Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.     

Construction and Functional Analysis of CRISPR/Cas9 Vector of <i>FAD2</i> Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil. In the synthesis pathway of soybean fatty acids, the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid. In this study, CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression. Firstly, the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed. Then, the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation, and the mutant plants were obtained. Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out. The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties. The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Silencing of Gm<Emphasis Type="Italic">FAD3</Emphasis> gene by siRNA leads to low α-linolenic acids (18:3) of <Emphasis Type="Italic">fad3</Emphasis>-mutant phenotype in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (Merr.)]

RNA interference (RNAi) has been recently employed as an effective experimental tool for both basic and applied biological studies in various organisms including plants. RNAi deploys small RNAs, mainly small interfering RNAs (siRNAs), to mediate the degradation of mRNA for regulating gene expression in plants. Here we report an efficient siRNA-mediated gene silencing of the omega-3 fatty acid desaturase (FAD3) gene family in a complex genome, the soybean (Glycine max). The FAD3 enzyme is responsible for the synthesis of alpha-linolenic acids (18:3) in the polyunsaturated fatty acid pathway. It is this fatty acid that contributes mostly to the instability of soybean and other seed oils. Therefore, a significant reduction of this fatty acid will increase the stability of the seed oil, enhancing the seed agronomical value. A conserved nucleotide sequence, 318-nt in length, common to the three gene family members was used as an inverted repeat for RNA interference. The RNAi expression cassette was driven by a seed-specific promoter. We show that the transgene-produced siRNA caused silencing of FAD3 that was comparable to the fad3 mutant phenotype and, furthermore, that such a silencing is stably inherited in engineered soybean lines. Since the pool size of the alpha-linolenic acids is small relative to the other polyunsaturated fatty acids in soybean, the significant reduction of this fatty acid suggests a role and great potential for the siRNA strategy in silencing gene families in a complex genome.     

Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with β‐oxidation of α‐linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value‐added soybeans.     

大豆ω-3脂肪酸脱氢酶基因GmFAD3C在酿酒酵母中的表达

α亚麻酸(ALA)被称为必需脂肪酸,对人体有一系列的保健作用。ω-3脂肪酸脱氢酶(FAD)催化亚油酸(LA)生成ALA。大豆种子油中ALA含量较高,为了研究大豆ω3FAD的功能,用RTPCR方法从大豆未成熟种子中扩增出GmFAD3C的cDNA,克隆到酵母表达载体p416中,并用醋酸锂法转化酿酒酵母营养缺陷型K601,经筛选鉴定,得到阳性克隆。气相色谱分析脂肪酸成分,发现工程菌产生了新的脂肪成分ALA,含量占总脂肪酸的3.1%,LA含量与对照相比相应地下降,证明该基因编码的蛋白具有催化18碳多不饱和脂肪酸(PUFA)底物LA在Δ15位脱氢生成ALA的ω3FAD功能,首次实现大豆ω-3脂肪酸脱氢酶基因在酿酒酵母K601p416系统中的表达,建立了一种新的高效低成本的FAD酵母表达系统。     

cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids

Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.     

Characterization of a new <Emphasis Type="Italic">GmFAD3A</Emphasis> allele in Brazilian CS303TNKCA soybean cultivar

Key Message

We molecularly characterized a new mutation in the GmFAD3A gene associated with low linolenic content in the Brazilian soybean cultivar CS303TNKCA and developed a molecular marker to select this mutation.

Abstract

Soybean is one of the most important crops cultivated worldwide. Soybean oil has 13% palmitic acid, 4% stearic acid, 20% oleic acid, 55% linoleic acid and 8% linolenic acid. Breeding programs are developing varieties with high oleic and low polyunsaturated fatty acids (linoleic and linolenic) to improve the oil oxidative stability and make the varieties more attractive for the soy industry. The main goal of this study was to characterize the low linoleic acid trait in CS303TNKCA cultivar. We sequenced CS303TNKCA GmFAD3A, GmFAD3B and GmFAD3C genes and identified an adenine point deletion in the GmFAD3A exon 5 (delA). This alteration creates a premature stop codon, leading to a truncated protein with just 207 residues that result in a non-functional enzyme. Analysis of enzymatic activity by heterologous expression in yeast support delA as the cause of low linolenic acid content in CS303TNKCA. Thus, we developed a TaqMan genotyping assay to associate delA with low linolenic acid content in segregating populations. Lines homozygous for delA had a linolenic acid content of 3.3 to 4.4%, and the variation at this locus accounted for 50.83 to 73.70% of the phenotypic variation. This molecular marker is a new tool to introgress the low linolenic acid trait into elite soybean cultivars and can be used to combine with high oleic trait markers to produce soybean with enhanced economic value. The advantage of using CS303TNKCA compared to other lines available in the literature is that this cultivar has good agronomic characteristics and is adapted to Brazilian conditions.

