Cryptic regulatory elements obtained from plants

Abstract

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated transgenic Nicotiana tabacum plant that expressed GUS activity either only in developing seed coats, or constitutively. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. Analysis of the region demonstrated that the seed coat-specificity of GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. This promoter is useful in controlling the expression of genes to the developing seed coat in plant seeds. Similarly, cloning and characterization of the cryptic constitutive promoter revealed the occurrence of several cryptic regulatory regions. These regions include promoter, negative regulatory elements, transcriptional enhancers, core promoter regions, and translational enhancers and other regulatory elements.

Description

[0001] This invention relates to cryptic regulatory elements within plants.BACKGROUND AND PRIOR ART[0002] Bacteria from the genus Agrobacterium have the ability to transfer specific segments of DNA (T-DNA) to plant cells, where they stably integrate into the nuclear chromosomes. Analyses of plants harbouring the T-DNA have revealed that this genetic element may be integrated at numerous locations, and can occasionally be found within genes. One strategy which may be exploited to identify integration events within genes is to transform plant cells with specially designed T-DNA vectors which contain a reporter gene, devoid of cis-acting transcriptional and translational expression signals (i.e. promoterless), located at the end of the T-DNA. Upon integration, the initiation codon of the promoterless gene (reporter gene) will be juxtaposed to plant sequences. The consequence of T-DNA insertion adjacent to, and downstream of, gene promoter elements may be the activation of reporter gene...

AI Technical Summary

Benefits of technology

[0124] Further analysis confirmed the presence of a regulatory sequence within the NdeI-SmaI fragment of the mRNA leader sequence that had a significant impact on the level of GUS specific activity expressed in all organs tested. Deletion of the NdeI-SmaI fragment from the T1275-GUS-nos gene (FIG. 15) resulted in about a 46-fold reduction in the amount of GUS specific activity that could be detected in leaves of transgenic tobacco cv Delgold (see Table 7). Similar results were also obserevd in the transgenic tobacco cultivar SRI and transgenic alfalfa (Table 7). Addition of the same fragment to a 35S-GUS-nos gene construct (FIG. 15) increased the amount of GUS specific activity by about 5-fold in transgenic tobacco and a higher amount in transgenic alfalfa (see Table 7). Increased GUS activity was observed in organs of tobacco and alfalfa plants tranformed with constructs containing NdeI-SmaI fragment (Table 8 and 9).

[0138] 4. Inhibition of seed coat maturation by expression of ribonuclease genes to allow for increased seed size, and to reduce the relative biomass of seed coats, and to aid in dehulling of seeds.

Problems solved by technology

A further deletion to 0.5 kb of the 5' flanking site resulted in complete loss of GUS activity.

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