Seed-Specific Expression of Apolipoprotein A-IMilano Dimer in Engineered Rice Lines

Apolipoprotein A-IMilano(ApoA-IM)has been shown to significantly reduce coronary atherosclerotic plaques.However,the preparation of cost-effective pharmaceutical formulations of ApoA-IM is limited by the high cost and difficulty of purifying the protein and producing the highly effective dimeric form.The aim of this study was to create an expression cassette that specifically drives the expression of dimeric ApoA-IM in the protein bodies of rice seeds.The ApoA-IM protein under control of the 13 kDa prolamin promoter is expressed exclusively in its dimeric form within the seeds,and immunocytochemical and immunogold analyses confirmed its expression in different caryopsis tissue such as seed coat,aleurone cell and endosperm,particularly in amyloplast and storage vacuoles.A plant-based ApoA-IM production system offered numerous advantages over current production systems,including the direct production of the most therapeutically effective dimeric ApoA-IM forms,long-term protein storage in seeds,and ease of protein production by simply growing plants.Therefore,seeds had the potential to serve as a costeffective source of therapeutic ApoA-IM.

Isolation and Structural Analysis of the Seed-Specific Promoter from Soybean

The promoter region (BCSP666) of b-conglycinin a-subunit gene from the genomic DNA of soybean Jilin 43 was isolatedby PCR method. Sequencing analysis showed that the cloned fragment BCSP666 had the similar structure to the soybeanseed-specific promoter b-conglycinin a'-subunit gene promoter and b-conglycinin b-subunit gene promoter, and it alsocontains many motifs that contribute to the seed-specific promoter activity. Based on this sequencing analysis, wededuced that promoter fragment BCSP666 had the seed-sepecific promoter activity. And then we constructed the seed-specific expression vector pBMI666 with the promoter fragment BCSP666 and D6-fatty acid desaturase gene fromMortierella isabellina. The D6-fatty acid desaturase is the rate-limiting enzyme of the desaturation of linoleic acid in theproduction of a human essential fatty acid, g-linolenic acid(GLA). The production of g-linolenic acid(GLA) was observedin soybean callus cells, which were transformed with this vector. This confirmed the activity of the activity fragmentBCSP666.

Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenic Brassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes

The seed-specific promoter and transit peptide were amplified and fused to the three genes phbA, phbB and phbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containing phbC and phbB, and pSCAB containing phbC, phbB and phbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. Transgenic Brassica napus H165 were obtained by Agrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.

Cloning and functional analysis of the promoter of allergen gene Ara h 1 from peanut

Peanut seeds are ideal bioreactors for the production of foreign recombinant proteins or secondary metabolites.Seed-specific promoters(SSPs)can direct the expression of genes specifically in seeds to avoid undesirable effects associated with constitutive expression.However,few SSPs have been identified in peanut.Previous studies have shown that some allergen-encoding genes encode seed storage proteins or exhibit seed-specific/preferential expression.In this study,we characterized allergen-encoding genes from across the genomes of Arachis species to explore seed-specific genes.We found that at least 9 out of 16 identified peanut allergen-encoding genes were expressed specifically in the seeds or were preferentially expressed.A 1493-bp promoter fragment of allergen gene Ara h 1(we named it AHSSP6)was isolated from cultivated peanut genome.cis-element analysis showed that three RY repeat elements which usually exsisted in seed or embryo specific promoter sequence were also present in AHSSP6 sequence.Histochemical analysis showed AHSSP6 could drive the expression of aβ-glucuronidase(GUS)reporter gene specifically in the seeds or cotyledon tissue of transgenic Arabidopsis,while not in other tissues.These findings indicated that these promoters of allergen genes were candidate SSPs,and AHSSP6 was a novel SSP which could be potentially utilized in peanut improvement.

Efficient LEC2 activation of OLEOSIN expression requires two neighboring RY elements on its promoter

As the main structural protein of oil body,OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here,we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN,responsible for seed specificity and LEC2 activation,was determined by 5'-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region. In transient expression assays,mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity,while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore,we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.