     

Effects of dietary supplementation with sea buckthorn (Hippophaë rhamnoides) seed and pulp oils on atopic dermatitis

A placebo-controlled, double-blind study was conducted to investigate the effects of seed and pulp oils of sea buckthorn (Hipphophae rhamnoides) on atopic dermatitis. Linoleic (34%), alpha-linolenic (25%), and oleic (19%) acids were the major fatty acids in the seed oil, whereas palmitic (33%), oleic (26%), and palmitoleic (25%) acids were the major fatty acids in the pulp oil. The study group included 49 atopic dermatitis patients who took 5 g (10 capsules) of seed oil, pulp oil, or paraffin oil daily for 4 months. During follow-up dermatitis improved significantly in the pulp oil (P < 0.01) and paraffin oil (P < 0.001) groups, but improvement in the seed oil group was not significant (P = 0.11). Supplementation of seed oil increased the proportion of alpha-linolenic acid in plasma neutral lipids (P < 0.01), and increases of linoleic, alpha-linolenic, and eicosapentaenoic acids in plasma phospholipids were close to significant (0.05 < P < 0.1). Pulp oil treatment increased the proportion of palmitoleic acid (P < 0.05) and lowered the percentage of pentadecanoic acid (P < 0.01) in both plasma phospholipids and neutral lipids. In the seed oil group, after 1 month of supplementation, positive correlations were found between symptom improvement and the increase in proportions of alpha-linolenic acid in plasma phospholipids (Rs = 0.84; P = 0.001) and neutral lipids (Rs = 0.68; P = 0.02). No changes in the levels of triacylglycerols, serum total, or specific immunoglobulin E were detected. In the pulp oil group, a significant (P < 0.05) increase in the level of high density lipoprotein cholesterol, from 1.38 to 1.53 mmol/L was observed.     

Heterologous expression of two <Emphasis Type="Italic">Glycine max</Emphasis> ω-3 fatty acid desaturases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

α-Linolenic acid (ALA, C 18:3^Δ9,12,15) has many important biological functions. ω-3 Fatty acid desaturase (FAD3), existing in cytosolic and plastidic compartments of higher plants, catalyzes linoleic acid (LA) desaturation to produce ALA. GmFAD3A-2 and GmFAD3C genes encoding cytosolic FAD3 from Qihuang 29 soybean were cloned and inserted into p416 vector and expressed in K601 yeast strain. Gas chromatography showed that the transformed yeast strains could produce ALA. The ALA accumulation levels for the strains transformed with GmFAD3A-2 or GmFAD3C genes were 0.77 ± 0.1 and 4.13 ± 0.4% of total fatty acids, respectively, while, as compared with that of the control, the contents of LA decreased from 14.34 ± 0.8 to 10.93 ± 0.0 and 7.85 ± 0.1%, respectively, implying that the GmFAD3C enzyme is more vigorous or stable, than GmFAD3A-2. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 629–634. This text was submitted by the authors in English.     

Cloning of fatty acid desaturase-coding sequence (<Emphasis Type="Italic">Lufad3</Emphasis>) from flax and its functional validation in rice

Flax contains very high levels of alpha-linolenic acid (57%) and a fatty acid desaturase 3-coding sequence (Lufad3) of flax has been amplified from the RNA isolated from developing seeds. The deduced amino acid sequence of LuFAD3 showed the presence of three histidine motifs, six membrane spanning domains and an endoplasmic reticulum (ER) retention signal KSK, indicating its plausible localization into the ER. Flax is not amenable for genetic transformation and not suitable for functional validation of Lufad3 gene. Hence, rice with well-developed genetic transformation has been selected as heterologous host system. Coding sequence of Lufad3 driven by maize Ubi1 promoter has been introduced into indica rice by Agrobacterium tumefaciens-mediated genetic transformation. Southern analysis of putative transformants (T ₀) revealed signals at variable lengths at >5 kbp indicating random integration of transgene into the genomes of different transformants. The Mendelian segregation observed for selectable marker gene hyg in both T₁ and T₂ generations confirmed stable inheritance and single-site integration of transgenes. As compared to untransformed control (UC), homozygous transgenic rice expressing Lufad3 showed higher levels of essential α-linolenic acid in leaves and seeds validating its functionality.     

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.     

食物中多不饱和脂肪酸在家蝇幼虫体内的富集与代谢及对其生长的影响

【目的】阐明家蝇 Musca domestica 幼虫对食物中各种多不饱和脂肪酸的富集能力以及代谢转化情况,并探究各种多不饱和脂肪酸对家蝇幼虫生长的影响。【方法】在基础饲料中添加不同浓度(3%, 6%和12%)的多不饱和脂肪酸(亚油酸、α-亚麻酸、花生四烯酸和二十二碳六烯酸)饲养经过脱脂传代培养的家蝇幼虫;提取家蝇幼虫的总脂肪酸,利用气相色谱仪进行检测和分析;测定统计幼虫体重,以分析多不饱和脂肪酸对家蝇幼虫生长的影响。【结果】亚油酸、α-亚麻酸和花生四烯酸在家蝇幼虫体内均能被富集,且它们的富集程度随着食物中多不饱和脂肪酸的添加浓度的升高而增加,其中亚油酸、α-亚麻酸和花生四烯酸在幼虫体内富集的最高含量(占体内总脂肪酸的比例)分别为21.93%, 16.13%和9.68%,而二十二碳六烯酸不能在家蝇幼虫体内富集,提示家蝇幼虫食物中添加的各种多不饱和脂肪酸经过代谢后并没有在其体内产生新的脂肪酸,而食物中添加的二十二碳六烯酸在家蝇幼虫体内被分解代谢后消除。饲喂α-亚麻酸及花生四烯酸后家蝇幼虫体重增长较为明显,其中6%α-亚麻酸添加组的幼虫体重显著高于对照组(取食脱脂饲料)和3%和12%α-亚麻酸添加组,3%和6%花生四烯酸添加组的幼虫体重显著高于对照组和12%花生四烯酸添加组。【结论】家蝇幼虫体内能够从食物中富集部分多不饱和脂肪酸,多不饱和脂肪酸碳链越长其富集程度越低直至不能富集,富集的多不饱和脂肪酸对家蝇幼虫生长有不同程度的影响。