A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana

Tonoplast intrinsic protein (TIP) is a subfamily of the aquaporin (AQP), also known as major intrinsic protein (MIP) family, and regulates water movement across vacuolar membranes. Some reports have implied that TIP genes are associated with plant tolerance to some abiotic stresses that cause water loss, such as drought and high salinity. In our previous work, we found that an expressed sequence tag (EST) representing a TIP gene in our Glycine soja EST library was inducible by abiotic stresses. This TIP was subsequently isolated from G. soja with cDNA library screening, EST assembly and PCR, and named as GsTIP2;1. The expression patterns of GsTIP2;1 in G. soja under low temperature, salt and dehydration stress were different in leaves and roots. Though GsTIP2;1 is a stress-induced gene, overexpression of GsTIP2;1 in Arabidopsis thaliana depressed tolerance to salt and dehydration stress, but did not affect seedling growth under cold or favorable conditions. Higher dehydration speed was detected in Arabidopsis plants overexpressing GsTIP2;1, implying GsTIP2;1 might mediate stress sensitivity by enhancing water loss in the plant. Such a result is not identical to previous reports, providing some new information about the relationship between TIP and plant abiotic stress tolerance.     

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA₃) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA₁ and real-time PCR quantitative assay analyses confirmed that the decrease of GA₁ in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice.     

Expressivity tag: a novel tool for increased expression in Escherichia coli

Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3′ deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag.The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression.     

Long-term retention of dummy acoustic transmitters in adult brown trout

A group of 36 1+ age class Salmo trutta were surgically implanted with dummy acoustic tags and monitored for 370 days. In total 13 tags were expelled throughout the experiment with an overall tag loss rate of c. 0^.035 tags per day. Fish length was the only explanatory variable which had a significant association with subsequent tag expulsion. The estimated probability of retaining a tag for a year for a fish of length 32 cm was 0.76, 34 cm was 0.60 and 36 cm was 0.38. The long-term tag loss patterns were examined and discussed.     

聚乙二醇对ELP[I]40相变温度的影响

目的:研究聚乙二醇(polyethylene glycol,PEG)对类弹性蛋白(elastin-like protein,ELP)ELP[I]40相变温度(inverse temperature transition,Tt)的影响.方法:设计并合成ELP[I]40基因(由40个(VPGIG)五肽单元串联组成),表达纯化后,检测不同浓度PEG条件下ELP[I]40的Tt.结果:在ELP[I]40终浓度为25 μmol/L时,PEG浓度为5％,10％,15％,20％,25％时分别使Tt由29℃降至26.5℃,22℃,15.2℃,8.8℃,2.5℃.结论:PEG可降低ELP的Tt,可通过PEG促进ELP重组蛋白分离纯化.     

Heterotetrameric forms of human phenylalanine hydroxylase: Co-expression of wild-type and mutant forms in a bicistronic system

Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~ 50% tetramer; ~ 10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~ 80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~ 95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH₄ supplementation.