Characterization of enhancer trap and gene trap harboring Ac／Ds transposon in transgenic rice

Insertion mutagenesis has become one of the most popular methods for gene functions analysis.Here we report a two-element Ac/Ds transposon system containing enhancer trap and gene trap for gene tagging in rice.The excision of Ds element was examined by PCR amplification.The excision frequency of Ds element varied from 0% to 40% among 20 F2 populations derived from 11 different Ds parents.Southern blot analysis revealed that more than 70% of excised Ds elements reinserted into rice genome and above 70% of the reinserted Ds elements were located at different positions of the chromosome in rice.The result of histochemical GUS analysis indicated that 28% of enhancer trap and 22% of gene trap tagging plants displayed GUS activity in leaves, roots,flowers or seeds.The GUS positive lines will be useful for identifying gene function in rice.

Functional genomics: Gene identification via T-DNA mediated gene trap tagging in plants

The fully sequenced genomes of Arabidopsis, rice, tomato, potato, ma ize, wheat, and soybean offer large amounts of information about cellular and de velopmental biology. It is a central challenge of genomics to use this informati on in discovering the function of proteins and identifying developmentally impor tant genes. Although classical genetic approaches to gene identification which r ely on disruption of a gene leading to a recognizable phenotype continues to be an extremely successful one, T-DNA mediated gene trap tagging which has been dev eloped that utilize random integration of reporter gene constructs has also prov en to be an extremely powerful tool in plant cellular developmental biology. In this review, how gene trap tagging, promoter trap tagging, and enhancer trap tag ging detection systems have been applied to plant biology is described and these gene identification techniques could be useful to the plant molecular biology a nd plant biotechnology community.

Conditional gene manipulation:Cre-ating a new biological era

To solve the problem of embryonic lethality in conventional gene knockouts, site-specific recombinase （SSR） systems （Cre-loxP, FIp-FRT, and φC31） have been used for tissue-specific gene knockout. With the combination of an SSR system and inducible gene expression systems （tetracycline and tamoxifen）, stage-specific knockout and transgenic expression can be achieved. The application of this ＂SSR＋inducible＂ conditional tool to genomic manipulation can be extended in various ways. Alternatives to conditional gene targeting, such as conditional gene trapping, multipurpose conditional alleles, and conditional gene silencing, have been developed. SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities. With these exciting achievements, we are moving towards a new era in which the whole genome can be manipulated as we wish.

Establishing a Gene Trap System Mediated by T-DNA(GUS) in Rice

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promotsriess β-glucuronidase （GUS） reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 （Oryza sativa ssp. japonica cv.）, mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistsnt （hygr） homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.