Comparative analysis of MIR168 promoters in three plant species.

MicroRNAs (miRNAs) play important roles in the regulation of gene expression by post-transcriptionally targeting mRNAs for cleavage or translational repression. miR168 is a key miRNA because it regulates the expression of the slicer protein ARGONAUTE1 (AGO1), which catalyzes mRNA cleavage. Interestingly, plant miR168s are highly evolutionarily conserved; however, it is unclear whether MIR168 promoter elements and expression patterns are also conserved. Here, we isolated MIR168 promoters from monocot rice and dicot grape genomes. To determine the expression pattern, different promoters were fused to a beta-glucoronidase reporter gene and the resulting constructs were then transformed in Arabidopsis. The results revealed clear differences in the MIR168 promoter sequence of monocot and dicot plant species. Moreover, the pattern of MIR168 promoter expression differed between monocots and dicots. These results suggest that, unlike that of miR168, the MIR168 promoter is not conserved in monocots and dicots.

shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells.

Here, we investigated the effects of blocking chicken telomerase reverse transcriptase (chTERT) in MDCC-MSB1 cells, using small-hairpin RNAs (shRNAs) to interfere with gene expression. shRNAs specific to chTERT mRNA were designed, cloned into DNA plasmid vectors, and transfected into MDCC-MSB1 cells. The transfected chTERT RNAs were expressed by the RNA polymerase machinery of the MDCC-MSB1 cells. mRNA expression in transfected MDCC-MSB1 cells was detected using real-time PCR. After transfection, telomerase activity was monitored via a modified telomeric repeat amplification protocol assay, and cell cycle analysis was performed using a flow cytometer. At 72 h after transfection, chTERT expression was considerably reduced in cells transfected with shRNA; the highest inhibition rate was 89%. Compared with the control group, telomerase activity was significantly reduced and the cells failed to progress to S phase. shRNA effectively reduced telomerase activity and prohibited the transition of MDCC-MSB1 cells from G2/M to S phase.

Comparative study of neural differentiation of bone marrow mesenchymal stem cells by different induction methods.

Neurogenic differentiation of bone marrow (BM) mesenchymal stem cells (MSCs) offers a new hope for patients with many neurological disorders. Several chemical inducers are employed to induce BMMSCs differentiation into nerve cells. In the present study, we compared different inducers [2-mercaptoethanol (BME), tretinoin (ATRA), dimethyl sulfoxide/butylated hydroxyanisole (DMSO/BHA), and indomethacin/3-isobutyl-1-methylxanthine (indomethacin/IBMX)] on the neurogenic differentiation of BMMSCs and aimed to identify a more efficient and safer method. The MSCs were first identified by their ability to adhere to plastic and by the expression of positive (CD44, CD90, and CD105) and negative (CD34) markers assessed by flow cytometry. The efficiency of the neurogenic differentiation was determined by assessing the mRNA and protein expression of nestin, microtubule-associated protein-2 (MAP2), neuron specific enolase (NSE), and glial fibrillary acidic protein (GFAP) by reverse transcription-polymerase chain reaction and western-blot, respectively. The effect of these inducers on cell viability was also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. This comprehensive study shows that indomethacin/IBMX is better than BME, DMSO/BHA, and ATRA both in terms of efficiency and safety, while BME suppressed the growth and proliferation of MSCs.

A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs.

Novel method to establish molecular identity using inter-simple sequence repeat markers in cotton (Gossypium hirsutum) cultivars.

Plant molecular identity (ID) is used to describe molecular characteristics of plants, which should contain all of the necessary information. Using inter-simple sequence repeat (ISSR) primers, molecular ID can be described in a way that reflects the polymerase chain reaction (PCR) conditions, annealing temperature, and the bands obtained in PCR amplification. A new complete molecular ID system is described in this study, which can be easily used and expanded to include more information. Using three cotton cultivars, we analyzed the products of PCR with ISSR primers and discussed the strategy for establishing their molecular ID. Using the segmented naming method, we designate the simple names and the full name systems of these three cultivars.

Molecular cloning and characterization of the pseudorabies virus UL31 gene.

We amplified a 816-bp sequence of the UL31 gene from the pseudorabies virus (PRV) Becker strain genome. Evidence that this was the UL31 gene was confirmed by cloning and sequencing. The PRV UL31 gene encodes a putative protein of 271-amino acid residues, which was designated the UL31 protein. Bioinformatic analysis indicated that PRV UL31 contains a conserved PHA03328 domain, closely related with the herpes virus nuclear egress lamina protein UL31 family and highly conserved among counterparts encoded by herpes UL31 genes. Nucleic acid sequence and amino acid sequence alignments demonstrated that PRV UL31 has a relatively higher homology with UL31 homologous proteins of subfamily Alphaherpesvirinae than other subfamilies. In addition, phylogenetic analysis showed that PRV UL31 has a close evolutionary relationship with members of the subfamily Alphaherpesvirinae, especially bovine herpesvirus 1 (BoHV-1), BoHV-5, equine herpesvirus 4 (EHV-4), EHV-9 and EHV-1. Antigen prediction demonstrated that several potential B-cell epitopes are located in PRV UL31. Additionally, secondary structure and three-dimension structure prediction revealed that PRV UL31 predominantly consists of α-helix. Taken together, these results provide insight on the function and mechanism of UL31 during PRV infection.

