Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics

Oxford University Press (OUP) - Tập 161 Số 1 - Trang 36-47 - 2012
Yaya Cui1,2, Shyam Barampuram1,2, Minviluz G. Stacey1,2, C. Nathan Hancock1,2, Seth D. Findley1,2, Mélanie Mathieu1,2, Zhanyuan Zhang1,2, Wayne A. Parrott1,2, Gary Stacey1,2
1Department of Biology and Geology, University of South Carolina, Aiken, South Carolina 29801 (C.N.H.)
2Divisions of Plant Sciences and Biochemistry, National Center for Soybean Biotechnology, University of Missouri, Columbia, Missouri 65211 (Y.C., S.B., M.G.S., S.F., M.M., Z.Z., G.S.); Department of Biology and Geology, University of South Carolina, Aiken, South Carolina 29801 (C.N.H.); and Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia 30602–7272 (W.A.P.)

Tóm tắt

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.

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Oxford University Press (OUP)

Tập 161 Số 1

36-47

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