Hiroshi Asao

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.

Transgenic Lettuce Producing a Candidate Protein for Vaccine against Edema Disease

Pig edema disease is a bacterial disease caused by Shiga toxin 2e-producing Escherichia coli belonging mainly to serotypes O138, O139, and O141. The B subunit of Shiga toxin 2e (Stx2eB) is a candidate protein for use in a vaccine against edema disease. We produced this protein in transgenic lettuce (Lactuca sativa), an edible plant that can be cultivated in a factory setting. In a transient expression system, we found that NtADH 5′-untranslated region (5′-UTR) functions as a translational enhancer in lettuce cells, and that Stx2eB accumulates most efficiently in the endoplasmic reticulum (ER) of lettuce cells. Stx2eB was produced in stable transgenic lettuce plants expressing a modified Stx2eB gene fused with the NtADH 5′-UTR and sequence encoding ER localization signals.

Wound-responsive cis-Element in the 5′-Upstream Region of Cucumber Ascorbate Oxidase Gene

The cucumber (Cucumis sativas) AAO1 gene (former name, Aso1) encodes an ascorbate oxidase that catalyzes the oxidation by molecular oxygen of ascorbic acid to dehydroascorbate. CsAAO1 mRNA concentrations rose rapidly after mechanical wounding of cucumbers. To study the wound-responsive expression of CsAAO1 in detail, we examined transgenic tobacco plants harboring a CsAAO1 promoter-β-glucuronidase fusion gene. CsAAO1 promoter activity in leaves of the tobacco was induced by wounding. Analysis of the regulatory properties of 5′-deleted promoter fragments showed that a putative wound-responsive cis-element (WRE) was located −736 to −707 bp from the translation initiation site. DNA binding factors that bound specifically to the putative WRE sequence were identified in tobacco nuclear extracts by gel retardation assays.

Somatic Hybridization Between Solanum melongena L. (Eggplant) and Solanum sanitwongsei Craib.

Eggplant (Solanum melongena L.) is an economically important nontuberous solanaceous crop in tropical and warm temperate regions. The origin of the eggplant is supposed to be in India. It is cultivated on about 1.31×106 ha worldwide, yielding 21.24 million t in 1999, with China (6.27×105ha, 11.03 million t), India (4.25×105ha, 6.10 million t), Turkey (0.33×105ha, 0.85 million t), Egypt (4.25×105ha, 0.57 milhon t) and Japan (0.15×105ha, 0.49 million t) being the main producers (FAO 1999).