Project description:Goal: High-throughput sequencing-by-synthesis (Illumina) RNA sequencing technology was carried out with an aim to gain deeper understanding of immune host protective mechanisms. Here, RNA-seq was applied to understand the differential gene expression profile of mice spleen following immunization with Brucella abortus S19∆per mutant (perosamine synthetase gene mutant of Brucella abortus S19) in comparison to mock immunized mice spleen (PBS inoculated). Methods: RNA-seq data of 15th day post immunized mice spleen (with Brucella abortus S19∆per) and PBS control mice were generated by deep sequencing ( in duplicate) using IlluminaNextSeq 500 . The sequence reads that passed quality filters were analyzed for transcript abundance using RSEM package (RNA-Seq by Expectation Maximization) (Li and Dewey, 2011). Breifly, the RSEM package generated a reference sequence based on given mouse transcript annotations (Mus_musculus.GrCm38.83.chr.gtf.gz). The Bowtie allignmet tool available within the package was used to calculate expected counts (number of mapped reads) using quality trimmed reads and reference sequence. Finally, the expected counts estimated by RSEM were fed into different DE package tools, such as DESeq2 (Love et al., 2014), edgeR (Robinson et al., 2010) and EBSeq (Leng et al., 2013) in order to identify differentially expressed genes across spleen samples (B. abortus S19∆per versus (vs) PBS control. Functional annotation of differently expressed genes were carried out using g:Profiler (Reimandet al., 2016; http://biit.cs.ut.ee/gprofiler/). Results: A mean of 37.58 million processed reads (range: 30.51 million to 51.79 million reads per individual RNA-seq library) were generated during the experiment. The expected counts generated by the RSEM package followed by differential analysis calculation using different DE packages identified a total of 1917 differentially expressed genes (DEGs), of which 968 and 949 genes were up- and down-regulated respectively. Functional annotation revealed 545 significantly enriched genes to be associated with immune system processes within the total 1917 differentially expressed genes. Further analysis revealed 21 genes showing significant expression were also in MHC-I and MHC-II antigen processing and presentation pathway during S19∆per immunization. Conclusions:The RNA-seq data revealed the coordinated up-regulation of MHC-I and MHC-II processing pathways providing insights into the molecular mechanism of immune protection conferred by B. abortus S19∆per in mice at day 15 post immunization and might aid in the development of new attenuated vaccine strains with improved efficacy.

Project description:BACKGROUND: Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value.FINDINGS: Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2 and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified.CONCLUSIONS: Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.

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