Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii.

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry.

Research Progress in Heterologous Crocin Production.

Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of ß-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Molecular and antimicrobial characterization of a group G anti-lipopolysaccharide factor (ALF) from Penaeus monodon.

Anti-lipopolysaccharide factors (ALFs) are important host-defense molecules of crustaceans. They all contain a lipopolysaccharide-binding domain (LBD) and some ALFs exhibit strong antimicrobial activity. In this research, a Group G ALF from Penaeus monodon (ALFPm11) was studied. It is an anionic peptide specifically having a cationic and highly amphipathic LBD, with five positively charged residues separated by aromatic residues. It was abundantly expressed in the hepatopancreas of P. monodon normally but the expression level in other tissues was relatively low or undetectable. However, in the shrimps challenged by Vibrio, expression of ALFPm11 could be detected in all tissues. Chemically synthesized ALFPm11-LBD displayed high inhibitory activity (minimum inhibition concentration≤ 4 µM) against various bacteria, e.g. Exiguobacterium sp. L33, Bacillus sp. T2, and Acinetobacter sp. L32. It also displayed apparent activity in the agar well diffusion assay. Furthermore, it could efficiently induce agglutination of both Gram-positive and Gram-negative bacteria and cause significant membrane permeabilization of the bacteria. As a comparative study, ALFPm11-LBD showed a better or equal antimicrobial function to ALFPm3-LBD which was reported to possess strong antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. Thus, this research found a new effective ALF in P. monodon and demonstrated its antimicrobial mechanism, suggesting its potential applications in the future.

Discovery of Geranylgeranyl Pyrophosphate Synthase (GGPPS) Paralogs from Haematococcus pluvialis Based on Iso-Seq Analysis and Their Function on Astaxanthin Biosynthesis.

Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7-9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis.

Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA.

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR.

Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological methods is an arduous task requiring extensive training on nematode taxonomy and years of experience. Thus, molecular methods for P. scribneri detection and identification were developed. Conventional and real-time polymerase chain reaction (PCR) assays with new species-specific primers were used in this study, which exclusively amplified DNA of P. scribneri but not DNA from other Pratylenchus spp. or non-Pratylenchus spp. tested. Compared with conventional PCR that was able to detect an equivalent to 1/4 of the DNA of a single nematode, real-time PCR was more sensitive and could amplify an equivalent to 1/128 of the DNA of one nematode. Both conventional and real-time PCR assays successfully identified P. scribneri and distinguished it from P. penetrans and P. neglectus isolated from field samples collected from various locations in North Dakota and Minnesota. The Blast-search based on the sequence information confirmed the reliability of the PCR assays for species identification. This is the first report of P. scribneri identification using a real-time PCR assay. The developed PCR assays are suitable for use in diagnostic laboratories and detection of field infestations with this nematode species.

Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius.

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10-4 ng of DNA template could be detected by conventional PCR and 1.92 × 10-4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.

First Report of the Spiral Nematode Helicotylenchus microlobus Infecting Soybean in North Dakota.

