We have investigated the use of the soybean seed coat as a bioreactor for the production of valuable proteins. In this system heterologous proteins are targeted to the hourglass cells. While seed coats currently have little commercial value, the introduction of high valued bio-products may generate new markets for Canadian industries and contribute towards the growth of rural economies.
The seed coat system is based upon a promoter responsible for the production of soybean peroxidase (SBP) in the hourglass cell layer of the seed coat. SBP protein is thought to be synthesized as a pre-protein containing N- and C-terminal propeptides responsible for the intracellular localization of the mature SBP to the vacuole. We used this promoter and these signals to direct the synthesis and targeting of horseradish peroxidase (HRPC) from Armoracia rusticana and two laccases (LCC1 and LCC4) from the fungus Trametes versicolor. The cDNA sequences were codon-optimized for soybean, and fused with the SBP N- and C-terminal propeptides. A HIS tag was added to the C-terminal end of the mature protein to facilitate purification. Expression of the chimeric genes was driven by the Ep 1.5 kb fragment cloned from soybean peroxidase promoter.
Fourteen lines of HRPC transgenics, 16 lines of LCC1 transgenics and 40 lines of LCC4 transgenics were generated by particle bombardment. The T 1 and T2 seeds of the transgenics were screened for peroxidase and laccase activity with the highest expressing lines further analysed by PCR and Western blots. The peroxidase activities measured in HRPC transgenics ranged from 0.02 to 3.09 U/mg protein while the laccase activities measured in laccase transgenics varied from 0 to 0.099 U/mg protein. In general these values are low compared to activities measured in the native species or in other transgenics. We have found that expression of HRPC and laccase as a trait is inheritable from T0 to T2 generation. The anti-LCC4, anti-HRPC antibodies were used to detect the expression of LCC4 and HRPC by Western blotting. A 35.4 kDa protein reactive with anti-HRPC and anti-HIS tag antibodies was found in the seed coat of an HRPC transgenic while a 77.6 kDa protein reactive with anti-LCC4 and anti-HIS tag antibodies was found in the seed coat of an LCC4 transgenic.
These results support the use of the soybean seed coat as a bioreactor for the production of foreign proteins. The factors which affect foreign protein expression are fully discussed and future directions have been proposed to optimize the system.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29764 |
| Date | January 2009 |
| Creators | Han, Shuyou |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 167 p. |
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