Green tides, dominated by Ulva, are on the rise worldwide, and invasive Ulva spp. tend to bloom in new habitats. Over the last decade, green tides have caused serious problems in all marine regions of China, especially the Yellow Sea, but there has been no study emphasizing alien species or new records of Ulva in China. In this study, based on a molecular investigation spanning over 10 years, we found six new records of Ulva in China and analysed their distribution patterns. Three northern temperate species – Ulva simplex, U. splitiana and U. partita – were also distributed in temperate marine areas in China. Meanwhile, three subtropical species – U. meridionalis, U. tepida and U. chaugulii – were found to be dominant in the green tides in the South China Sea, and have also successfully exploited temperate areas, especially U. meridionalis which has a wide distribution in China. U. meridionalis has an unusual morphology distinct from the other three types reported in Ulva: along the middle of the longitudinal axis, both sides of the thallus are connected, forming a thin sheet in the middle with bilateral tubular structures, like a bow-tie in transverse view. We found a rapid Ulva bioinvasion and we provide fundamental new data for the future monitoring and risk assessment of green tides in China.

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Ulva prolifera is a green macroalgae with an extremely high growth rate that can accumulate biomass with considerable protein content. To set up an available seaweed expression system, a prior step is to isolate endogenous and strong constitutive promoters. For this reason, the full-length genomic actin1 gene from U. prolifera (Upactin1) was cloned and its 5′ flanking sequence was obtained by genome walking. The Upactin1 open reading frame consisted of 1134 nucleotides encoding 377 amino acid residues. Besides 4 exons and 3 introns in the coding region, an extra leader intron was identified in the 5′ untranslated region. According to quantitative GUS assays based on transient expression, the promoter activity of the Upactin1 5′ flanking region was found to be several times higher than that of the widely-used cauliflower mosaic virus 35S (CaMV35S) in all tested species of Ulva. In addition, precise deletion of the leader intron led to a significant decrease of promoter strength in U. prolifera, and almost entire loss of strength in U. linza and U. pertusa. To our knowledge, this is the first report to prove function of a leader intron in algae. The 5′ flanking region of Upactin1 was shown to be a much stronger promoter than the foreign CaMV35S, and its activity was highly dependent on the presence of the leader intron. We propose that the Upactin1 promoter could serve as an endogenous and strong constitutive element for genetic engineering of U. prolifera.