Spiral nematodes (Helicotylenchus spp.) are common plant-parasitic nematodes in fields of many crops. In June 2015, two soil samples were collected from a soybean field in Richland County, ND. Nematodes were extracted from soil using the sugar centrifugal flotation method (Jenkins, 1964). Plant-parasitic nematodes were identified to genus based on morphological features and counted. Both samples contained spiral nematodes from 1,500 to 3,300 per kilogram of soil. In June and August 2016, 10 soil samples were collected from the same field. Nematodes were extracted, and nine of the samples had spiral nematodes ranging from 125 to 3,065 per kilogram of soil. One soil sample with 1,500 spiral nematodes per kilogram was used to inoculate two soybean cultivars Sheyenne and Barnes each in four replicates. After 15 wk of growth at 22°C in a greenhouse room, the population of spiral nematodes was found to have increased greatly. The final density was 9,300 ± 1,701 spiral nematodes per kilogram of soil for Sheyenne and 9,451 ± 2,751 for Barnes. The reproductive factor in Sheyenne and Barnes was 6.2 and 6.3, respectively, indicating that this spiral nematode infects and reproduces well on these two soybean cultivars. Infected soybean roots had small brown lesions on the surface. Individual spiral nematodes were handpicked and examined morphologically and molecularly for species identification. Morphological measurements of adult females (n = 15) included body length (mean = 708.5 µm, range = 600.0-812.0 µm), stylet (27.6, 26.0-29.0), body width (28.3, 25.0-33.0), lip region end to posterior end of pharyngeal glands (142.5, 130.0-152.0), anal body width (15.8, 14.0-17.5), tail length (20.3, 15.0-25.0), tail annules (11.6, 10.0-14.0), a (25.0, 21.4-27.1), b (5.0, 4.4-5.7), c (35.4, 30.2-41.7), c' (1.3, 1.0-1.6), and V (61.8%, 60.0-63.7). The spiral nematode was identified as Helicotylenchus microlobus according to morphological and morphometric characteristics (Subbotin et al., 2015). DNA was extracted from single nematodes (n = 8) using the Proteinase K method (Kumari and Subbotin, 2012). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers rDNA2/rDNA1.58S (Cherry et al., 1997). The PCR products were then purified and sequenced. The consensus ITS rDNA sequence (accession no. KY271078, 822 bp) that was deposited into the GenBank shared 99% identity with two isolates of H. microlobus from California (KM506860.1 and KM506859.1) and one isolate of H. microlobus from Spain (KM506862.1) (Subbotin et al., 2015). It had only 91% sequence identity with seven isolates of H. pseudorobustus (KM506875.1, KM506880.1, KM506876.1, KM506874.1, KM506872.1, KM506879.1, and KM506878.1) from California, Switzerland, and New Zealand, a spiral nematode species very closely related to H. microlobus in morphology. The molecular tests confirmed the identity of this spiral nematode as H. microlobus. The H. microlobus nematode was reported as one of the most commonly observed spiral nematodes in soil samples in the state of Minnesota, and all 13 soybean cultivars tested except Hawkeye were rated as hosts (Taylor, 1960). To our knowledge, this is the first report of H. microlobus in North Dakota.

De novo transcriptome analysis in radish (Raphanus sativus L.) and identification of critical genes involved in bolting and flowering.

BACKGROUND: The appropriate timing of bolting and flowering is pivotal for reproductive success in Brassicaceae crops including radish (Raphanus sativus L.). Although several flowering regulatory pathways had been described in some plant species, no study on genetic networks of bolting and flowering regulation was performed in radish. In this study, to generate dataset of radish unigene sequences for large-scale gene discovery and functional pathway identification, a cDNA library from mixed radish leaves at different developmental stages was subjected to high-throughput RNA sequencing (RNA-seq). RESULTS: A total of 54.64 million clean reads and 111,167 contigs representing 53,642 unigenes were obtained from the radish leaf transcriptome. Among these, 50,385 unigenes were successfully annotated by BLAST searching against the public protein databases. Functional classification and annotation indicated that 42,903 and 15,382 unique sequences were assigned to 55 GO terms and 25 COG categories, respectively. KEGG pathway analysis revealed that 25,973 unigenes were classified into 128 functional pathways, among which 24 candidate genes related to plant circadian rhythm were identified. Moreover, 142 potential bolting and flowering-related genes involved in various flowering pathways were identified. In addition, seven critical bolting and flowering-related genes were isolated and profiled by T-A cloning and RT-qPCR analysis. Finally, a schematic network model of bolting and flowering regulation and pathways was put forward in radish. CONCLUSIONS: This study is the first report on systematic identification of bolting and flowering-related genes based on transcriptome sequencing and assembly in radish. These results could provide a foundation for further investigating bolting and flowering regulatory networks in radish, and facilitate dissecting molecular genetic mechanisms underlying bolting and flowering in Brassicaceae vegetable crops.

Molecular characterization of the basic helix-loop-helix (bHLH) genes that are differentially expressed and induced by iron deficiency in Populus.

