Genetics Identification And Behavior Characterization Of Autonomous Helitron Transposons In Maize

Helitron transposons are DNA transposons discovered computationally,confirmed by experiment,and occur ubiquitously in most sequenced genomes of various eukaryotes which includes the fungi,plants and animals.Lacking of structure features of classic DNA transposons,such as TIRs and TSDs,makes it less feasible to identify Helitrons by either genetic approaches or bioinformatics applications.Recent evidence suggests that Helitrons may transpose mainly by either cut-and-paste or rolling-circle-replication（RCR）mechanisms.It is obviously essential to clone the yet mysterious autonomous Helitrons for elucidating the actual transposition mechanism（s）in plants.Maize is known for its abundancy,numerous family members and genome fraction of Helitron transposons,which are results of higher genetic activities of Helitron transposons driven by autonomous Helitrons.This research is aiming to establish maize Helitron transposon activity reporting systems and extensively screen the candidates bearing an autonomous Helitron transposition activity from maize inbred lines in addition to evaluate their genetic performance,elucidate the gene structure of a Hel and all bona fide transposition mechanisms.Aiming to identify autonomous Helitrons in maize inbred lines,an artificial d Hel reporter system was constructed to illustrate the transposition activities of autonomous Helitrons in maize.In brief,a green fluorescent protein（GFP）packaged in defective Helitron transposons,which inserted in C1,an indispensable transcription factor and member of the regulatory complex activating structural genes in aleurone tissue pigmentation of maize kernels.The autonomous Helitron transposons can be cloned subsequently in segregation populations and further analyzed for the detailed molecular characterization of their novel sequence structure,the genetic base of either rolling-circle-replication or cut-and-paste transposition mechanism,particular repairing mechanism upon excision and sequence preference at new insertion sites.Tackling these questions will provide theoretical basis,research scheme and technical means for genome engineering editing with Helitron transposons.Through the Helitron transposition activity detection and reporting system,the main research progress is as follows:1.Four families,Hel1-4,Hel1613,Hel1158 and Hel A2 were selected out by sequences alignments from public database and in-house collection of Helitron transposons.The most conserved terminal sequences of both 5’and 3’ends of each family were used as consensus border sequences of the tagged GFP gene to unify the d Hel-GFP transposable elements.Four primary insertion sites were identified for embedding the d Hel-GFP elements from the functional analysis of the C1 promoter region.After genetic transformation and hybridization with different inbred lines,Purple-spotted kernel observed in hybrid populations.The phenotypes are differed from each other among transgenic lines bearing constructs of various d Hel inserted at the same site in C1 gene or the same d Hel inserted at different sites in C1 gene.Molecular characterization is performed among genetic confirmed transgenic lines where GFP expression is segregated along with purple-pigmented kernels,resulting from a leaky expression of C1 gene driven by the d Hel inserted promoter.2.The leaves of all transgenic lines of seedlings contain variant C1 transcripts by RACE（rapid amplification of c DNA end）.Transcription analysis indicated that the insertion of different d Hels into gene promoter or exon would lead to multiple distinct m RNA transcripts generated from transgene in the host genome or numbers of alternative splicing transcripts encoded by transgene of which the same d Hel inserted at the same site.These results indicate that the insertional d Hels in the C1 promoter region are the directly causative factors leading to the polychromatic transcripts of the host gene,a novel pathway for gene expression regulation and manupilation.3.Precise excision products of d Hel-GFP transposition have been detected from the leaf tissue of the seedlings in F₁ hybrids of transgenic lines（S3-Hel A2、S3-Hel1158、S1-Hel1-4、S4-Hel1-4）with corresponding c1 tester line,although simple somatic excision events were failed to be detected in all primary transgenic lines.It was suggested that there might be an autonomous Helitron in c1 tester lines to catalyze the d Hel-GFP excision from insertion site via cut-and-paste mechanism.4.To elucidate whether d Hel-GFP produces circular intermediate products during transposition,we performed nested RT-PCR on genomic DNA samples with d Hel-GFP excision.Either the amplicons of precisely repairing product of d Hel-GFP simple excision upon transposition or the lack of detectable amplicons of circular d Hel-GFP intermediate resulting from the proposed RCR transposition favors the presence of autonomous Helitrons in the corresponding c1 tester lines and the cut-and-paste transposition mechanism in tested somatic tissues.Very likely,the d Hel-GFP elements undergoing transposition via cut-and-paste mechanism may avoid from the rolling-circle-replication mechanism.Our results strongly suggest that the newly constructed reporter system is feasible to detect the genetic activity of autonomic Helitron at molecular level in maize.Sequence features of d Hel itself together with the flanking regions impact the excision activity of d Hel and the regulation of the d Hel on the transcription level of the host gene.The d Hel-GFP elements transpose mainly via cut-and-paste mechanism rather than the rolling-circle-replication one catalyzed by a Hels.Our current studied may lay the ground for a Hel cloning through genetic approaches,create valuable new germplasms for exploring the transposition mechanisms and shed light on the genetic contributions of Helitrons to the gene structure variation and genome evolution in maize.