Reduction in Carotenoid Levels in the Marine Diatom Phaeodactylum tricornutum by Artificial MicroRNAs Targeted Against the Endogenous Phytoene Synthase Gene

Abstract

MicroRNAs (miRNAs) are key regulators of gene expression in eukaryotes where they can function to downregulate expression levels or functioning of messenger RNAs (mRNAs) that are targeted by mature miRNAs displaying sequence homology. The 'active' mature miRNA forms are short RNAs which are processed from longer precursor miRNA molecules that have a stem-loop structure. While artificial miRNAs have been developed for gene knockdown experiments in a range of eukaryotes, it is not known whether artificial or endogenous miRNAs can functionally knockdown mRNA levels in the model marine diatom Phaeodactylum tricornutum. Here, we investigate the potential use of artificial microRNAs (amiRNAs) for targeted gene knockdowns in P. tricornutum, by generation of transformants harbouring a transgene cassette for the generation of amiRNAs designed to target the endogenous phytoene synthase (PSY) gene. In P. tricornutum, the amiRNA stem-loop precursor was processed to produce a mature amiRNA that successfully targeted the PSY mRNA and reduced PSY mRNA levels. As the PSY gene is a key component of the carotenoid biosynthetic pathway, the levels of carotenoids in the P. tricornutum amiRNA knockdown lines were reduced relative to untransformed control lines. This study demonstrates that artificial miRNAs can be successfully deployed for gene knockdown experiments in the model diatom P. tricornutum, providing a powerful tool for future metabolic engineering and synthetic biology experimentation in this model marine diatom.

Rapid Genomic and Genetic Changes in the First Generation of Autotetraploid Lineages Derived from Distant Hybridization of Carassius auratus Red Var. (♀) × Megalobrama amblycephala (♂)

“…The pairing of homologous chromosomes is defective in F 1 hybrids (diploids) because of divergence in the number and structure of chromosomes, but each homologous chromosome can have its own pairing partner through genome doubling in somatic or germ cells (Wu et al 2001 ; Paterson et al 2004 ). In our previous research, fertile allotetraploid hybrids (4nRB) (RRBB, 4n = 148) were obtained in the first generation of RCC (RR, 2n = 100) ( ♀ ) × BSB (BB, 2n = 48) ( ♂ ); these hybrids exhibited abnormal chromosomal behavior during meiosis and generated autodiploid gametes with two sets of RCC-derived chromosomes (RR, 2n = 100) (Liu et al 2007 ; Qin et al 2014a , 2015a ). Thus, autodiploid sperm can fertilize autodiploid ova, resulting in 4nRR formation (RRRR, 4n = 200) (Qin et al 2014b ).…”

Modulation of lipid biosynthesis by stress in diatoms

“…This technique allowed clarification of the role of urease in the urea cycle and also improved the molecular toolkit for diatom engineering. Other mechanisms for the targeted knockdown/out of endogenous genes include RNA silencing using RNAi and antisense RNA techniques [134], and high-throughput artificial-micro RNA (armiRNA) techniques [135]. …”

“…Overexpression of P. tricornutum malic enzyme (PtME) resulted in markedly increased lipid production in transgenic cells [135]. The total lipid content was enhanced 2.5-fold and reached 57.8% of dry cell mass with a growth rate similar to that of wild-type.…”

Genetic and metabolic engineering in diatoms

“…Today, only the first two of these approaches have been used in diatoms but we expect a substantial increase over the next few years. Methods based on the knock-down of gene expression have been developed for diatoms, making it possible to link genes to particular functions: RNA interference (RNAi) [14,41,42] and more recently the artificial miRNAs [43]. The proof of concept of RNAi was in 2009 with the GUS reporter gene [14].…”