KEY MESSAGE: Two Populus bHLH genes ( PtFIT and PtIRO ) were cloned and characterized. The iron deficiency tolerance may be regulated by the PtFIT -dependent response pathway in Populus. Five orthologs of eight Arabidopsis basic helix-loop-helix (bHLH) genes responding to iron deficiency in Populus were analyzed. Open reading frame (ORF) regions of two bHLH genes (PtFIT and PtIRO) were isolated from the iron deficiency tolerant (PtG) and susceptible (PtY) genotypes of Populus tremula 'Erecta'. Gene sequence analyses showed that each of the two genes was identical in PtG and PtY. Phylogenetic analysis revealed that PtFIT was clustered with the bHLH genes regulating iron deficiency responses, while PtIRO was clustered with another group of the bHLH genes regulating iron deficiency responses in a FIT-independent pathway. Tissue-specific expression analysis indicated that PtFIT was only detected in the root among all tested tissues, while PtIRO was rarely detected in all tested tissues. Real-time PCR showed that PtFIT was up-regulated in roots under the iron-deficient condition. A higher level of PtFIT transcripts was detected in PtG than in PtY. Pearson Correlation Coefficient calculations indicated a strong positive correlation (r = 0.94) between PtFIT and PtIRT1 in PtG. It suggests that the iron deficiency tolerance of PtG may be regulated by the PtFIT-dependent response pathway. The PtFIT-transgenic poplar plants had an increased expression level of PtFIT and PtIRT1 responding to iron deficiency. One PtFIT-transgenic line (TL2) showed enhanced iron deficiency tolerance with higher chlorophyll content and Chl a/b ratio under iron deficiency than the control plants, indicating that PtFIT is involved in iron deficiency response in Populus. The results would provide useful information to understand iron deficiency response mechanisms in woody species.

Improvement of Carotenoids' Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies.

Canthaxanthin is an important antioxidant with wide application prospects, and ß-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of ß-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of ß-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of ß-carotene ketolase in Escherichia coli. In particular, the production of soluble ß-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from ß-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of ß-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different ß-carotene ketolase. This study not only laid a foundation for further research on the improvement of ß-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

Secretory Expression and Application of Antilipopolysaccharide Factor 3 in Chlamydomonas reinhardtii.

Anti-lipopolysaccharide factor is a class of antimicrobial peptides with lipopolysaccharide-binding structural domains, which has a broad antimicrobial spectrum, high antimicrobial activities, and broad application prospects in terms of the aquaculture industry. However, the low yield of natural antimicrobial peptides and their poor expression activity in bacteria and yeast have hindered their exploration and utilization. Therefore, in this study, the extracellular expression system of Chlamydomonas reinhardtii, by fusing the target gene with the signal peptide, was used to express anti-lipopolysaccharide factor 3 (ALFPm3) from Penaeus monodon in order to obtain highly active ALFPm3. Transgenic C. reinhardtii T-JiA2, T-JiA3, T-JiA5, and T-JiA6, were verified using DNA-PCR, RT-PCR, and immunoblot. Additionally, the IBP1-ALFPm3 fusion protein could be detected not only within the cells but also in the culture supernatant. Moreover, the extracellular secretion containing ALFPm3 was collected from algal cultures, and then its bacterial inhibitory activity was analyzed. The results showed that the extracts from T-JiA3 had an inhibition rate of 97% against four common aquaculture pathogenic bacteria, including Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio parahaemolyticus. The highest inhibition rate of 116.18% was observed in the test against V. anguillarum. Finally, the minimum inhibition concentration (MIC) of the extracts from T-JiA3 to V. harveyi, V. anguillarum, V. alginolyticus, and V. parahaemolyticus were 0.11 µg/µL, 0.088 µg/µL, 0.11 µg/µL, and 0.011 µg/µL, respectively. This study supports the foundation of the expression of highly active anti-lipopolysaccharide factors using the extracellular expression system in C. reinhardtii, providing new ideas for the expression of highly active antimicrobial peptides.

Enhancement of ß-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene ß-cyclase.

ß-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of ß-carotene and α-carotene. Aiming to develop the microalgae with enhanced ß-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of ß-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant ß-carotene), confirming the superior activity of CrtY on ß-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased ß-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

A chromosome-level genome assembly for the astaxanthin-producing microalga Haematococcus pluvialis.

