Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics    

Abstract

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southernblot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean.

Insertional Mutagenesis Using Tnt1 Retrotransposon in Potato

“…The Tnt1 element in potato showed similar activity and behavior to that documented in other plant species, including activation by tissue culture, stability during sexual reproduction, and preferential transposition into genic regions (Courtial et al, 2001;d'Erfurth et al, 2003;Mazier et al, 2007;Cui et al, 2013). We examined several factors that may affect the Tnt1 retrotransposition.…”

“…Tissue culture-induced retrotransposition of Tnt1 was previously reported in several plant species (Courtial et al, 2001;d'Erfurth et al, 2003;Mazier et al, 2007;Cui et al, 2013). We wanted to confirm whether tissue culture can also induce Tnt1 transposition in the potato genome.…”

“…The transposed Tnt1 copy numbers of the regenerants derived from CT1 ranged from one to more than 20, which is similar to the numbers observed in other plant species. The transposition events ranged from four to more than 30 in M. truncatula (d'Erfurth et al, 2003), zero to 26 in Arabidopsis (Courtial et al, 2001), and four to 19 in soybean (Cui et al, 2013). A total of 1,667 insertions was recovered from 70 Tnt1-harboring lines, averaging 24 insertions per line.…”

“…Most remarkably, the Tnt1 retrotransposon, originally identified in tobacco (Nicotiana tabacum; Grandbastien et al, 1989), has been successfully used in insertional mutagenesis in several heterologous species, including Arabidopsis (Courtial et al, 2001), Medicago truncatula (d 'Erfurth et al, 2003), lettuce (Lactuca sativa; Mazier et al, 2007), and soybean (Glycine max; Cui et al, 2013). Tnt1 was used to generate approximately 12,000 independent lines that represent over 300,000 insertions in M. truncatula (Tadege et al, 2008;Cheng et al, 2011).…”

The Hairless Stem Phenotype of Cotton (Gossypium barbadense) Is Linked to aCopia-Like Retrotransposon Insertion in aHomeodomain-Leucine ZipperGene (HD1)

“…Some important trichome developmental and patterningrelated genes include GLABRA1 (GL1), GLABRA2 (GL2), basic helix-loop-helix protein (GL3), ENHANCER OF GLABRA3 (EGL3), and WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1) in Arabidopsis (Larkin et al 1994;Payne et al 1999;Esch et al 2003;Zhang et al 2003;Wang et al 2004;Humphries et al 2005;Zhao et al 2008;Cui et al 2013;Zhou et al 2014). Their homologous genes in cotton also have been identified as critical for trichome development.…”