Modification And Application Of Soybean Transposon Tgm9

Tgm9 is an active DNA transposon discovered in soybean genome, belonging to CACTA family, it is similar to snapdragon transposon Tam1 and maize transposon En/Spm in structure. The total length of Tgm9 is about 20.5kb, the length of it's 5'-TR (terminal region) is about 400bp, the length of it's 3'-TR is about 700bp; and there are two transposase gene GmTNP1 and GmTNP2, which encode two proteins related to the transposition.In soybean unstable line T322, Tgm9 is inserted into the second intron of DFR2 which encods dihydroflavonol-4-reductase 2 in anthocyanin biosynthesis pathway. The insertion of Tgm9 leads to the loss of DFR2 protein, so the pigment synthesis is blocked, and soybean flowers are white. When Tgm9 jumped out from the gene, the synthesis of DFR2 protein recovered, and soybean plants produce wild type purple flowers. When the Tgm9 jumped out and inserted into new sites, it will cause new mutations. Moreover, Tgm9 provides a potential tool for soybean gene cloning through transposon tagging technique.The main objectives of this work are modifying the Tgm9 structure, and transforming it to cv. Williams82 whose genome is fully sequenced. It is hoped that this work would facilitate creating a soybean insertional mutant library in future. For these purposes, I firstly constructed two sets of constructs, one containing modified non-autonomous transposons dTgm9s and the other containing two transposase genes. And then, I transformed them to cv. Williams82 respectively. The experiments were preformed as following:1? Soybean transposon modification:I planed to established a two-line transposon jumping systems through plant transformation:one is a transposon system containing modified dTgm9, and the other is a transposase system containing transposase expression cassettes. The binary vectors applied for transposon and transposase systems are named as transposon and transposase plasmids, respectively.(1) Construction of the transposon plasmids:I cloned the 5'-TR and 3'-TR of Tgm9 from T322. and ligated them together to get a simplified transposon dTgm9. Then dTgm9 was inserted between the 35S promoter (p35S) and the coding sequence of reporter gene F3'5'H to create the expression cassette p35S::dTgm9::F3'5'H::nosT on binary vector pLM-B001.Three types of dTgm9s differing in length were made, so there were three kinds of transposon plasmids constructed:pLM-B001_dTgm9-1_F3'5'H:dTgm9-1(2.5 kb 5'-TR+2.0 kb 3'-TR)pLM-B001_dTgm9-2_F3'5'H:dTgm9-2 (1.5 kb 5'-TR+2.0 kb 3'-TR)pLM-B001_dTgm9-3_F3'5'H:dTgm9-3 (0.5 kb 5'-TR+1.0 kb 3'-TR)(2) Construction of the transposase plasmid:The encoding sequences of GmTNP1 and GmTNP2 were amplified by PCR and RT-PCR, respectively. Next, two expression cassettes pUbi7::GmTNP1:nosT and p35S::GmTNP2::nosT were made and inserted into the same vector. The binary vector used here also was pLM-B001.2?Agrobacterium-mediated transformation of soybean:Firstly, I transformed the positive control vector, transposon plasmid and transposase plasmid into the Agrobacterium tumefaciens strain GV3101. In this study. Agrobacterium-mediated cotyledonary-node transformation method used in sequenced line Williams82, and then the explants followed by co-cultivation, shoot induction, shoot elongation and rooting culture stage, successively.Eventually, under the glufosinate selection pressure, by Agrobacterium-mediated, in positive control vector transformed plants, there were 200 explants, and plant emergence rate was about 0.5%. In this conversion process, because my operation was not standardized, resulting in Agrobacterium tumefaciens and fungi pollution was more serious. At last, from the initial to final, there was only 1 emergence, so as not to 0.5% real rate of emergence. In transposon plasmid transformed plants, there were 400 explants, and plant emergence rate was about 3%. In transposase plasmid transformed plants, explants were still at emergence stage, so the emergence rate was not sure.Identification of the herbicide resistance of the leaves of transgenic soybean plants using 135 mg/L Basta in To lines, and there were no transgenic plants in positive control vector transformed plants, and in transposon plasmid transformed plants, there were 10 transgenic plants.