Function analysis of transcription factor OSR1 regulating osmotic stress resistance in maize.

BACKGROUND: Maize is a major cereal crop world widely, however, the yield of maize is frequently limited by dehydration and even death of plants, which resulted from osmotic stress such as drought and salinity. Dissection of molecular mechanisms controlling stress tolerance will enable plant scientists and breeders to increase crops yield by manipulating key regulatory components. METHODS: The candidate OSR1 gene was identified by map-based cloning. The expression level of OSR1 was verified by qRT-PCR and digital PCR in WT and osr1 mutant. Electrophoretic mobility shift assay, transactivation activity assay, subcellular localization, transcriptome analysis and physiological characters measurements were conducted to analyze the function of OSR1 in osmotic stress resistance in maize. RESULTS: The osr1 mutant was significantly less sensitive to osmotic stress than the WT plants and displayed stronger water-holding capacity, and the OSR1 homologous mutant in Arabidopsis showed a phenotype similar with maize osr1 mutant. Differentially expressed genes (DEGs) were identified between WT and osr1 under osmotic stress by transcriptome analysis, the expression levels of many genes, such as LEA, auxin-related factors, PPR family members, and TPR family members, changed notably, which may primarily involve in osmotic stress or promote root development. CONCLUSIONS: OSR1 may serve as a negative regulatory factor in response to osmotic stress in maize. The present study sheds new light on the molecular mechanisms of osmotic stress in maize.

Boundary lubrication by associative mucin.

Mucus lubricants are widely distributed in living organisms. Such lubricants consist of a gel structure constructed by associative mucin. However, limited tribological studies exist on associative mucin fluids. The present research is the first to investigate the frictional behavior of a typical intact vertebrate mucin (loach skin mucin), which can recover the gel structure of mucus via hydrophobic association under physiological conditions (5-10 mg/mL loach skin mucin dissolved in water). Both rough hydrophobic and hydrophilic polydimethylsiloxane (PDMS) rubber plates were used as friction substrates. Up to 10 mg/mL loach skin mucin dissolved in water led to a 10-fold reduction in boundary friction of the two substrates. The boundary-lubricating ability for hydrophilic PDMS decreased with rubbing time, whereas that for hydrophobic PDMS remained constant. The boundary-lubricating abilities of the mucin on hydrophobic PDMS and hydrophilic PDMS showed almost similar responses toward changing concentration or sodium dodecyl sulfate (SDS). The mucin fluids reduced boundary friction coefficients (µ) only at concentrations (c) in which intermucin associations were formed, with a relationship shown as µ ∼ c(-0.7). Destroying intermucin associations by SDS largely impaired the boundary-lubricating ability. Results reveal for the first time that intermolecular association of intact mucin in bulk solution largely enhances boundary lubrication, whereas tightly adsorbed layer plays a minor role in the lubrication. This study indicates that associated mucin should contribute considerably to the lubricating ability of biological mucus in vivo.

[Secretable eukaryotic expression of recombinant chicken Iglambda and preparation of monoclonal antibodies against chicken Iglambda].

AIM: To construct the eukaryotic expression vector for chicken Iglambda light chain, to express it on COS7 cells and to prepare the monoclonal antibodies against chicken Iglambda. METHODS: The cDNA of chicken Iglambda light chain with signal peptide sequence was amplified and then inserted into eukaryotic expression plasmid pcDNA3 after double enzyme cutting. The constructed recombinant vector was transfected into COS7 cells by lipofectamin and the secretable eukaryotic expression of chicken Iglambda light chain was verified by Western blot. The monoclonal antibodies (mAbs) against chicken Iglambda light chain were prepared by immunizing BALB/c mice with 2 x 10(6) chicken B cells and by cell fusion technology. RESULTS: The eukaryotic expression vector was successfully constructed. Western blot demonstrated that chicken Iglambda light chain existed in the cultural supernatant. The hybridoma lines secreting anti-Iglambda mAbs were screened by indirect ELISA. The specific reactivity between anti-Iglambda mAbs and recombinant chicken Iglambda light chain was detected by Western blot. CONCLUSION: The secreted recombinant chicken Iglambda light chain and anti-Iglambda mAbs provide a basis for further study of the functions of chicken Iglambda.