The green microalga Haematococcus pluvialis can synthesize high amounts of astaxanthin, which is a valuable antioxidant that has been utilized in human health, cosmetics, and aquaculture. To illustrate detailed molecular clues to astaxanthin yield, we performed PacBio HIFI along with Hi-C sequencing to construct an improved chromosome-level haplotypic genome assembly with 32 chromosomes and a genome size of 316.0 Mb. Its scaffold N50 (942.6 kb) and contig N50 (304.8 kb) have been upgraded remarkably from our previous genome draft, and a total of 32,416 protein-coding genes were predicted. We also established a high-evidence phylogenetic tree from seven representative algae species, with the main aim to calculate their divergence times and identify expanded/contracted gene families. We also characterized genome-wide localizations on chromosomes of some important genes such as five BKTs (encoding beta-carotene ketolases) that are putatively involved in astaxanthin production. In summary, we reported the first chromosome-scale map of H. pluvialis, which provides a valuable genetic resource for in-depth biomedical investigations on this momentous green alga and commercial astaxanthin bioproduction.

Development and cross-species/genera transferability of microsatellite markers discovered using 454 genome sequencing in chokecherry (Prunus virginiana L.).

Chokecherry (Prunus virginiana L.) (2n = 4x = 32) is a unique Prunus species for both genetics and disease-resistance research due to its tetraploid nature and X-disease resistance. However, no genetic and genomic information on chokecherry is available. A partial chokecherry genome was sequenced using Roche 454 sequencing technology. A total of 145,094 reads covering 4.8 Mbp of the chokecherry genome were generated and 15,113 contigs were assembled, of which 11,675 contigs were larger than 100 bp in size. A total of 481 SSR loci were identified from 234 (out of 11,675) contigs and 246 polymerase chain reaction (PCR) primer pairs were designed. Of 246 primers, 212 (86.2 %) effectively produced amplification from the genomic DNA of chokecherry. All 212 amplifiable chokecherry primers were used to amplify genomic DNA from 11 other rosaceous species (sour cherry, sweet cherry, black cherry, peach, apricot, plum, apple, crabapple, pear, juneberry, and raspberry). Thus, chokecherry SSR primers can be transferable across Prunus species and other rosaceous species. An average of 63.2 and 58.7 % of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2 % of amplifiable chokecherry primers amplified DNA from other rosaceous species. Using random genome sequence data generated from next-generation sequencing technology to identify microsatellite loci appears to be rapid and cost-efficient, particularly for species with no sequence information available. Sequence information and confirmed transferability of the identified chokecherry SSRs among species will be valuable for genetic research in Prunus and other rosaceous species. Key message A total of 246 SSR primers were identified from chokecherry genome sequences. Of which, 212 were confirmed amplifiable both in chokecherry and other 11 other rosaceous species.

The Histone Acetyltransferase HpGCN5 Involved in the Regulation of Abiotic Stress Responses and Astaxanthin Accumulation in Haematococcus pluvialis.

The histone acetyltransferases (HATs), together with histone deacetylases, regulate the gene transcription related to various biological processes, including stress responses in eukaryotes. This study found a member of HATs (HpGCN5) from a transcriptome of the economically important microalgae Haematococcus pluvialis. Its expression pattern responding to multiple abiotic stresses and its correlation with transcription factors and genes involved in triacylglycerols and astaxanthin biosynthesis under stress conditions were evaluated, aiming to discover its potential biological function. The isolated HpGCN5 was 1,712 bp in length encoding 415 amino acids. The signature domains of Acetyltransf_1 and BROMO were presented, as the GCN5 gene from Arabidopsis and Saccharomyces cerevisiae, confirming that HpGCN5 belongs to the GCN5 subfamily of the GNAT superfamily. The phylogenetic analysis revealed that HpGCN5 is grouped with GNAT genes from algae and is closer to that from higher plants, compared with yeast, animal, fungus, and bacteria. It was predicted that HpGCN5 is composed of 10 exons and contains multiple stress-related cis-elements in the promoter region, revealing its potential role in stress regulation. Real-time quantitative PCR revealed that HpGCN5 responds to high light and high salt stresses in similar behavior, evidenced by their down-regulation exposing to stresses. Differently, HpGCN5 expression was significantly induced by SA and Nitrogen-depletion stresses at the early stage but was dropped back after then. The correlation network analysis suggested that HpGCN5 has a strong correlation with major genes and a transcription factor involved in astaxanthin biosynthesis. Besides, the correlation was only found between HpGCN5 and a few genes involved in triacylglycerols biosynthesis. Therefore, this study proposed that HpGCN5 might play a role in the regulation of astaxanthin biosynthesis. This study firstly examined the role of HATs in stress regulation and results will enrich our understanding of the role of HATs in microalgae.