Xinzheng Li

Superfamily of ankyrin repeat proteins in tomato.

The ankyrin repeat (ANK) protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, no detailed information concerning this family is available for tomato (Solanum lycopersicum) due to the limited information on whole genome sequences. In this study, we identified a total of 130 ANK genes in tomato genome (SlANK), and these genes were distributed across all 12 chromosomes at various densities. And chromosomal localizations of SlANK genes indicated 25 SlANK genes were involved in tandem duplications. Based on their domain composition, all of the SlANK proteins were grouped into 13 subgroups. A combined phylogenetic tree was constructed with the aligned SlANK protein sequences. This tree revealed that the SlANK proteins comprise five major groups. An analysis of the expression profiles of SlANK genes in tomato in different tissues and in response to stresses showed that the SlANK proteins play roles in plant growth, development and stress responses. To our knowledge, this is the first report of a genome-wide analysis of the tomato ANK gene family. This study provides valuable information regarding the classification and putative functions of SlANK genes in tomato.

Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans

Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ5, 8, 11, 14) and eicosapentaenoic acid (EPA; 20:5Δ5, 8, 11, 14, 17) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

Creation and validation of a widely applicable multiple gene transfer vector system for stable transformation in plant

Multiple gene transfer (MGT) technology has become a powerful tool for basic and applied plant biology research in recent years. Despite some notable successes in obtaining plant lines harbouring multiple transgenes, these methods are still generally unwieldy and costly. We report here a straightforward and cost effective strategy, utilizing commonly available restriction enzymes for the transfer of multiple genes into plants, hence greatly widening the accessibility of MGT. This methodology exploits the specific ‘nested’ arrangement of a pair of isocaudomer restriction enzymes (for example XbaI—AvrII–XbaI) so that through the alternate use of these two enzymes in a reiterative fashion multiple genes/constructs (up to five in this study) could be ‘stacked’ together with ease. In a proof-of-concept experiment, we constructed a plant transformation vector containing three reporter gene expression cassettes flanked by two matrix attachment region sequences. The expression of all three genes was confirmed in transgenic Arabidopsis thaliana. The usefulness of this technology was further validated by the construction of a plant transformation vector containing five transgenes for the production of eicosapentaenoic acid (EPA, C20∆5,8,11,14,17), a polyunsaturated essential fatty acid found in fish oils that is beneficial for health. In addition, we constructed four more vectors, incorporating one seed specific and three promoters conferring constitutive expression. These expression cassettes are flanked by a different isocaudomer pair (AvrII—SpeI–AvrII) and four other unique restriction sites, allowing the exchange of promoters and terminators of choice.

Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana

IgASE1, a C18 Δ9-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and α-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the Δ8 desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants. The transgenic Arabidopsis were hypersensitive to abscisic acid (ABA) during seed germination, post-germination growth, and seedling development. They had elevated leaf ABA levels under well-watered and dehydrated conditions and their stomata were more sensitive to ABA. Exogenous application of eicosadienoic acid and eicosatrienoic acid can mimic ABA and drought responses in the wild type plants, similar to that found in the transgenic ones. The transcript levels of genes involved in the biosynthesis of ABA (NCED3, ABA1, AAO3) as well as other stress-related genes were upregulated in this transgenic line upon osmotic stress (300mM mannitol). Taken together, these results indicate that these two eicosapolyenoic acids or their derived metabolites can mitigate the effects of drought in transgenic Arabidopsis, at least in part, through the action of ABA.

Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton

We have isolated four genes encoding a Δ9 elongase,a Δ8 desaturase,a Δ5 desaturase,and a Δ15 desaturase from Isochrysis galbana,Euglena gracilis,Mortierella alpina,and Arabidopsis thaliana respectively.Using a multigene transfer tech-nology that we developed,these genes were stacked together in the plant expression vector pCambia2300.Each gene contained its own CaMV35S promoter and Tnos terminator.This plant expression vector was then transferred into cotton by Agrobacte-rium-mediated transformation method.Transgenic cotton seedlings were first identified by screening them based on kanamycin-containing media and followed by PCR with gene-specific primers of the four transgenes.Finally,these transgenic plants were subjected to gas liquid chromatography analysis for their fatty acid composition and the results showed that the contents of arachidonic acid(ARA,20:4Δ5,8,11,14) and eicosapentaenoic acid(EPA,20:5Δ5,8,11,14,17) were 1.0% and 5.0% respectively in the leaves of the transgenic plants,indicating that the four genes were expressed in cotton.Therefore,our data clearly demonstrated the feasibility for the heterologous production of EPA in cotton and this will lay a foundation for the production of VLCPUFAs,including EPA and DHA in cotton seed through transgenic technology in the future.

Production of eicosapentaenoic acid (EPA, 20:5n-3) in maize (Zea mays L.) through the alternative ∆8 desaturation pathway mediated by particle bombardment

Very long chain polyunsaturated fatty acids (VLCPUFAs), especially eicosapentaenoic acid (EPA, 20:5∆5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6∆4,7,10,13,16,19) have demonstrated important roles in a number of aspects of human health. Currently, our primary dietary sources for these fatty acids are from marine fish. Producing VLCPUFAs in oilseed crop by metabolic engineering was considered to provide an alternative, sustainable sources. Here, three heterologous genes, ∆9 elongase (∆9-Elo) of Isochrysis galbana, ∆8 desaturase (∆8-Des) of Euglena gracilis, ∆5 desaturase (∆5-Des) of Mortierella alpina, were co-transformed into maize inbred line Qi319 using a particle bombardment transformation method to produce EPA through the alternative ∆8 desaturation synthetic pathway. A total of 144 herbicide resistant lines were obtained with an average transformation efficiency of 7.95%, of which 98 lines contain the Bar genes with the positive transformation efficiency of 4.74%. In addition, 60 of the 98 positive transgenic lines were identified to contain all three transgenes, ∆9-Elo, ∆8-Des, and ∆5-Des. The fatty acid composition of the leaves from the 60 transgenic lines were subjected to gas liquid chromatography analysis and the results showed that the amounts of EPA reached 1.99% in an individual leaf. These data demonstrate the feasibility for the heterologous production of EPA in maize and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA, in maize by metabolic engineering in the future.

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.