Correction to: CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L).

An amendment to this paper has been published and can be accessed via the original article.

CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean (Glycine max.L).

BACKGROUND: Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis. RESULTS: Here we demonstrated an efficient sequence specific integration/mutation of FAD2-2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2-2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2-2 loci. The integration of FAD2-2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis. CONCLUSION: High frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.

The morphological changes and molecular biomarker responses in the liver of fluoride-exposed Bufo gargarizans larvae.

The goal of the current study was to evaluate the negative influences of fluoride on liver of Bufo gargarizans larvae. B. gargarizans larvae were treated with 42.4mgF-/L for 0, 24, 48 and 72h at Gosner stage 37. The morphological changes and responses of molecular biomarkers involved in lipid metabolism, oxidative stress and apoptosis were examined in liver. Disappearance of cell boundaries, degeneration of hepatic parenchyma cells and significant increase in the number of melanomacrophage centres and the quantity of lipid droplets were found in the liver treated with 42.4mgF-/L for 72h. In addition, in the relative expression of acetyl CoA carboxylase 1 (ACC-1), fatty acid elongase 1 (FAE-1), sterol carrier protein 2 (SCP-2), and carnitine palmitoyltransferase-1 (CPT-1), decrease was observed after 24, 48 and 72h of 42.4mgF-/L exposure. Furthermore, the transcript levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were downregulated in tadpoles exposed for 24, 48 and 72h to 42.4mgF-/L, while the transcript level of heat shock protein 90 (HSP90) was upregulated at 42.4mgF-/L for 72h. Also, mRNA expression of Bcl-2-associated transcription factor 1(BCLAF1) and thyroid hormone receptors (TRα and TRß) was significantly upregulated in tadpoles treated with 42.4mgF-/L for 72h. Therefore, our results suggested that the liver injury induced by fluoride might result from disruption of lipid metabolism, oxidative damage and apoptosis.

The effect of cadmium exposure on diversity of intestinal microbial community of Rana chensinensis tadpoles.

Cadmium is a natural and widely distributed toxicant, and can be commonly found in environment. Intestinal microbiota plays a very important role in maintaining its host's health. The effects of cadmium on the intestinal microbiota composition and stability of amphibians are little known. We exposed Rana chensinensis (R. chensinensis) embryos to different concentrations of cadmium (0, 112 and 448 µg Cd L-1) until they reached Gosner stage 38, and analyzed their microbial communities using 16S rRNA amplicon sequencing. By measures of both alpha and beta diversity, intestinal microbial communities were significantly differentiated in 448 µg Cd L-1 exposure groups. Cadmium exposure significantly altered the intestinal microflora diversity and composition of R. chensinensis. At the phylum level, it is worth noting that Fusobacteria and Spirochaetae were not detected in 448 µg Cd L-1 exposure groups. Firmicutes rapidly decreased in 448 µg Cd L-1 exposure group. At the genus level, Succinispira (Firmicutes), Desulfovibrio (Proteobacteria) and Fusobacterium (Fusobacteria) vanished in 448 µg Cd L-1 exposure groups. Our results demonstrate that cadmium exposure changed the composition and decreased the community diversity of intestinal microbiota of R. chensinensis tadpoles. Our study may provide a new framework based on intestinal microbiota to evaluate the response of amphibians to environmental chemicals pollution.

Transcriptome analyses reveal molecular mechanisms that regulate endochondral ossification in amphibian Bufo gargarizans during metamorphosis.

BACKGROUND: A developmental transition from aquatic to terrestrial existence is one of the most important events in the evolution of terrestrial vertebrates. Amphibian metamorphosis is a classic model to study this transition. The development of the vertebrate skeleton can reflect its evolutionary history. Endochondral ossification serves a vital role in skeletal development. Thus, we sought to unravel molecular mechanisms that regulate endochondral ossification during Bufo gargarizans metamorphosis. METHODS: The alizarin red-alcian blue double staining method was used to visualize the skeletal development of B. gargarizans during metamorphosis. RNA sequencing (RNA-seq) was used to explore the transcriptome of B. gargarizans in four key developmental stages during metamorphosis. Real-time quantitative PCR (RT-qPCR) was used to validate the expression patterns of endochondral ossification related genes. RESULTS: Endochondral ossification increased gradually in skeletal system of B. gargarizans during metamorphosis. A total of 137,264 unigenes were assembled and 44,035 unigenes were annotated. 10,352 differentially expressed genes (DEGs) were further extracted among four key developmental stages. In addition, 28 endochondral ossification related genes were found by searching for DEG libraries in B. gargarizans. Of the 28 genes, 10 genes were validated using RT-qPCR. CONCLUSIONS: The exquisite coordination of the 28 genes is essential for regulation of endochondral ossification during B. gargarizans metamorphosis. GENERAL SIGNIFICANCE: The present study will not only provide an invaluable genomic resource and background for further research of endochondral ossification in amphibians but will also aid in enhancing our understanding of the evolution of terrestrial vertebrates.