Characterization Of Genetic Behaviors Of Maize TED Transposon And Construction Of TED/dTED Two-Element Systems

TED,an autonomous transposon,has been recently cloned in maize（Zea mays）.Although both belong to the Mutator superfamily,TED is different from the founding member Mu DR or another maize Mutator superfamily autonomous transposon Jittery in the sequence and genetic behaviors.Therefore,it has important theoretical and practical significance to further analyze its structure and function and develop its unique application potential.The main findings are as follows:1.The TED full-length cDNA sequences of 3098 bp were obtained by RACE technology,and the predictedTEDA transposase consisted of 825 amino acids and includes multiple conserved functional domains unique to the Mutator superfamily.At the same time,the molecular biology experiments found that the 3′-TIR of the TED transposon did not transcript any products in maize seedling leaves.2.When trying to clone the TED and Jittery genomic sequences or their full-length cDNAs in E.coli,we have encountered a lot of failure,especially with TED genome sequence and its cDNA.Properly prolonging the recovery time of the bacterial solution prior to plating would increase the number of colonies and the number of TED sequence-positive clones.However,it was difficult to maintain the complete sequences cloned in E.coli for a long time due to the frequent accumulation of various sequence variations.In vitro experiments showed that direct application of TED nucleic acid solution had no effect on bacterial growth,so the harshness in cloning process would occur after the introduction of recombinant DNA plasmid into the bacterial cell.In addition,the cloning experiments of multiple dTED and f TED sequences showed that the harshness in cloning was not related to the leaky expression of the transposase or the TIR sequences at both ends of the transposon,and there was no obvious"toxic"segment in the TED genomic sequence.3.Using C1,Bz1 and GFP as marker gene,we have constructed five transformation vectors of modified dTED（dTED^*）elements flanked with TSD of varied length and sequence composition.Those constructs were used for generated transgenic lines via Agrobacterium-mediated transformation of maize immature embryos.Combined with phenotypic screening and molecular biology characterization,single-copy T-DNA transgenic lines with stable genetic performance were identified,and multiple sets of maize TED/dTED two-element systems were established from genetic crosses with tester lines（generated from specific genetic experiments）for several generations.4.The reciprocal crosses of transgenic lines with specific tester lines showed that,in the maize endosperm aleurone layer,both transposition activities of 0-bp-TSD and 3-bp-TSD dTED^*elements were lower than all other three dTED^*elements flanked with 9-bp,10-bp or 15-bp TSD.In the germinal cells of transgenic lines with 9 bp TSD dTED^*element,the Bz’reverse mutation rate was comparable to that of the reported autonomous TED at bz-m175 site(around 1×10^-4).Furthermore,sequence analysis of PCR amplicons showed that somatic excision products from different types of TSD transgenic lines are rich of sequence variation,among which,precisely repaired excision product（fp0）was merely detected from the 9-bp,10-bp and 15-bp TSD transgenic lines.In germinal cells,in addition to the Bz’reverse mutations,some bz-s mutants were found to be mutations concentrated at the 3’-end of the transformed dTED^*element and its downstream,including deletions,inversions and other sequence rearrangement complex difficult to clarify.In addition,sequence analysis of the isolated dTED^*flanking sequences showed that the newly generated TSD of the transposed dTED^*at the reinsertion site were quite conserved（9 bp in length）with no regard to their primary TSD length or sequence composition.In summary,this study has improved the understanding of the genetic characteristics of Mutator superfamily transposons,and created a large number of maize transposon genetic materials,which laid a solid foundation for further research on the behavior and regulation of transposons.