Enhanced resistance to soybean cyst nematode in transgenic soybean via host-induced silencing of vital Heterodera glycines genes.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most economically damaging pathogen affecting soybean production worldwide. Host-induced gene silencing provides a promising approach to confer resistance to plant parasitic nematodes. In the present study, we produced stable transgenic soybean plants individually harboring the inverted repeats of three essential H. glycines genes, Hg-rps23, Hg-snb1, and Hg-cpn1, and evaluated their resistance to SCN infection. Molecular characterization confirmed the stable integration of the hairpin double stranded (ds) RNA in host plants. Inoculation assays with SCN race 3 showed significant reduction of female index (FI, 11.84 ~ 17.47%) on the roots of T4 transgenic plants, with 73.29 ~ 81.90% reduction for the three RNA interference (RNAi) constructs, compared to non-transformed plants (NT, 65.43%). Enhanced resistance to SCN race 3 was further confirmed in subsequent generations (T5) of transgenic soybean. Moreover, when inoculated with SCN race 4 which was considered highly virulent to most of soybean germplasms and varieties, transgenic soybean plants also exhibited reduced FIs (9.96 ~ 23.67%) and increased resistance, relative to the NT plants (46.46%). Consistently, significant down-regulation in transcript levels of the Hg-rps23, Hg-snb1, Hg-cpn1 genes were observed in the nematodes feeding on the transgenic roots, suggesting a broad-spectrum resistance mediated by the host-mediated silencing of vital H. glycines genes. There were no significant differences in morphological traits between transgenic and NT soybean plants under conditions with negligible SCN infection. In summary, our results demonstrate the effectiveness of host-induced silencing of essential H. glycines genes to enhance broad-spectrum SCN resistance in stable transgenic soybean plants, without negative consequences on the agronomic performance.

Efficient identification of genomic insertions and flanking regions through whole-genome sequencing in three transgenic soybean events.

Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in the development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported three transgenic soybean events harboring the inverted repeats of the soybean mosaic virus NIb (nuclear inclusion b) gene, exhibiting significantly enhanced resistance to multiple Potyvirus strains. To facilitate safety assessment and event-specific detection, we identified the transgene insertion sites and flanking sequences of the events L120, L122, and L123 using whole-genome sequencing. More than 14.48 Gb sequence data (13 × coverage) were generated using the Illumina HiSeq Xten platform for each event. The sequence reads corresponding to boundaries of inserted T-DNA, and associated native flanking sequences were identified by bioinformatic comparison with the soybean reference genome (Wm82.a2.v1) and the transformation vector sequence. The results indicated that two T-DNA insertions occurred in L120, on Chr07 and Chr13, while L122 and L123 showed single insertions, on Chr02 and Chr06, respectively. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established using specific PCR primers, and PCR amplification followed by sequencing of PCR products further confirmed the putative insertion loci and flanking regions in the transgenic lines. Our results demonstrate the efficacy and robustness of whole-genome sequencing in identifying the genomic insertions and flanking regions in GM crops. Moreover, the characterization of insertion loci and the establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.

Elastin-like polypeptide and γ-zein fusions significantly increase recombinant protein accumulation in soybean seeds.

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of ß-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.

Enhanced tolerance to Phytophthora root and stem rot by over-expression of the plant antimicrobial peptide CaAMP1 gene in soybean.

BACKGROUND: Antimicrobial peptides play important roles in both plant and animal defense systems. Moreover, over-expression of CaAMP1 (Capsicum annuum antimicrobial protein 1), an antimicrobial protein gene isolated from C. annuum leaves infected with Xanthomonas campestris pv. vesicatoria, confers broad-spectrum resistance to hemibiotrophic bacterial and necrotrophic fungal pathogens in Arabidopsis. Phytophthora root and stem rot (PRR), caused by the fungus Phytophthora sojae, is one of the most devastating diseases affecting soybean (Glycine max) production worldwide. RESULTS: In this study, CaAMP1 was transformed into soybean by Agrobacterium-mediated genetic transformation. Integration of the foreign gene in the genome of transgenic soybean plants and its expression at the translation level were verified by Southern and western blot analyses, respectively. CaAMP1 over-expression (CaAMP1-OX) lines inoculated with P. sojae race 1 exhibited enhanced and stable PRR tolerance through T2-T4 generations compared with the wild-type Williams 82 plants. Gene expression analyses in the transgenic plants revealed that the expression of salicylic acid-dependent, jasmonic acid-dependent, and plant disease resistance genes (R-genes) were significantly up-regulated after P. sojae inoculation. CONCLUSIONS: These results indicate that CaAMP1 over-expression can significantly enhance PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways. This provides an alternative approach for developing soybean varieties with improved tolerance against soil-borne pathogenic PRR.

