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BackgroundOil in the form of triacylglycerols (TAGs) is quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase (DGAT) is considered the rate-limiting enzyme for TAG accumulation. Chlorella, a unicellular eukaryotic green alga, has attracted much attention as a potential feedstock for renewable energy production. However, the function of DGAT1 in Chlorella has not been reported.ResultsA full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Chlorella ellipsoidea. The 2,142 bp open reading frame of this cDNA, designated CeDGAT1, encodes a protein of 713 amino acids showing no more than 40% identity with DGAT1s of higher plants. Transcript analysis showed that the expression level of CeDGAT1 markedly increased under nitrogen starvation, which led to significant triacylglycerol (TAG) accumulation. CeDGAT1 activity was confirmed in the yeast quadruple mutant strain H1246 by restoring its ability to produce TAG. Upon expression of CeDGAT1, the total fatty acid content in wild-type yeast (INVSc1) increased by 142%, significantly higher than that transformed with DGAT1s from higher plants, including even the oil crop soybean. The over-expression of CeDGAT1 under the NOS promoter in wild-type Arabidopsis thaliana and Brassica napus var. Westar significantly increased the oil content by 8–37% and 12–18% and the average 1,000-seed weight by 9–15% and 6–29%, respectively, but did not alter the fatty acid composition of the seed oil. The net increase in the 1,000-seed total lipid content was up to 25–50% in both transgenic Arabidopsis and B. napus.ConclusionsWe identified a gene encoding DGAT1 in C. ellipsoidea and confirmed that it plays an important role in TAG accumulation. This is the first functional analysis of DGAT1 in Chlorella. This information is important for understanding lipid synthesis and accumulation in Chlorella and for genetic engineering to enhance oil production in microalgae and oil plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-0995-5) contains supplementary material, which is available to authorized users.

BackgroundThe lipid content of microalgae is regarded as an important indicator for biodiesel. Many attempts have been made to increase the lipid content of microalgae through biochemical and genetic engineering. Significant lipid accumulation in microalgae has been achieved using biochemical engineering, such as nitrogen starvation, but the cell growth was severely limited. However, enrichment of lipid content in microalgae by genetic engineering is anticipated. In this study, GmDof4 from soybean (Glycine max), a transcription factor affecting the lipid content in Arabidopsis, was transferred into Chlorella ellipsoidea. We then investigated the molecular mechanism underlying the enhancement of the lipid content of transformed C. ellipsoidea.ResultsWe constructed a plant expression vector, pGmDof4, and transformed GmDof4 into C. ellipsoidea by electroporation. The resulting expression of GmDof4 significantly enhanced the lipid content by 46.4 to 52.9%, but did not affect the growth rate of the host cells under mixotrophic culture conditions. Transcriptome profiles indicated that 1,076 transcripts were differentially regulated: of these, 754 genes were significantly upregulated and 322 genes were significantly downregulated in the transgenic strains under mixotrophic culture conditions. There are 22 significantly regulated genes (|log2 ratio| >1) involved in lipid and fatty acid metabolism. Quantitative real-time PCR and an enzyme activity assay revealed that GmDof4 significantly up-regulated the gene expression and enzyme activity of acetyl-coenzyme A carboxylase, a key enzyme for fatty acid synthesis, in transgenic C. ellipsoidea cells.ConclusionsThe hetero-expression of a transcription factor GmDof4 gene from soybean can significantly increase the lipid content but not affect the growth rate of C. ellipsoidea under mixotrophic culture conditions. The increase in lipid content could be attributed to the large number of genes with regulated expression. In particular, the acetyl-coenzyme A carboxylase gene expression and enzyme activity were significantly upregulated in the transgenic cells. Our research provides a new way to increase the lipid content of microalgae by introducing a specific transcription factor to microalgae strains that can be used for the biofuel and food industries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0128-4) contains supplementary material, which is available to authorized users.

Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5′-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO3. The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.

In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat - Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th . intermedium . Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th . intermedium , 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th . intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a Js genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (Js, J and St) in Th . intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th . bessarabicum . Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the Js genome, within a single chromosome, and among different genomes in Th . intermedium . Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different genomes in polyploid plants.

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR 3 was amplified from the dissected alien chromosome of TAi-27, TcDR 2 and TcDR 3 were from cDNA of Th. intermedium, AcDR 3 was from cDNA of TAi-27, FcDR 2 was from cDNA of 3B-2, AR 2 was from genomic DNA of TAi-27 and TR 2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR 3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR 3 as the probe showed that there is only one copy of ACR 3 in the genome of Th. intermedium and TAi-27, but it is absent in 3B-2. The ACR 3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR 3 was constitutively expressed in Th. intermedium and TAi-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAi-27.

Gamma-linolenic acid (γ-linolenic acid, GLA; C18:3 Δ6, 9, 12) belongs to the omega-6 family and exists primarily in several plant oils, such as evening primrose oil, blackcurrant oil, and borage oil. Δ6-desaturase is a key enzyme involved in the synthesis of GLA. There have been no previous reports on the genes encoding Δ6-desaturase in blackcurrant (Ribes nigrum L.). In this research, five nearly identical copies of Δ6-desaturase gene-like sequences, named RnD8A, RnD8B, RnD6C, RnD6D, and RnD6E, were isolated from blackcurrant. Heterologous expression in Saccharomyces cerevisiae and/or Arabidopsis thaliana confirmed that RnD6C/D/E were Δ6-desaturases that could use both α-linolenic acids (ALA; C18:3 Δ9,12,15) and linoleic acid (LA; C18:2 Δ9,12) precursors in vivo, whereas RnD8A/B were Δ8-sphlingolipid desaturases. Expression of GFP tagged with RnD6C/D/E showed that blackcurrant Δ6-desaturases were located in the mitochondrion (MIT) in yeast and the endoplasmic reticulum (ER) in tobacco. GC-MS results showed that blackcurrant accumulated GLA and octadecatetraenoic acids (OTA; C18:4 Δ6,9,12,15) mainly in seeds and a little in other organs and tissues. RT-PCR results showed that RnD6C and RnD6E were expressed in all the tissues at a low level, whereas RnD6D was expressed at a high level only in seeds, leading to the accumulation of GLA and OTA in seeds. This research provides new insights to our understanding of GLA synthesis and accumulation in plants and the evolutionary relationship of this class of desaturases, and new clues as to the amino acid determinants which define precise enzyme activity.

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114-174, 206-257, and 258-276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114-276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.