An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10^-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE^* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE^*. Promoter SPL-57 showed activity comparable to that of permE^*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE^*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
Conjugation, Genetic ; Glucuronidase/genetics* ; Promoter Regions, Genetic ; Streptomyces rimosus/genetics*

OBJECTIVETo construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.

METHODSThree heparanase-shRNA structures were designed based miR30. The targeting fragments were obtained by PCR, then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA, which was identified by PCR and sequencing. The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA, pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses, with which A375 cells were infected. Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein.

RESULTSThe lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group.

CONCLUSIONThe lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.

Genetic Vectors ; Glucuronidase ; genetics ; Lentivirus ; genetics ; MicroRNAs ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the relationship between heparanase 1 (Hpa1) expression and the development of human pancreatic cancer.

METHODSReal-time PCR and Western blot methods were used the expression of Hpa1 in 37 cases of pancreatic cancer were analyzed quantitatively. The relationship between the expression of Hpa1 and the clinicopathological features of pancreatic cancer was analyzed.

RESULTSThe expression of Hpa1 mRNA in pancreatic cancer was 23.53 +/- 4.13, in pancreatic tissues was 4.08 +/- 2.14 and in pancreatic cancer adjacent tissue was 16.93 +/- 3.06 by real-time PCR detected (P < 0.01). The expression of Hpa1 protein in normal tissue was 0.36 +/- 0.14, in pancreatic cancer adjacent tissue was 1.21 +/- 0.37 and in pancreatic cancer tissue was 1.76 +/- 0.28 by Western blot detected (P < 0.05). The expression of Hpa1 mRNA and Hpa1 protein in pancreatic cancer tissue were both higher than pancreatic cancer adjacent tissue and normal tissue. The expression of Hpa1 was significant associated with TNM grade and Invasion to nerves or vascellum lymph node metastasis separately (P < 0.05). But no relationship between histological differentiation and the tumor size (P > 0.05).

CONCLUSIONSThe Hpa1 overexpressed in pancreatic cancer tissues and its abnormal expression may be involved in the oncogenesis and development of pancreatic cancer.

Adult ; Aged ; Female ; Glucuronidase ; biosynthesis ; genetics ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms ; enzymology ; pathology ; RNA, Messenger ; genetics

Genetic transformation is an effective method to improve breeding objective traits of orchids. However, there is little information about genetic transformation of Cymbidium sinensis. Rhizomes from shoot-tip culture of C. sinensis cv. 'Qijianbaimo' were used to establish a practical transformation protocol of C. sinensis. Pre-culture time, concentration and treating methods of acetosyringone, concentration of infection bacteria fluid (OD600), infection time, and co-culture time had significant effects on β-glucuronidase (GUS) transient expression rate of C. sinensis cv. 'Qijianbaimo' rhizome. The GUS transient expression rate of rhizome was the highest (11.67%) when rhizomes pre-cultured for 39 d were soaked in bacterium suspension (OD600 = 0.9) supplemented with 200 μmol/L acetosyringone for 35 min, followed by culturing on co-culture medium supplemented with 200 μmol/L acetosyringone for 7 d. Under this transformation conditions, 3 transgenic plantlets, confirmed by GUS histochemical assay and PCR, were obtained from 400 regenerated plantlets, and the genetic transformation rate was 0.75%. This proved that it was feasible to create new cultivars by the use of Agrobacterium-mediated genetic transformation in C. sinense.
Agrobacterium ; Coculture Techniques ; Genetic Engineering ; Glucuronidase ; Orchidaceae ; genetics ; Plants, Genetically Modified ; genetics ; Polymerase Chain Reaction ; Transformation, Genetic

The aim of the studies was to investigate klotho gene effect on the endothelial dysfunction of spontaneously hypertensive rats (SHR). In this study, ten SHR and ten normal Wistar rats, all 22 week old, were prepared. After given intraperitoneal anesthesia, the rats' brains, lungs, hearts, kidneys and aortas were removed. The identification was made by means of real-time polymerase chain reaction (Real-time PCR) and Enzyme-Linked Immunosorbent Assay (ELISA). Compared with the normal group, the klotho mRNA and protein in SHR were less than those in the control group with normal corresponding values, while Endothelin-1 (ET-1)'s mRNA and protein were more than those of normal group. The analysis of the correlation of mRNA and protein in heart and aorta revealed that klotho gene was negatively correlated to ET-1. The results showed that klotho significantly decreased in SHR, which might be influenced by hypertension-induced damage on the endothelial function.
Aging ; genetics ; Animals ; Endothelin-1 ; genetics ; metabolism ; Endothelium, Vascular ; physiopathology ; Glucuronidase ; genetics ; metabolism ; Hypertension ; genetics ; physiopathology ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR

