Download Homologous Promoter Use in Genetic Modification

ISB News Report
September 2005
Homologous Promoter Use in Genetic Modification
Keerti S. Rathore & Ganesan Sunilkumar
Genetic modification generally requires stable transgene expression at a desired level in the transgenic organism, without
adversely affecting native gene activities. The concept of gene stacking to introduce multiple agronomic traits requires
coordinated expression of several genes. In addition, second and third generation biotechnology products require controlled
expression of several transgenes. These applications necessitate a heterologous and/or a homologous promoter to drive the
expression of one or more transgenes. For certain applications, a homologous promoter may be a better choice since it is
likely to provide a more precise level of developmental and spatial control and its activity may be stronger in the native
environment.
The benefits of homologous promoter use were demonstrated recently in a study on the characterization of a cotton αglobulin promoter, which showed that this promoter resulted in a significantly higher level of gusA gene expression in
cotton, compared to that of two other heterologous systems, Arabidopsis and tobacco1. However, it is generally believed
that homologous promoters should be avoided, as they can lead to transgene and/or the resident gene/transgene silencing25
. This notion mainly stems from studies conducted by two independent groups who demonstrated that, in some tobacco
lines, reintroduction of a heterologous promoter into a transgenic plant that contains a previously introduced copy of that
promoter silenced transgenes driven by each promoter6-8. Methylation of the promoters was found to be the basis of their
inactivation.
If homology-dependent promoter inactivation is based on a general mechanism, it should apply not only to the multiple uses
of a heterologous promoter but also to the single use of a homologous promoter. There are several reports on the successful
use of homologous promoters for transgene expression (see references in ref. 9). However, to our knowledge, no systematic
study has been conducted to specifically address the negative impact of a transgenic, homologous promoter on the activity
of the endogenous promoter. Inactivation of a transgenic and/or resident promoter is a serious concern in agricultural
biotechnology that requires a thorough investigation.
We have directly addressed this important issue in transgenic lines that were created to enhance the levels of oleic acid
in cottonseed9. The promoter region isolated from a cotton α-globulin B gene was used to drive an antisense construct
of a ∆-12 desaturase gene in cotton. The seeds from several antisense lines exhibited increased levels of oleic acid and a
concomitant decrease in the levels of linoleic acid. These lines exhibiting the antisense-mediated phenotype, as well as some
transgenic lines that did not exhibit the high-oleate phenotype, provided a suitable resource to study the impact of the use of
a homologous promoter on the activity of an endogenous promoter.
The level of the α-globulin B protein in the seed is expected to accurately reflect the activity of its promoter. Therefore, we
examined the quantity of this protein by estimating the intensity of the 52 kDa band on a Coomassie Brilliant Blue-stained
SDS-PAGE gel. The levels of α-globulin B protein in the seeds from four selected, highest-oleate lines were compared with
control seeds. The controls consisted of non-transgenic cottonseeds as well as seeds from two different null segregant plants
derived from one of the high-oleate lines. Total proteins were extracted from a pooled sample of 10 seeds from each plant
and the quantitation was performed on three replicate protein extracts that were fractionated on three different gels. The αglobulin B protein band was quantified from the digital photographs of the gels using AlphaEaseTM software. The levels of
α-globulin B protein in the high-oleate lines were not significantly different from control seeds. Therefore, the endogenous
promoter as well as the transgenic promoter in high-oleic acid lines were functioning normally.
As with any transformation experiment, we obtained a few lines that did not exhibit the transgenic phenotype, i.e., higher
levels of oleic acid in the seeds. One reason for the absence of the transgenic phenotype may be that the transgene was not
expressed due to inactivation of the promoter. In this scenario, if the promoter homology-based mutual silencing mechanism
was in operation, the possibility exists that the corresponding endogenous promoter was also silenced. To test this possibility,
the levels of α-globulin B protein in the seeds from eight transgenic lines, which showed little or no increase in seed oleic
acid, were compared with control seeds. The levels of α-globulin B protein were not significantly different between the
seeds of control and transgenic lines that exhibited wild-type levels of oleic acid. These results, taken together, show that the
ISB News Report
September 2005
seed-specific, α-globulin promoter can be used effectively for genetic modification of cottonseed without interfering with
the activity of the corresponding endogenous promoter9.
Previous studies6,8 that led to the notion of promoter homology-mediated silencing were based on observations on a few,
isolated transgenic events containing multiple T-DNA inserts or rearranged T-DNA structures. These events were subjected
to detailed characterizations in subsequent investigations by each group. It is possible that the extensive nature of these
series of studies may have helped create a perception that promoter homology causes silencing problems. In our study, we
have directly addressed this issue and found that introduction of a homologous promoter, per se, does not cause silencing of
either the transgenic or resident promoter.
References
1.
Sunilkumar G et al. (2002) Transgenic Res. 11, 347-359
2.
Finnegan J & McElroy D. (1994) Biotechnol. 12, 883-888
3.
4.
De Wilde C et al. (2000) Plant Mol. Biol. 43, 347-359
Halpin C et al. (2001) Plant Mol. Biol., 47, 295-310
5.
Potenza C, Aleman L & Sengupta-Gopalan C (2004) In Vitro Cell. Dev. Biol.-Plant 40, 1-22
6.
Matzke MA et al. (1989) EMBO J., 8, 643-649.
7.
Matzke MA, Neuhuber F & Matzke AJM. (1993) Mol. Gen. Genet. 236, 379-386
8.
Vaucheret H (1993) C. R. Acad. Sci. Paris 316, 1471-1483
9.
Sunilkumar G et al. (2005) Plant Biotechnology Journal 3, 319-330
Keerti S. Rathore & Ganesan Sunilkumar
Institute for Plant Genomics & Biotechnology
Texas A&M University, College Station, TX 77843-2123
[email protected]