RNA sequencing (RNA-Seq) is the leading, routine, high-throughput, and cost-effective next-generation sequencing (NGS) approach for mapping and quantifying transcriptomes, and determining the transcriptional structure. The transcriptome is a complete collection of transcripts found in a cell or tissue or organism at a given time point or specific developmental or environmental or physiological condition. The emergence and evolution of RNA-Seq chemistries have changed the landscape and the pace of transcriptome research in life sciences over a decade. This chapter introduces RNA-Seq and surveys its recent food and agriculture applications, ranging from differential gene expression, variants calling and detection, allele-specific expression, alternative splicing, alternative polyadenylation site usage, microRNA profiling, circular RNAs, single-cell RNA-Seq, metatranscriptomics, and systems biology. A few popular RNA-Seq databases and analysis tools are also presented for each application. We began to witness the broader impacts of RNA-Seq in addressing complex biological questions in food and agriculture.
ATP citrate lyase (ACL) catalyzes the ATP‐dependent conversion of citrate to the fatty acid precursor, acetyl‐CoA. ACL presence in yeasts has been associated with their ability to accumulate lipids (i.e., oleaginous phenotype), but little is known about the regulation of this enzyme in oleaginous yeasts. In the model oleaginous yeast Yarrowia lipolytica, ACL is a heterodimer comprised of a catalytic and a regulatory subunit, encoded by the ACL1 and ACL2 genes, respectively. From the earlier studies, it was shown that the loss of ACL1 resulted in lower lipid levels and altered fatty acid profiles. However, the regulation of ACL expression and activity during lipogenesis has not been studied. To better understand the role, ACL plays during lipogenesis in Y.lipolytica, we generated antibodies against its two subunits (i.e., Acl1 and Acl2). We also constructed strains that lack Acl2 (i.e., acl2Δ) and strains that overexpress Acl1 and Acl2 either alone or in combination. Preliminary experiments showed that the overexpression of Acl1 increased the protein levels of Acl2. We are currently analyzing the effects of Acl2 overexpression and the time‐dependent regulation of Acl1 and Acl2.
The cotton crop is economically important and primarily grown for its fiber. Although the genus Gossypium consists of over 50 species, only four domesticated species produce spinnable fiber. However, the genes determine the molecular phenotype of fiber, and variation in their expression primarily contributes to associated phenotypic changes. Transcriptome analyses can elucidate the similarity or variation in gene expression (GE) among organisms at a given time or a circumstance. Even though several algorithms are available for analyzing such high-throughput data generated from RNA Sequencing (RNA-Seq), a reliable pipeline that includes a combination of tools such as an aligner for read mapping, an assembler for quantitating full-length transcripts, a differential gene expression (DGE) package for identifying differences in the transcripts across the samples, a gene ontology tool for assigning function, and enrichment and pathway mapping tools for finding interrelationships between genes based on their associated functions are needed. Therefore, this chapter first introduces the cotton crop, fiber phenotype, transcriptome, then discusses the basic RNA-Seq pipeline and later emphasizes various transcriptome analyses studies focused on genes associated with fiber quality and its attributes.
The PAH1 gene encodes for phosphatidate (PA) phosphatase which catalyzes the dephosphorylation of PA to produce diacylglycerol that can be further converted to triacylglycerol (TAG). Our recent studies in the oleaginous yeast Yarrowia lipolytica showed that the pah1Δ mutation caused a significant decrease in total lipid content and TAG levels during the lipogenic phase, where cell growth ceases and lipid biosynthesis predominates. Also, the expression of the Pah1 protein is regulated under these conditions. To gain a better understanding of this regulation, integrative vectors that drive the expression of PAH1 under the control of its native promoter and either the LIP2 or the PEX20 terminator were constructed and transformed into pah1Δ cells. The expression of Pah1 in the transformants was confirmed by immunoblot analysis using antibodies directed against the protein, and its biological function was examined by analyzing the PA phosphatase activity, lipid content, and TAG levels. The results showed that the levels of the Pah1 protein expressed under theLIP2 and the PEX20 terminators were similar to the wild type levels. Also, the vector expressed Pah1 was able to restore the TAG levels in pah1Δ cells. The constructed vectors will be further used to study the function of Pah1 by mutating its catalytic motif using site‐directed mutagenesis. We hypothesize that the catalytically inactive Pah1 protein will not complement the phenotype of the pah1Δ cells, thereby confirming that the catalytic activity of Pah1 is responsible for the phenotypical changes observed in the pah1Δ cells.
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