Evolution And Utilization Of The Transposon Tgm9

Soybean [Glycine max(L.)Merr.] is the most important legume crop,which is a great source of feed in animal,human protein and oils.Soybean production costs have changed dramatically over the past 20 years,so it is necessary to improve the yield and quality of soybean.In this study,we mainly introduced the endogenous transposon Tgm9 of soybean into soybean Williams 82 genome by transgenic technology.Then we can construct a soybean germplasm database using Tgm9 independent activity.This work provided a good foundation for the function of unknown genes in the soybean genome.The highly active endogenous transposon,Tgm9,is a 20,548-bp CACTA-Like transposable element.It was isolated from the second intron of the dihyroflavonol-4-reductase 2(DFR2)gene of the soybean T322 W4 locus.The W4 locus controls the pigment formation in flowers and hypocotyls.The insertion of Tgm9 altered pigment accumulation in the petals results in the variegated flowers.When the element is excised from DFR2,the petals return to wild-type purple color.Tgm9 contains 5’ and 3’ terminal inverted repeat(TIR)sequence and two ORFs,similar to other autonomous transposons of CACTA families.ORF1 encodes transposase Gm TNP2(1063aa)and ORF2 encodes transposase Gm TNP1(755aa).Sequence alignment and phylogenetic tree analysis,these results revealed that Tgm9,Tgmt* and Tgm-Express1 have high homology,so they could be from the same progenitor.On the other hand high homology seen in CACTA transposase from various species has supported their monophyletic origin.En/Spm and Tgm9 have high homology,so Tgm9 can be also used for transposon tagging system as utilized previously in case of En/Spm elements.In this study,the modified transposon Tgm9 was introduced into the soybean Williams 82 genome by agrobacterium-mediated soybean transformation system.Acetosyringone(AS)promoted transformation efficiency of soybean,so we used different AS concentrations to investigated the optimum transformation conditions in the experiment.The result showed when the AS concentration was 300 mmol / L,the regeneration and emergence rate can reach maximum(21.55±12.89)% and 5.81%,respectively.We identificated T0 plants using glufosinate and PCR technology and obtained these transgenic plants: 2 report gene,10 transposon and 9 transposase.To screened T1 generation of 2 transposon lines transgenic plants and 1 transposase line transgenic plants,We obtained 4 transposon and 3 transposase homozygous transgenic plants in T3 generation.Then we want to obtain heterzygotes by crossing the transposase with transposon transgenic plants.we failed to get heterozygotes plant by PCR identification F1 plants.In summary,we found Tgm9,Tgmt* and Tgm-Express1 may originate from a common ancestor and CACTA transposase from various species has supported their monophyletic origin.We obtained transposon and transposase transgenic plants and these transgenic plants can be applied to construct soybean germplasm banks and hybridization.