A novel transformation method was firstly established using glass beads in Dunaliella salina (D. salina). The results showed that the GUS gene, a reporter gene, was successfully expressed in D. salina. Cells of D. salina presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized including the transformation consecutive time, rotate speed, concentration of the plasmid and PEG 6000. The experiment indicated that this fit together can obtain the best results for D. salina transformation: adding 150 microL PEG and 90 microL plasmid DNA to 800 microL culture of D. salina (10(6) cells/mL) containing 300 mg glass beads, swirling 12 seconds under the rotate speed 2400r/min. This newly method can be used as a potential tool in the research of D. salina gene engineering with the advantage of more simpleness, convenience, quickness and less expense.
Chlorophyta ; genetics ; DNA ; chemistry ; genetics ; Genetic Engineering ; methods ; Glass ; Glucuronidase ; genetics ; metabolism ; Histocytochemistry ; Microspheres ; Plasmids ; genetics ; Polyethylene Glycols ; chemistry ; Time Factors ; Transformation, Genetic ; genetics

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.
Arachis/genetics* ; Fabaceae/genetics* ; GTP-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Salt Stress ; Stress, Physiological/genetics* ; Tobacco/genetics*

OBJECTIVETo determine DNA methylation status of ZIC1 and KLOTHO gene in colorectal carcinomas and its relationship with clinicopathological features of patients.

METHODSThe mRNA expression of ZIC1 and KLOTHO genes in colorectal carcinomas was detected by real-time quantitative RT-PCR, and the promoter methylation status was detected by methylation specific PCR (MSP). The relationship of ZIC1 and KLOTHO methylation status with clinicopathological features of colorectal carcinoma was analyzed.

RESULTThe mRNA expression levels of ZIC1 and KLOTHO genes were significantly down-regulated in tumor tissues when compared to adjacent nontumor tissues (P<0.001). ZIC1 and KLOTHO methylation was detected in 80.0%(20/25) and 76.0%(19/25) of colorectal tumor tissues, respectively, and the both positive rate was 64.0%(16/25).

CONCLUSIONThe down-regulated expression of ZIC1 and KLOTHO in colorectal carcinoma may relate to promoter methylation. The detection of methylation of ZIC1 and KLOTHO gene potentially provides biomarkers for diagnosis of colorectal carcinoma.

Adult ; Aged ; Colorectal Neoplasms ; genetics ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Glucuronidase ; genetics ; Humans ; Male ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; genetics

OBJECTIVETo construct short hairpin RNA (shRNA) expression vectors of RNA for specific silencing of heparanase (HPA) gene.

METHODSThe genomic sequence of HPA gene was retrieved from GenBank and the cDNA encoding shRNA for HPA gene silencing was designed. Five specific interference sequences and a random negative control sequence were inserted into the vector pGPU6/GFP/Neo. After verification by restriction enzyme digestion and sequence analysis, the recombinant vectors were transfected into MDA-MB-231 cells via lipofectamine. Fluorescent quantitative PCR and Western blotting were employed to detect the expression of HPA gene expressions in the transfected cells at the mRNA and protein levels, respectively.

RESULTSBoth restriction analysis and sequencing confirmed correct construction of the shRNA vectors. Transfected with the specific siRNA vectors HPSE-1 and HPSE-5 resulted in significantly decreased expression level of HPA protein in MDA-MB-231 cells, while negative control vector produced no significant changes in HPA expressions.

CONCLUSIONWe have obtained two shRNA vectors which can significantly down-regulate HPA expressions in MDA-MB-231 cells, which facilitates further investigation of the role HPA may play in the invasiveness and metastasis of human breast cancer.

Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Female ; Gene Expression ; Gene Targeting ; methods ; Genetic Vectors ; Glucuronidase ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Transfection

To improve the efficiency of targeted gene replacement (TGR), a dual screen (DS) system with gusA gene as negative selective marker (GUS-DS) was developed in Magnaporthe oryzae. First, we tested the endogenous beta-glucuronidase (GUS) activities of 78 fungal strains. All tested strains were GUS-, only with 3 exceptions. Whereas, after the gusA being introduced in, M. oryzae, Fusarium oxysporum and Colletotrichum lagenarium acquired high GUS activities. The gusA is thus usable as a selective maker in fungal species. With gusA as the negative marker, HPH gene as the positive marker, and the peroxisomal targeting signal receptor genes MGPEX5 and MGPEX7 as 2 instances of target genes, we established the GUS-DS system. After transformation, we collected the transformants from hygromycin B screen media and then tested the GUS activities of them. The GUS- ones were selected as potential mutants and checked in succession by PCR and Southern blotting to identify the true mutants and calculate the efficiency of GUS-DS. As a result, GUS-DS improved the screen efficiency for delta mgpex5 from 65.8% to 90.6%, and for delta mgpex7 from 31.2% to 82.8%. In addition, we established a multiple PCR (M-PCR) method for mutant confirmation. By amplifying the different regions at the targeted locus, M-PCR differentiated the wild type, the ectopic transformants and the mutants effectively and rapidly, and had the same reliability as Southern blotting. In conclusion, GUS-DS and M-PCR are useful tools to improve the efficiency of TGR and would be helpful for fungal genomics.
Escherichia coli ; enzymology ; genetics ; Gene Expression Regulation, Enzymologic ; Genes, Fungal ; Glucuronidase ; genetics ; Magnaporthe ; genetics ; Mutagenesis, Insertional ; methods ; Mutation ; Recombination, Genetic ; Transformation, Genetic