Abstract
Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.
Highlights
Eukaryotic microalgae are a very diverse group of organisms that have been reported to play vital roles in the fixation of CO2, O2 production, and climate change [1,2]
We constructed two vectors that were controlled by the endogenous tub promoter and terminator with sizes of 6124 and 6580bp, respectively, and we named the vectors pSimple-tub-expressing the green fluorescent protein (eGFP) and pEASY-tub-neomycin phosphotransferase II-encoding G418-resistant gene (nptII) (Figure 2C)
Thissaamlgpalehas the ability to grow under autotrophic, mixotrophic, and heterotrophic conditions, and it can synthesize and accumulate a significant amouFnitgFuoirfgeuf6ar.ettQ6y.uaQacnuitdaifinictniafttiicroantcieoolnflutohlfaetrhnlpeytnI[Ip2gt7IIe]n.geEenisnetatihbnrltiehsehrteirenagntrsagfnoesrnmfoeartmnictastnootfosTlos.fmfToi.nrmuTsin.tuhmsroitnhuurgoshuwrgehoalru-etlaidml-teiinmPcCereRPa.CseR.the 3pfo.orD3teX.inDsatcniiuatshslcsuovipasoslhnuiyoecneoaf et.his alga in biotechnological applications, and it could serve as a molecular model
Summary
Eukaryotic microalgae are a very diverse group of organisms that have been reported to play vital roles in the fixation of CO2, O2 production, and climate change [1,2]. Many groups of microalgae have great potential to produce a variety of commercially valuable carbon compounds, including lipids, starch, and carbohydrates, and they are well known for their ability to produce long-chain fatty acids, such as polyunsaturated fatty acids [3,4,5]. These microalgae with rapid growth rates have been considered as possible sources of next-generation energy fuels and high-value products. Information on transgene expression and genetic engineering for understanding molecular mechanisms and strain development is very limited in the Tribonema genus
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