Construction of recombinant plasmid vector for gene expression is a key step in making transgenic plants and important to study gene function and plant genetic engineering. A right choice of gene construction method can be cost-effective and achieve more diverse recombinant plasmids. In addition to the traditional methods in construction of plant gene expression vectors, such as Gateway technology, three DNA method and one step cloning, a few novel methods have been developed in recent years. These methods include oligonucleotide synthesis-based construction of small fragment gene expression vectors via competitive connection; construction of small RNA expression vector using pre-microRNA; recombination-fusion PCR method which inserts DNA fragments of multiple restriction sites into the target vector; and insertion of a DNA fragment into any region of a linear vector via In-Fusion Kit. Construction of complex vectors with many fragments uses sequence and ligation-independent cloning method, Gibson isothermal assembly or Golden Gate assembly. This paper summarizes our working experience in the area of recombinant vector construction and reports from others with an intention to disseminate ideas about currently widely used DNA recombination methods for plant transformation.
Cloning, Molecular ; DNA ; Gene Expression ; Genes, Plant ; Genetic Engineering ; methods ; Genetic Vectors ; Plants, Genetically Modified ; Plasmids ; Polymerase Chain Reaction

Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins (MIPs) that facilitate water transport across biological membranes. Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. We focused on GhPIP1;2 which belongs to the PIP subfamily and GhgammaTIP1 which belongs to the gammaTIP group of the TIP subfamily. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1;2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post anthesis. The high and preferential expression of GhPIP1;2 and GhgammaTIP1 suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion. Also, the effects of Ca2+ on aquaporins in salinity-stressed plants were studied. Researchers treated the protoplasts and plasma membrane with NaCl or CaCl2, alone or in combination. Under saline conditions, osmotic water permeability (Pf) values decreased in protoplasts and plasma membrane vesicles, and the same reduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein abundance. Two different actions of Ca2+ were observed. Increase in free cytosolic calcium concentrations associated with stress perception may lead to aquaporin closure, however, the extra-calcium would lead to an upregulation of aquaporins. Meanwhile, experiments have demonstrated HvPIP2;1, one of barley aquaporins, has a higher water and CO2 transport activity. The goal of our plant aquaporin research is to determine the key aquaporin species responsible for water and CO2 transport, and to improve plant water relations, stress tolerance, CO2 uptake or assimilation, and plant productivity.
Aquaporins ; physiology ; Cell Enlargement ; Cotton Fiber ; Gossypium ; metabolism ; physiology ; Photosynthesis ; physiology ; Plant Proteins ; physiology ; Sodium Chloride ; pharmacology ; Stress, Physiological ; physiology

Objective To screen the non-nucleoside compounds against HIV-1 reverse transcriptase by molecular modeling and bioactivity assay. Methods Surflex-Dock module of Tripos SYBYL software was used to simulate the binding pattern of 22 000 compounds in SPECS database with the active pocket of HIV-1 reverse transcriptase. Based on the simulation results, the interaction mode between the above compounds and the crystal structure of HIV-1 reverse transcriptase was analyzed. The compounds with higher docking scores and better binding pattern were determined by anti-HIV-1 ac tivities test in vitro. Results The virtual screening results showed that the docking conformation of 1- (4-fluorophenyl) -3- [2- (1H-indol-3-yl) ethyl] thiourea was similar to the embedded ligand in Rilpivirine crystal structure. 1- ( 4-fluorophenyl) -3- [ 2- ( 1H-indol-3-yl) ethyl] thiourea was held together with the key residue Lys101 in docking pocket of HIV-1 reverse transcriptase by hydrogen bonds, and hadπ-πstacking action together with the conservative residue Trp229 and the aromatic residue Tyr181 respectively. The bioassay in vitro results showed that when the proliferation rate of C8166 lymphocyte syncytium infected by HIV-1ⅢB arrived 50% ( EC50) , the concentration of 1- ( 4-fluorophenyl) -3- [ 2- ( 1H-indol-3-yl) ethyl] thiourea was 5.45μg/mL. Conclusion Molecule docking technology is an effective approach to reducing the screening of candidate compounds with micromolecular activity, and can be used to predict the interaction mode between the compound and the target receptor. In the study, active compound 1- (4-fluorophenyl) -3- [2- (1H-indol-3-yl) ethyl] thiourea has been screened out by molecule docking technology.

To enrich the resource pool of endophytic fungi from plants which produce taxol, a taxol-producing endophytic fungus TMS-26 was isolated from the stem of Taxus Media. The result of high performance liquid chromatography (HPLC) showed that TMS-26 extract exhibited similar chromatographic peaks and retention time (4.545 min) with authentic taxol. Then mass spectrometry (MS) analysis further confirmed that TMS-26 extracts contained the same mass peaks with authentic taxol ((M+Na)+=876). These indicated that the isolated endophytic fungus TMS-26 can produce taxol. According to the morphological characteristics, the molecular analysis of 18S rDNA and internal transcribed spacer nuclear rDNA gene sequence, the fungus was identified as Aspergillus fumigatus TMS-26.