Cloning and Function Analysis of ω-3-Fatty Acid Desaturase 8 Gene from Perilla frutescens / 中国生物化学与分子生物学报

ω-3-Fatty acid desaturase 8 (FAD8), as a dehydrogenase enzyme, plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids, which is helpful to enhance the freezing tolerance of plants. However, it remains unclear whether the expression level of FAD8 in Perilla frutescens is regulated by low temperature. Based on transcriptome data, the FAD8 gene was cloned, characterized and then successfully expressed in tobacco Nicotiana tabacum. The gene was designated as PfFAD8 and has a full-length coding sequence of 1 317 bp coding for 438 amino acids with a predicted molecular weight of 50 kD and a theoretical isoelectric point of 9. 13. Our research indicated that the expression of PfFAD8 in Perilla frutescens was increased under the freezing stress. To further confirm this result, a 35S::PfFAD8 vector were constructed and transformed into N. tabacum by Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco leaves that over-expressed the PfFAD8 gene exhibited significantly higher unsaturated fatty acids (UFA) such as linoleic (C18:2) and palmitic acid (C16:0) content and advanced freezing tolerance. Moreover, PfFAD8 overexpression in transgenic tobacco leaves increases malondialdehyde (MDA) and proline (PRO) content, and enhances defense enzymes activities of superoxide dismutase (SOD) and catalase (CAT) to some extent under the cold condition, which might prevent the decline of UFA. Taken together, PfFAD8 overexpression in Perilla frutescens might be involved in the desaturation process of lipids leading to increased membrane stability and/ or induction of other genes related to freezing tolerance by octadecanoid pathway or lipid peroxidation products. Thus, PfFAD8 overexpression could be useful in the production of freeze-tolerant varieties of N. tabacum.

Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz.

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.

How Machine Learning Methods Helped Find Putative Rye Wax Genes Among GBS Data.

The standard approach to genetic mapping was supplemented by machine learning (ML) to establish the location of the rye gene associated with epicuticular wax formation (glaucous phenotype). Over 180 plants of the biparental F2 population were genotyped with the DArTseq (sequencing-based diversity array technology). A maximum likelihood (MLH) algorithm (JoinMap 5.0) and three ML algorithms: logistic regression (LR), random forest and extreme gradient boosted trees (XGBoost), were used to select markers closely linked to the gene encoding wax layer. The allele conditioning the nonglaucous appearance of plants, derived from the cultivar Karlikovaja Zelenostebelnaja, was mapped at the chromosome 2R, which is the first report on this localization. The DNA sequence of DArT-Silico 3585843, closely linked to wax segregation detected by using ML methods, was indicated as one of the candidates controlling the studied trait. The putative gene encodes the ABCG11 transporter.

Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2-Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2-Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

[Analysis of the peanut transgenic offspring with depressing AhFAD2 gene].

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

Analysis of the peanut transgenic offspring with depressing AhFAD2 gene / 生物工程学报

The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.

OeFAD8, OeLIP and OeOSM expression and activity in cold-acclimation of Olea europaea, a perennial dicot without winter-dormancy.

MAIN CONCLUSION: Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships between OeFAD8, OeOSM , and OeLIP19 and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation. Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.

Variation in seed fatty acid composition and sequence divergence in the FAD2 gene coding region between wild and cultivated sesame.

Sesame germplasm harbors genetic diversity which can be useful for sesame improvement in breeding programs. Seven accessions with different levels of oleic acid were selected from the entire USDA sesame germplasm collection (1232 accessions) and planted for morphological observation and re-examination of fatty acid composition. The coding region of the FAD2 gene for fatty acid desaturase (FAD) in these accessions was also sequenced. Cultivated sesame accessions flowered and matured earlier than the wild species. The cultivated sesame seeds contained a significantly higher percentage of oleic acid (40.4%) than the seeds of the wild species (26.1%). Nucleotide polymorphisms were identified in the FAD2 gene coding region between wild and cultivated species. Some nucleotide polymorphisms led to amino acid changes, one of which was located in the enzyme active site and may contribute to the altered fatty acid composition. Based on the morphology observation, chemical analysis, and sequence analysis, it was determined that two accessions were misnamed and need to be reclassified. The results obtained from this study are useful for sesame improvement in molecular breeding programs.