Part:BBa_K5160002
E8 promoter
Overview
In order to ensure the stability of sweet protein expression in transgenic tomatoes, we chose the tomato E8 fruit-ripening-specific promoter instead of the 35S promoter. This tissue-specific promoter can not only accumulate the expression products of target genes in certain organs or tissue sites and increase the regional expression, but also avoid unnecessary waste of plant nutrients.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Biology
The E8 promoter has a highly specific expression pattern in tomato fruit. It is mainly activated at specific stages of tomato fruit development, driving preferential expression of downstream genes in fruit tissues. The E8 promoter is significantly more active in fruit compared to other tissues such as roots, stems and leaves. It can be utilized to achieve precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in tomato fruits, avoiding unwanted effects in other tissues. This is important for improving the quality and characteristics of tomato fruits.
During fruit ripening, the E8 promoter can initiate the expression of a series of genes that are involved in physiological processes such as fruit color change, texture softening, and flavor substance synthesis. These changes are the result of the mutual regulation of the E8 promoter and ethylene.
There are at least two major regions in the E8 promoter sequence that contribute to its transcriptional regulation. One is the upstream region from -2181 to -1088, which contains ethylene-responsive transcriptional elements. When ethylene concentration is elevated, the expression of the E8 promoter and ethylene synthesis genes increases simultaneously, forming a positive feedback regulatory mechanism that further promotes fruit ripening. And in the absence of ethylene synthesis, another region comes into play. The sequence from -409 to -263 of the transcription start site is sufficient for ripening-specific transcription.
Design
Specific Expression
Plants have a typical division of labor in which different organs perform different functions. In the plant body, roots are responsible for drawing water and inorganic salts downward, leaves are responsible for photosynthesis to provide energy, and fruits are responsible for synthesizing products and storing them. The E8 promoter is one of the most broadly characterized ripening-specific tomato promoters, and it exhibits strong conservation among different tomato varieties. Therefore, it enables the specific expression of thaumatin within the tomato fruit without affecting the normal functioning of roots and leaves.
To ensure that the sweet protein is expressed only in tomato fruits, we used the full-length sequence of the E8 promoter of 2209 bp constructed into a binary expression vector. This sequence contains an ethylene-responsive transcription element that facilitates efficient expression of the sweet protein.
Stable Expression
Tomato fruit ripening is a highly coordinated process involving multiple changes in color, texture, and flavor. To achieve these changes, precise regulation of specific genes is required. Epigenetic modifications can be used as a mechanism to rapidly respond to environmental and developmental signals, adjusting gene expression patterns to meet the demands of fruit ripening, most commonly manifested as methylation modifications of DNA.
Methylation of the DNA itself may physically hinder the binding of transcription factors to genes. At the same time, DNA modified by methylation may be bound by MBD proteins, which recruit other modifying factors, resulting in the formation of dense, inactive chromatin that leads to failure of DNA unlinking during transcription.
In tomato, DNA methylation is very common. On the other hand, there is a natural SlDML2-mediated active DNA demethylation in tomato, a process that counteracts the effects of methylation modifications. SlDML2 acts as a DNA demethylase that activates the expression of ripening-related genes such as RIN, ethylene, and pigment synthesis-related genes. Due to regulation by ethylene, genes determined by the E8 promoter are more likely to be transcribed during tomato fruit ripening.
Construction
We inserted the genes thaumatin and brazzein downstream of the E8 promoter and constructed two plasmids, pCAMBIA1301_Thaumatin and pCAMBIA1301_Brazzein, which were transformed into Agrobacterium GV3101.
Characterized
Translation validation
We used specific primers for pcr and proved the success of our expression vector into Agrobacterium GV3101 by agarose gel electrophoresis (Fig 5). The callus tissue was then infested with positive bacteria, and when the callus tissue grew to a certain extent, it was inoculated in rooting medium.
Expression at DNA level
The callus tissues were cultivated on rooting medium. After the plants had grown leaves, we harvested the leaves to test whether our genes were present in the tomato genome. The results of the analysis showed that the DNA of both thaumatin and brazzein could be detected (Fig 8).
Expression at protein level
We took leaf, flower, and fruit samples and extracted the proteins for WB assay. Eventually we only succeeded in detecting the presence of positive bands around 28 kD and 10 kD in fruits, but not in leaves and flowers (Figs 7,8,9). This result suggests that Thaumatin (28.36 kD) and Brazzein (9.75 kD) were successfully expressed in our transgenic tomato under the initiation of the E8 promoter. This is a milestone in our experimental process, indicating that we successfully achieved specific expression of sweet proteins in tomato fruits.
Using thaumatin as an example, we analyzed the efficacy of protein expression from the E8 promoter. We determined the concentration of thaumatin expressed with the 35S promoter and the E8 promoter in transgenic tomatoes by immunoenzymatic assay (ELISA). By comparison, we obtained that the average level of thaumatin expressed under the induction of the constitutive promoter 35S was 11.1598 mg/L; While the expression level using the fruit ripening-specific promoter was 11.0591mg/L. Statistically, there was no significant difference, suggesting that the expression level of the fruit-specific promoter E8 had already reached the level of expression of the strong promoter 35S ( Fig 10). Compared with 35S, the E8 promoter not only ensured fruit-specific expression and avoided burdening other parts of the tomato plant, but also was able to ensure the high expression level of thaumatin.
Application Prospects
Based on the above results, it can be concluded that our designed E8 promoter has shown excellent specific expressions and we can ensure that thaumatin and brazzein are efficiently expressed only in fruits, and in the future, there is an opportunity to industrialize this production model.
References
[1]An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin, Original Paper, Published: 11 January 2013.
[2]Jill Deikman, Randy Kline, Robert L. Fischer, Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum) , Plant Physiology, Volume 100, Issue 4, December 1992.
[3]M. L. Kneissl, J. Deikman, The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers, Plant Physiology, Volume 112, Issue 2, October 1996.
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