The study of Agrobacterium transformation technique to transform antisense of the Flavanone 3-Hydroxylase (F3H) gene in Lotus (Nelumbo nucifera Geartn. cv. Buntharik) using to silence F3H gene with RNA interference

Abstract

The silencing of flavanone 3-hydroxylase (F3H) gene results in the inhibition of anthocyanin pathway, which is involved in color production in flowering plants. Therefore, the transformation of antisense of F3H gene in plasmid pJA8F3H by Agrobacterium to silence F3H in lotus “Buntharik” (Nelumbo nucifera Geartn.) with RNA interference (RNAi) technique was studied. The plasmid pJA8F3H contains a selectable marker gene, phosphinotricin acetyl transferase (bar gene), conferring resistance to the herbicide Basta® (glufosinate ammonium). It was found that the optimum concentration of Basta® was 10mg/L and the appropriate concentration of the antibiotic Augmentin was 500 mg/L to eliminate Agrobacteria strain GV3101. The RT-PCR analysis of F3H gene was determined by primer F3H sense F and F3H sense R, which are specific to 300 nucleotides of the F3H gene. The F3H gene expression analysis showed that the expression in the lotus plants that received the plasmid transfer was reduced compared to the plants that did not undergo gene transfer, which is consistent with the qRT-PCR results. The relative gene expression of the F3H was found that the control plants had an expression value of 1 whereas the five transgenic plants that received the plasmid transfer showed an average expression value of only 0.149. Therefore, F3H gene transfer in the form of RNAi is another option for changing and improving the color of flowering plants in the future.