We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P sac7d -hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P sac7d -hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His 6 -tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (⌬herA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

Solar-driven interfacial evaporation with heat localization is an efficient method for large-scale water purification. However, due to the high latent heat of water evaporation and dilute solar flux (1 kW m–2), the solar steam productivity is low. Here, the latent heat of water evaporation was reduced because of the capillary water state in wood channels. We constructed a wood-based 3D solar evaporator via regulating the hydrophilicity of a surface of burnt wood and adjusting the height of the wood above a water surface. Capillary water was formed in the light absorption layer, resulting in the latent heat decrease from 2444 to 1769 J g–1. A high evaporation rate of 1.93 kg m–2 h–1 under one sun irradiation (1 kW m–2) was achieved. Together with the environmental energy-harvesting ability, the evaporation rate reached 3.91 kg m–2 h–1 (per occupied area), which is among the best values ever reported. More importantly, the 3D solar evaporator works efficiently in a water collection device, yielding 2.2 times more water than that of a common interfacial evaporator.

Soybean (Glycine max L.) seeds showing serious symptoms from rotted pods were collected from fields during the harvesting period (July to August of 2020) in Taihu Farm, Jingzhou City, Hubei Province, China. Fusarium strains were frequently encountered during fungal isolation. According to the morphology and pre-pathogenicity tests, six strains showing variable effects on the seeds were selected for identification based on morphology and multi-locus phylogenetic analysis of the internal transcribed spacer region of the rDNA (ITS), translation elongation factor (EF-1α), calmodulin (CAM), beta-tubulin (TUB), and partial RNA polymerase second largest subunit (RPB2), and to evaluate the pathogenic abilities on seed, root, and pod. The results indicated that the strains contained two species (Fusarium fujikuroi and F. proliferatum) in the Fusarium fujikuroi species complex (FFSC) and two species (F. luffae and F. sulawense) from the Fusarium incarnatum-equiseti species complex (FIESC). The two species of FFSC were more aggressive than those of FIESC on soybean seed, root, and pod. Among the strains, F. proliferatum YZU 201408 exhibited the most pathogenicity on all tests with 72.2–90% disease severity. Keywords: Fusarium, Glycine max L., Morphology, Pathogenicity, Phylogenetic analysis

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP-hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins.

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.