Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics

Zhanyuan Zhang,Melanie Mathieu,Seth Findley,Yaya Cui,Shyam Barampuram,C Nathan Hancock,Minviluz G Stacey,Gary Stacey,Wayne A Parrott

doi:10.1104/pp.112.205369

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. Southern-blot 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.

Highlights

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species
Our findings demonstrate that Tnt1 is an attractive and efficient system that can be used for large-scale insertion mutagenesis in soybean
Tnt1 transposes very efficiently in the legume model plant M. truncatula and several other plants, including tobacco, Arabidopsis, and lettuce (Courtial et al, 2001; d’Erfurth et al, 2003; Tadege et al, 2005, 2008; Mazier et al, 2007; Iantcheva et al, 2009), it was important to evaluate its utility in soybean for two reasons

Summary

Introduction

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Insertional mutagenesis has been successfully used to study gene function in both model and crop plant species (Cowperthwaite et al, 2002; Alonso et al, 2003; An et al, 2003; Fladung et al, 2004; Tadege et al, 2008; Mathieu et al, 2009). Originally isolated from tobacco, has been successfully used in several heterologous hosts, including Medicago truncatula (d’Erfurth et al, 2003; Tadege et al, 2005, 2008; Iantcheva et al, 2009), Arabidopsis (Lucas et al, 1995; Courtial et al, 2001), and lettuce (Lactuca sativa; Mazier et al, 2007) These studies demonstrate that retrotransposons transpose preferentially into gene-rich regions, making them highly mutagenic. Our findings demonstrate that Tnt is an attractive and efficient system that can be used for large-scale insertion mutagenesis in soybean

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