHO1 restricted enzymes along with our donor vector pENTR 2B. The reaction was incubated for 1 hour in 37° water bath. The reaction was then run on 1% agarose