Effects of exogenous 5-aminolevulinic acid on PIP1 and NIP aquaporin gene expression in seedlings of cucumber cultivars subjected to salinity stress.

Aquaporins play a direct role in plant water relation under salt stress, but the effects of 5-aminolevulinic acid (ALA) on aquaporin gene expression in salt-treated plants remain unknown. This study investigated the potential effects of exogenous ALA (50 mg/dm3) on aquaporin expression levels under salt stress (75 mM NaCl) in the salt-sensitive (Jinchun No.4) and the relatively salt-tolerant cucumber (Jinyou No.1) seedlings. The expressions of cucumber PIP aquaporin gene (CsPIP1:1) and cucumber NIP aquaporin gene (CsNIP) were analyzed in 20-day-old seedling leaves at 2, 4, 8, 16, and 24 h after ALA treatment. After treatment with saline alone and ALA alone, CsPIP1:1 and CsNIP gene expression levels in the 2 cucumber cultivars increased to maximum at 2 h. The aquaporin gene expression in salt-treated cucumber seedling leaves was considerably higher than that in leaves subjected to exogenous ALA. Further, the aquaporin expression levels in Jinchun No.4 were higher than those in Jinyou No.1, reaching 5.20- and 2-fold induction levels, respectively. After treatment with both ALA and NaCl, the CsNIP gene expression was downregulated in both the cucumber cultivars, while that of CsPIP1:1 decreased at 2 h and then increased to 3.8-fold in Jinchun No.4. In Jinyou No.1, CsPIP1:1 gene expression gradually increased to 2.3-fold at 4 h, followed by a decline in expression. The results indicated that ALA might delay and counteract the upregulated expression of CsPIP1:1 and CsNIP genes in cucumber seedlings under NaCl stress. Thus, salt tolerance of cucumber seedlings might be enhanced by ALA application.

Molecular characterization of the pseudorabies virus UL2 gene.

A 948-bp sequence of the UL2 gene was amplified from the pseudorabies virus (PRV) Becker strain genome using polymerase chain reaction, and the gene identity was confirmed through further cloning and sequencing. Bioinformatic analysis indicated that the PRV UL2 gene encodes a putative polypeptide with 315-amino acid residues. Its encoding protein, designated UL2, has a conserved uracil-DNA glycosylase (UDG)_F1 domain, which is closely related to the herpesvirus UDG family and is highly conserved among its counterparts encoded by UDG genes. Multiple nucleic acid and amino acid sequence alignments suggested that the product of PRV UL2 has a relatively higher homology with UL2-like proteins of Alphaherpesvirinae than that of other subfamilies of Herpesviridae. In addition, phylogenetic analysis showed that PRV UL2 had a close evolutionary relationship with members of Alphaherpesvirinae, especially members of the genus Varicellovirus of bovine herpesvirus 1 and bovine herpesvirus 5. Antigen prediction indicated the presence of several potential B-cell epitopes in PRV UL2. In addition, secondary structure and 3-dimensional structure prediction revealed that PRV UL2 consisted predominantly of an α-helix. Taken together, these results provide molecular biological insight for the further study of the function and mechanism of UL2 during PRV infection.

Molecular cloning and characterization of the pseudorabies virus US1 gene.

Using polymerase chain reaction, a 1050-bp sequence of the US1 gene was amplified from the pseudorabies virus (PRV) Becker strain genome; identification of the US1 gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV US1 gene encodes a putative polypeptide with 349 amino acids. The encoded protein, designated PICP22, had a conserved Herpes_IE68 domain, which was found to be closely related with the herpes virus immediate early regulatory protein family and is highly conserved among the counterparts encoded by Herpes_IE68 genes. Multiple nucleic acid sequence and amino acid sequence alignments suggested that the product of PRV US1 has a relatively higher homology with ICP22-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV US1 has a close evolutionary relationship with members of the genus Varicellovirus, especially Equid herpes virus 1 (EHV-1), EHV-4 and EHV-9. Antigen prediction indicated that several potential B-cell epitopes are located in PICP22. Also, subcellular localization analysis demonstrated that PICP22 is predominantly located in the cytoplasm, suggesting that it might function as a cytoplasmic-targeted protein.