抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

The number of sequenced plant genomes and associated genomic resources is growing rapidly with the advent of both an increased focus on plant genomics from funding agencies, and the application of inexpensive next generation sequencing. To interact with this increasing body of data, we have developed Phytozome (http://www.phytozome.net), a comparative hub for plant genome and gene family data and analysis. Phytozome provides a view of the evolutionary history of every plant gene at the level of sequence, gene structure, gene family and genome organization, while at the same time providing access to the sequences and functional annotations of a growing number (currently 25) of complete plant genomes, including all the land plants and selected algae sequenced at the Joint Genome Institute, as well as selected species sequenced elsewhere. Through a comprehensive plant genome database and web portal, these data and analyses are available to the broader plant science research community, providing powerful comparative genomics tools that help to link model systems with other plants of economic and ecological importance.

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Phytozome: a comparative platform for green plant genomics

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-313X.2008.03492.x

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(13)00087-5.pdf

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

/pdf/the-chlamydomonas-genome-reveals-the-evolution-of-key-animal-2n5q5e35gv.pdf

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

Mitochondria isolated from spinach (Spinacia oleracea) leaves oxidized glycine with a stoichiometry of CO2 evolution to O2 uptake of 2 : 1. In the absence of added substrate, the mitochondria exhibited an extremely low endogenous rate of O2 uptake.

/pdf/stoichiometry-of-carbon-dioxide-release-and-oxygen-uptake-14dm91o24o.pdf

Stoichiometry of carbon dioxide release and oxygen uptake during glycine oxidation in mitochondria isolated from spinach (Spinacia oleracea) leaves.

The invention provides the disclosure of a novel plant/algae/cyanobacteria photosynthesis, biomass production, and productivity pathway involving low carbon dioxide inducible (LCI) proteins. According to the invention, the activity of one or more LCI proteins may be modulated to increase the same under conditions where such proteins are typically repressed. According to the invention, modulation of LCI protein activity was able to increase biomass production by as much as 80% under elevated CO 2 conditions. The invention includes methods, and genetically modified plants/algae/cyanobacteria, cells, plant parts and tissues.

Modulation of low carbon dioxide inducible proteins (lci) for increased biomass production and photosynthesis

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO2 assimilation was determined by CO2 exchange studies at three, limiting CO2 concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO2 assimilation did not respond to the varying photon flux densities because of CO2 limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 μmol photons m(-2) s(-1), indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO2 assimilation responded to external CO2 concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO2 into the cells supplies most of the CO2 for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO2 concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO2 exchange method to determine whether inorganic carbon accumulation and photosynthetic CO2 assimilation compete for energy at low photon flux densities proved inconclusive.

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in [~]1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures. One sentence summaryCo-targeting two genes by co-electroporation of CRISPR/Cas9 RNPs and ssODN repair templates allows concomitant genome editing to create a selectable marker gene and to introduce precise modifications in another gene of interest.

https://biorxiv.org/content/10.1101/2021.03.26.437214v1.full.pdf

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

This paper reports on a microfluidic device capable of screening carbon dioxide (CO2) conditions for microalgal cell growth. The device mainly consists of a microfluidic cell culture (MCC) unit, a gas concentration gradient generator (CGG), and an in-line cell growth optical measurement unit. The MCC unit is structured with multiple aqueous-filled cell culture channels at the top layer, multiple CO2 flow channels at the bottom layer, and a commercial hydrophobic gas semipermeable membrane sandwiched between the two channel layers. The CGG unit provides different CO2 concentrations to support photosynthesis of microalgae in the culture channels. The integration of the commercial gas semipermeable membrane into the cell culture device allows rapid mass transport and uniform distribution of CO2 inside the culture medium without using conventional agitation-assisted convection methods, because the diffusion of CO2 from the gas flow channels to the culture channels is fast over a small length scale. In addition, automated in-line monitoring of microalgal cell growth is realized via the optical measurement unit that is able to detect changes in the light intensity transmitted through the cell culture in the culture channels. The microfluidic device also allows a simple grayscale analysis method to quantify the cell growth. The utility of the system is validated by growing Chlamydomonas reinhardtii cells under different low or very-low CO2 levels below the nominal ambient CO2 concentration.

Martin H. Spalding

Papers

Stoichiometry of carbon dioxide release and oxygen uptake during glycine oxidation in mitochondria isolated from spinach (Spinacia oleracea) leaves.

Modulation of low carbon dioxide inducible proteins (lci) for increased biomass production and photosynthesis

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Microfluidic chip for automated screening of carbon dioxide conditions for microalgal cell growth