Overexpression of the chitinase gene CmCH1 from Coniothyrium minitans renders enhanced resistance to Sclerotinia sclerotiorum in soybean.

Pathogenic fungi represent one of the major biotic stresses for soybean production across the world. Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen that is responsible for significant yield losses in soybean. In this study, the chitinase gene CmCH1, from the mycoparasitic fungus Coniothyrium minitans, which infects a range of ascomycetous sclerotia, including S. sclerotiorum and S. minor, was introduced into soybean. Transgenic plants expressing CmCH1 showed higher resistance to S. sclerotiorum infection, with significantly reduced lesion sizes in both detached stem and leaf assays, compared to the non-transformed control. Increased hydrogen peroxide content and activities of defense-responsive enzymes, such as peroxidase, superoxide dismutase, phenylalanine ammonia lyase, and polyphenoloxidase were also observed at the infection sites in the transgenic plants inoculated with S. sclerotiorum. Consistent with the role of chitinases in inducing downstream defense responses by the release of elicitors, several defense-related genes, such as GmNPR2, GmSGT-1, GmRAR1, GmPR1, GmPR3, GmPR12, GmPAL, GmAOS, GmPPO, were also significantly upregulated in the CmCH1-expressing soybean after inoculation. Collectively, our results demonstrate that overexpression of CmCH1 led to increased accumulation of H2O2 and up-regulation of defense-related genes and enzymes, and thus enhanced resistance to S. sclerotiorum infection while showing no detrimental effects on growth and development of soybean plants.

Introduction of the harpinXooc-encoding gene hrf2 in soybean enhances resistance against the oomycete pathogen Phytophthora sojae.

Phytophthora root and stem rot (PRR) caused by an oomycete pathogen Phytophthora sojae is one of the most devastating and widespread diseases throughout soybean-producing regions worldwide. The diversity and variability of P. sojae races make effective control of the pathogen challenging. Here, we introduced an elicitor of plant defense response, the harpinXooc-encoding hrf2 gene from the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola into soybean and evaluated resistance to P. sojae infection. Molecular analysis confirmed the integration and expression of hrf2 in the transgenic soybean. After inoculation with P. sojae, non-transformed control (NC) plants exhibited typical PRR symptoms, including necrotic and wilting leaves, and plant death, whereas most of the transgenic plants showed slightly chlorotic leaves and developed normally. Through T3 to T5 generations, the transgenic events displayed milder disease symptoms and had higher survival rates compared to NC plants, indicating enhanced and stable resistance to P. sojae infection, whereas without P. sojae inoculation, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants. Moreover, after inoculation with P. sojae, significant upregulation of a set of plant defense-related genes, including salicylic acid- and jasmonic acid-dependent and hypersensitive response-related genes was observed in the transgenic plants. Our results indicate that hrf2 expression in transgenic soybean significantly enhanced resistance to P. sojae by eliciting multiple defense responses mediated by different signaling pathways. The potential functional role of the hrf2 gene in plant defense against P. sojae and other pathogens makes it a promising tool for broadening disease resistance in soybean.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1.

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the ß-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

Over-expression of the Pseudomonas syringae harpin-encoding gene hrpZm confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean.

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T2-T4 generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.

RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean.

KEY MESSAGE: Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302 bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

Morpho-Agronomic and Biochemical Characterization of Accessions of Tiger Nut (Cyperus esculentus) Grown in the North Temperate Zone of China.