Expressed genes in regenerating rat liver after partial hepatectomy.

AIM: To reveal the liver regeneration (LR) and its control as well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers. METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of them showed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressed in 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.

Identification of expressed genes in regenerating rat liver in 0-4-8-12 h short interval successive partial hepatectomy.

AIM: To identify the genes differentially expressed in the regenerating rat liver of 0-4-8-12 h short interval successive partial hepatectomy (SISPH) and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements screened from subtractive cDNA libraries were made into a cDNA microarray (cDNA chip). Extensive gene expression analysis following 0-4-8-12 h SISPH was conducted by microarray. RESULTS: One hundred and eighty-three elements were selected, which were either up- or down-regulated more than 2-fold at one or more time points after SISPH. Cluster analysis and generalization analysis showed that there were five distinct temporal patterns of gene expression. Eighty-six genes were unreported, associated with liver regeneration (LR). CONCLUSION: Microarray analysis shows that the down regulated genes are much more than the up-regulated ones in SISPH; the numbers of genes expressed consistently are fewer than that expressed immediately; the genes expressed in high abundance are much fewer than that increased 2-5-fold. The comparison of SISPH with partial hepatectomy (PH) shows that the expression trends of most genes in SISPH and in PH are similar, but the expression of 43 genes is specifically altered in SISPH.

Large scale of identification of differentially expressed genes in the regenerating rat liver after SISPH.

Extensive gene expression analysis was carried out after a 0, 4, 36, 72, 96 h short interval successive partial hepatectomy (SISPH) was performed. A total of 185 elements were identified as differing by more than two-fold in their expression levels at one or more time points. Of these 185 elements, 103 were up-regulated, 82 were down-regulated and 86 elements were unreported genes. Quite a few genes were previously unknown to be involved in liver regeneration (LR). Using cluster and general analysis, we found that the genes at five time points of the SISPH share eight different types of different expression profiles and eight distinct temporal induction or suppression patterns. A comparison of the gene expression in SISPH with that after PH found that 41 genes were specifically altered in SISPH, and 144 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but they were present in different amounts at the different time points. The conclusions are that (i) microarrays combined with suppressive subtractive hybridization (SSH) can effectively identify genes involved in LR on a large scale; (ii) more genes were up-regulated than down-regulated; (iii) there are fewer abundantly expressed genes than those with increased levels of 2-5 fold.

Gene expression differences of regenerating rat liver in a short interval successive partial hepatectomy.

AIM: To identify the genes expressed differentially in the regenerating rat liver in a short interval successive partial hepatectomy (SISPH), and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements selected from subtractive cDNA libraries were conformed to a cDNA microarray (cDNA chip). An extensive gene expression analysis following 0-36-72-96-144 h SISPH was performed by microarray. RESULTS: Two hundred and sixteen elements were identified either up- or down-regulated more than 2-fold at one or more time points of SISPH. By cluster analysis and generalization analysis, 8 kinds of ramose gene expression clusters were generated in the SISPH. Of the 216 elements, 111 were up-regulated and 105 down-regulated. Except 99 unreported genes, 117 reported genes were categorized into 22 groups based on their biological functions. Comparison of the gene expression in SISPH with that after partial hepatectomy (PH) disclosed that 56 genes were specially altered in SISPH, and 160 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but in various amount and at different time points. CONCLUSION: Genes expressed consistently are far less than that intermittently; the genes strikingly increased are much less than that increased only 2-5 fold; the expression trends of most genes in SISPH and in PH are similar, but the expression of 56 genes is specifically altered in SISPH. Microarray combined with suppressive subtractive hybridization can in a large scale effectively identify the genes related to liver regeneration.

Identification and characterization of 177 unreported genes associated with liver regeneration.

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression profiles were studied by cluster and generalization analyses. Among them, 177 genes were identified unreported and up- or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2-10 folds, and 16 genes were either up- or down-regulated at different time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chromosomes. The cluster and generalization analyses showed that the gene expression profiles are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same profiles are similar, and those expressed in different profiles have less similarity. However, the types, characteristics and functions of the 177 genes remain to be further studied.