T-DNA insertional mutagenesis for functional genomics in rice

Plant J. 2000 Jun;22(6):561-70. doi: 10.1046/j.1365-313x.2000.00767.x.

Abstract

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Blotting, Southern
  • DNA, Bacterial / genetics*
  • Genes, Reporter
  • Genome, Plant*
  • Glucuronidase / genetics
  • Glucuronidase / metabolism
  • Mutagenesis, Insertional*
  • Oryza / genetics*
  • Oryza / metabolism
  • Plants, Genetically Modified
  • Promoter Regions, Genetic

Substances

  • DNA, Bacterial
  • T-DNA
  • Glucuronidase