Identification and isolation of Mu-flanking fragments from maize

a.
Agricultural College, Yangzhou University, Yangzhou 225009, China
b.
National Maize Improvement Center of China, China Agricultural University, Beijing 100094, China
c.
Corn Research Institute, Henan Agricultural University, Zhengzhou 450002, China
d.
Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana IL61801, USA

More Information

Corresponding author: E-mail address: mxu@cau.edu.cn (Mingliang Xu)

摘要

- /
- /
- /
Abstract: Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertionMutator (Mu) is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2) enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Top1. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.
- Mutator transposon /
- flanking fragment /
- AFLP /
- maize

HTML全文

参考文献(28)

[1]	Alleman, M., Freeling, M. Genetics, 112 (1986),pp. 107-119
[2]	Bennetzen, J.L. Curr. Top Microbiol. Immunol., 204 (1996),pp. 195-229
[3]	Bennetzen, J.L., Springer, P.S., Cresse, A.D. Crit. Rev. Plant Sci., 12 (1993),pp. 57-95
[4]	Bensen, R.J., Johal, G.S., Crane, V.C. et al. Plant Cell, 7 (1995),pp. 75-84
[5]	Brutnell, T.P. Transposon tagging in maize Funct. Integr. Genomics, 2 (2002),pp. 4-12
[6]	Cresse, A.D., Hulbert, S.H., Brown, W.E. et al. Genetics, 140 (1995),pp. 315-324
[7]	Das, L., Martienssen, R.A. Plant Cell, 7 (1995),pp. 287-294
[8]	den Broeck, D.V., Maes, T., Sauer, M. et al. Plant J., 13 (1998),pp. 121-129
[9]	Edwards, D., Coghill, J., Batley, J. et al. Biotechniques, 32 (2002),pp. 1090-1097
[10]	Edwards, K., Johnstone, C., Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis Nucleic Acids Res, 19 (1991),p. 1349
[11]	Frey, M., Stettner, C., Gierl, A. A general method for gene isolation in tagging approaches: amplification of insertion mutagenised sites (AIMS) Plant J., 13 (1998),pp. 717-721
[12]	Hanley, S., Edwards, D., Stevenson, D. et al. Plant J., 22 (2000),pp. 557-566
[13]	Lisch, D. Trends Plant Sci., 7 (2002),pp. 498-504
[14]	Liu, Y.G., Mitsukawa, N., Oosumi, T. et al. Plant J., 8 (1995),pp. 457-463
[15]	Liu, Y.G., Whittier, R.F. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking Genomics, 25 (1995),pp. 674-681
[16]	Lunde, C.F., Morrow, D.J., Roy, L.M. et al. Progress in maize gene discovery: a project update Funct. Integr. Genomics., 3 (2003),pp. 25-32
[17]	Maes, T., Keukeleire, P.D., Gerats, T. Plant tagnology Trends Plant Sci., 4 (1999),pp. 90-96
[18]	Martienssen, R.A. Functional genomics: probing plant gene function and expression with transposons Proc. Natl. Acad. Sci. USA, 95 (1998),pp. 2021-2026
[19]	May, B.P., Liu, H., Vollbrecht, E. et al. Maize-targeted mutagenesis: a knockout resource for maize Proc. Natl. Acad. Sci. USA, 100 (2003),pp. 11541-11546
[20]	Ochman, H., Gerber, A.S., Hartl, D.L. Genetic application of an inverse polymerase chain reaction Genetics, 120 (1988),pp. 621-623
[21]	Robertson, D.S. Mutat. Res., 51 (1978),pp. 21-28
[22]	Settles, A.M., Latshaw, S., McCarty, D.R. Molecular analysis of high-copy insertion sites in maize Nucleic Acids Res, 32 (2004),p. e54
[23]	Souer, E., Quattrocchio, F., de Vetten, N. et al. A general method to isolate genes tagged by a high copy number transposable element Plant J., 7 (1995),pp. 677-685
[24]	Vos, P., Hogers, R., Bleeker, M. et al. AFLP: a new technique for DNA fingerprinting Nucleic Acids Res., 23 (1995),pp. 4407-4414
[25]	Walbot, V. Saturation mutagenesis using maize transposons Curr. Opin. Plant Biol., 3 (2000),pp. 103-107
[26]	Xu, M.L., Huaracha, E., Korban, S.S. Genome, 44 (2001),pp. 63-70
[27]	Xu, M.L., Li, X.Q., Korban, S.S. AFLP-based detection of DNA methylation Plant Mol. Biol. Rep., 18 (2000),pp. 361-368
[28]	Yephremov, A., Saedler, H. Display and isolation of transposon-flanking sequences starting from genomic DNA or RNA Plant J., 21 (2000),pp. 495-505