Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystin synthetase (mcy) gene clusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers and TaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Continuous salt stress-induced long non-coding RNAs and DNA methylation patterns in soybean roots.

BACKGROUND: Environmental stimuli can activate a series of physiological and biochemical responses in plants accompanied by extensive transcriptional reprogramming. Long non-coding RNAs (lncRNAs), as versatile regulators, control gene expression in multiple ways and participate in the adaptation to biotic and abiotic stresses. RESULTS: In this study, soybean seedlings were continuously cultured for 15 days with high salinity solutions started from seed germination. Strand-specific whole transcriptome sequencing and stringent bioinformatic analysis led to the identification of 3030 long intergenic non-coding RNAs (lincRNAs) and 275 natural antisense transcripts (lncNATs) in soybean roots. In contrast to mRNAs, newly identified lncRNAs exhibited less exons, similar AU content to UTRs, even distribution across the genome and low evolutionary conservation. Remarkably, more than 75% of discovered lncRNAs that were activated or up-regulated by continuous salt stress mainly targeted proteins with binding and catalytic activities. Furthermore, two DNA methylation maps with single-base resolution were generated by using reduced representation bisulfite sequencing, offering a genome-wide perspective and important clues for epigenetic regulation of stress-associated lncRNAs and protein-coding genes. CONCLUSIONS: Taken together, our findings systematically demonstrated the characteristics of continuous salt stress-induced lncRNAs and extended the knowledge of corresponding methylation profiling, providing valuable evidence for a better understanding of how plants cope with long-term salt stress circumstances.

Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae.

BACKGROUND: Kaempferol is a flavonol with broad bioactivity of anti-oxidant, anti-cancer, anti-diabetic, anti-microbial, cardio-protective and anti-asthma. Microbial synthesis of kaempferol is a promising strategy because of the low content in primary plant source. METHODS: In this study, the biosynthesis pathway of kaempferol was constructed in the budding yeast Saccharomyces cerevisiae to produce kaempferol de novo, and several biological measures were taken for high production. RESULTS: Firstly, a high efficient flavonol synthases (FLS) from Populus deltoides was introduced into the biosynthetic pathway of kaempferol. Secondly, a S. cerevisiae recombinant was constructed for de novo synthesis of kaempferol, which generated about 6.97 mg/L kaempferol from glucose. To further promote kaempferol production, the acetyl-CoA biosynthetic pathway was overexpressed and p-coumarate was supplied as substrate, which improved kaempferol titer by about 23 and 120%, respectively. Finally, a fed-batch process was developed for better kaempferol fermentation performance, and the production reached 66.29 mg/L in 40 h. CONCLUSIONS: The titer of kaempferol in our engineered yeast is 2.5 times of the highest reported titer. Our study provides a possible strategy to produce kaempferol using microbial cell factory.

Genomic analyses reveal the stepwise domestication and genetic mechanism of curd biogenesis in cauliflower.

Cauliflower (Brassica oleracea L. var. botrytis) is a distinctive vegetable that supplies a nutrient-rich edible inflorescence meristem for the human diet. However, the genomic bases of its selective breeding have not been studied extensively. Herein, we present a high-quality reference genome assembly C-8 (V2) and a comprehensive genomic variation map consisting of 971 diverse accessions of cauliflower and its relatives. Genomic selection analysis and deep-mined divergences were used to explore a stepwise domestication process for cauliflower that initially evolved from broccoli (Curd-emergence and Curd-improvement), revealing that three MADS-box genes, CAULIFLOWER1 (CAL1), CAL2 and FRUITFULL (FUL2), could have essential roles during curd formation. Genome-wide association studies identified nine loci significantly associated with morphological and biological characters and demonstrated that a zinc-finger protein (BOB06G135460) positively regulates stem height in cauliflower. This study offers valuable genomic resources for better understanding the genetic bases of curd biogenesis and florescent development in crops.

Target Specificity of the CRISPR-Cas9 System in Arabidopsis thaliana, Oryza sativa, and Glycine max Genomes.

Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana, Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana, rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its applications in the future.

Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches.

The flavonoid extract from Erigeron breviscapus, breviscapine, has increasingly been used to treat cardio- and cerebrovascular diseases in China for more than 30 years, and plant supply of E. breviscapus is becoming insufficient to satisfy the growing market demand. Here we report an alternative strategy for the supply of breviscapine by building a yeast cell factory using synthetic biology. We identify two key enzymes in the biosynthetic pathway (flavonoid-7-O-glucuronosyltransferase and flavone-6-hydroxylase) from E. breviscapus genome and engineer yeast to produce breviscapine from glucose. After metabolic engineering and optimization of fed-batch fermentation, scutellarin and apigenin-7-O-glucuronide, two major active ingredients of breviscapine, reach to 108 and 185 mg l-1, respectively. Our study not only introduces an alternative source of these valuable compounds, but also provides an example of integrating genomics and synthetic biology knowledge for metabolic engineering of natural compounds.

Graphitic carbon nitride/phosphorus-rich aluminum phosphinates hybrids as smoke suppressants and flame retardants for polystyrene.

Graphitic carbon nitride/organic aluminum hypophosphites (g-C3N4/OAHPi) hybrids, i.e., CPDCPAHPi and CBPODAHPi, were synthesized by esterification and salification reactions, and then incorporated into polystyrene (PS) to prepare composites through a melt blending method. Structure and morphology characterizations demonstrated the successful synthesis of PDCPAHPi, BPODAHPi and their hybrids. The g-C3N4 protected OAHPi from external heat and thus improved the thermal stability of OAHPi. Combining g-C3N4 with OAHPi contributed to reduction in peak of heat release rate, total heat release and smoke production rate of PS matrix. Reduced smoke released has also been demonstrated by smoke density chamber testing. Additionally, introduction of the hybrids led to decreased release of flammable aromatic compounds. These properties improvement could be attributed to gas phase action and physical barrier effect in condensed phase: phosphorus-containing low-energy radicals generated from OAHPi effectively captured high-energy free-radicals evolved from PS; g-C3N4 nanosheets retarded the permeation of heat and the escape of volatile degradation products. Therefore, g-C3N4/OAHPi hybrids will provide a potential strategy to reduce the fire hazards of PS.