Tiger nut (Cyperus esculentus L.) has recently attracted increasing interest from scientific and technological communities because of its potential for serving as additional source of food, oil, and feed. The present study reports morphology and biochemical characterization of 42 tiger nut accessions collected from China and other counties performed in the 2020 and 2021 growing seasons at Nongan, Jilin Province. Assessment of variability of 14 agronomic traits including plant height, maturation, leaf width, tilling number, color, size, and shape: 100-tuber weight showed a wide range of phenotypic variation. The color, size, and shape and maturation of the tubers, as well as the leaf width, were the most distinct characteristics describing variation among the accessions. Compositional analyses of major nutritional components of the tubers reveals that this crop could be a source of high-value proteins, fatty acids, and carbohydrates. Specifically, tiger nut tubers contained high levels of starch, oil, and sugars, and significant amounts of fiber, Ca, P, and Na. Furthermore, the tubers appeared to be a good source of proteins as they contain 16 amino acids, including the essential ones. Amino acid profiles were dominated by aspartic acid followed by glutamic acid, leucine, alanine, and arginine. Overall, these results demonstrated that tiger nut is well adapted to the temperature and light conditions in the north temperate zone of China, even with a shorter growth season. The tiger nut accessions collected here exhibited wide variations for agronomical and biochemical traits, suggesting potential for potential for breeding improvement by maximizing the fresh tuber and grass yield based on the optimal selection of genetic characteristics in climate and soil conditions of northern China.

Complete mitochondrial genome sequence and comparative analysis of the cultivated yellow nutsedge.

As a monocotyledonous plant in family Cyperaceae, yellow nutsedge (Cyperus esculentus L.) is unique in accumulating a substantial amount of oil in underground tubers and provides a model system for studying oil accumulation in nonseed tissues. However, no data on the mitochondrial and nuclear genome sequences of this species are available, which greatly limits our understanding of its evolutionary characteristics and some essential biological mechanisms. In the present study, we report the first complete mitochondrial genome sequence of the cultivated yellow nutsedge. The analysis of the genome showed that the yellow nutsedge mitochondrial genome is 1,002,696 bp in size and encodes 62 genes consisting of 36 protein-coding genes (PCGs), 20 transfer RNA (tRNA) genes, and six ribosomal RNA (rRNA) genes. Compared with other angiosperms, yellow nutsedge mitochondrial genome contains much higher percentage of noncoding sequences (95.36%). Sixteen plastid-derived fragments were identified to be strongly associated with mitochondrial genes including one intact plastid-related gene (ndhH). Comparative analysis with seven other sequenced plant mitochondrial genomes revealed that two syntenic gene clusters, rps3-rpl16 and rps12-nad3, are highly conserved in all plant mitochondrial genomes, and the mitochondrial genome of yellow nutsedge is more similar to those of monocotyledons in the gene order. Phylogenetic analysis based on 13 shared protein-encoding genes in eight plant species showed that yellow nutsedge is evolutionarily more closely related to monocotyledonary species. Overall, the species-specific features of the cultivated yellow nutsedge mitochondrial genome provide additional information for the evolutionary and comparative genomic studies in the yellow nutsedge and other Cyperus species of the Cyperaceae family.

Genome-wide analysis and expression profiling under heat and drought treatments of HSP70 gene family in soybean (Glycine max L.).

Heat shock proteins (HSPs) perform a fundamental role in protecting plants against abiotic stresses. Previous studies have made great efforts in the functional analysis of individual family members, but there has not yet been an overall analysis or expression profiling of the HSP70 gene family in soybeans (Glycine max L.). In this study, an investigation of the soybean genome revealed 61 putative HSP70 genes, which were evaluated. These genes were classified into eight sub-families, denoted I-VIII, based on a phylogenetic analysis. In each sub-family, the constituent parts of the gene structure and motif were relatively conserved. These GmHSP70 genes were distributed unequally on 17 of the 20 chromosomes. The analysis of the expression profiles showed that 53 of the 61 GmHSP70 genes were differentially expressed across the 14 tissues. However, most of the GmHSP70s were differentially expressed in a tissue-specific expression pattern. Furthermore, the expression of some of the duplicate genes was partially redundant, while others showed functional diversity. The quantitative real-time PCR (qRT-PCR) analysis of the 61 soybean HSP70 genes confirmed their stress-inducible expression patterns under both drought and heat stress. These findings provide a thorough overview of the evolution and modification of the GmHSP70 gene family, which will help to determine the functional characteristics of the HSP70 genes in soybean growth and development.

[High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts].

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

Changes in oleic Acid content of transgenic soybeans by antisense RNA mediated posttranscriptional gene silencing.

The Delta-12 oleate desaturase gene (FAD2-1), which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71%) and a reduction in palmitic acid (to <3%) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.