RESUMO
This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 µg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 µg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors.
Assuntos
Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Lepidópteros/fisiologia , Nicotiana/genética , Folhas de Planta/genética , Agrobacterium tumefaciens/genética , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/biossíntese , Endotoxinas/biossíntese , Expressão Gênica , Vetores Genéticos , Proteínas Hemolisinas/biossíntese , Herbivoria , Larva/fisiologia , Controle Biológico de Vetores , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Nicotiana/metabolismo , Transformação Genética , TransgenesRESUMO
The objective of this study was to observe the effects of human umbilical cord mesenchymal stem cells (UCMSCs) on the proliferation and apoptosis of endometriotic cells. Endometriotic cells and UCMSCs were primarily cultured in vitro. In the experimental group, a UCMSC and endometriotic cell non-contact co-culture system was established. The control group consisted of 1 x 10(5) endometriotic cells cultured alone. The proliferation and apoptosis of endometriotic cells were respectively detected using the MTT method and flow cytometry. The mRNA expression level of the tensin homologue gene (PTEN) in endometriotic cells was detected by reverse transcription-polymerase chain reaction amplification. Compared with the control group, the proliferation of endometriotic cells in the experimental group was clearly inhibited (P < 0.05) and time-dependent (P < 0.05). In addition, the number of apoptotic cells were significantly increased (P < 0.05), and the amount of cells, which entered S phase from G1 phase, decreased significantly. Furthermore, the mRNA expression level of the PTEN gene in the experimental group was significantly higher than in the control group (P < 0.05). These results suggest that UCMSCs might inhibit the proliferation of human endometriotic cells in vitro and promote their apoptosis by upregulating the expression of PTEN.
Assuntos
Apoptose , Proliferação de Células , Endometriose/patologia , Endométrio/citologia , Células-Tronco Mesenquimais/citologia , Adulto , Células Cultivadas , Técnicas de Cocultura , Endométrio/patologia , Feminino , Humanos , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Cordão Umbilical/citologiaRESUMO
Beauveria bassiana is a soil fungus that parasitizes arthropod species, and is used to control the Asian corn borer in Northeast China. In this study, B. bassiana was investigated in Xiaoxian County and Baicheng City, and the results were compared with those of Gongzhuling City, where the fungus was not applied. Using the inter-simple sequence repeat (ISSR) molecular marker technique, 198 isolates were extracted from Asian corn borer and other insect cadavers, and soil and air, and two released strains were analyzed to trace the infection source. In Xiaoxian and Baicheng populations, artificially released B. bassiana subpopulations were more abundant than indigenous fungi, and the released strains were the main cause of disease in those areas. Artificial B. bassiana displayed positive effect on overwintering of Asian corn borers in corn straw stacks in Xiaoxian County. Indigenous populations in Gongzhuling City showed higher genetic variation. In summary, we identified a significant correlation between genetic distance and geographic distance (P < 0.01).
Assuntos
Beauveria/classificação , Beauveria/genética , Repetições de Microssatélites , Mariposas/microbiologia , Animais , Evolução Molecular , Fluxo Gênico , Humanos , Técnicas de Amplificação de Ácido Nucleico , Polimorfismo GenéticoRESUMO
This study aimed to explore how tumor-associated fibroblasts (TAFs) promote the proliferation and angiogenesis of tumor cells via the paracrine mechanism in vitro. Conditioned media (CM) of ovarian TAFs and normal fibroblasts (NFs) were collected. Ovarian cancer cells (OCCs) were treated with 2 mL TAFs-CM and NFs-CM in experimental and control groups, respectively; 20 mM SB431512, a specific small molecule inhibitor of transforming growth factor-ß (TGF-ß), was added in the experimental group as the intervention group. The cell cycle was determined in each group. mRNA expressions of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF), and protein expressions of α-SMA and VEGF were detected in each group. Proliferation of OCCs was significantly promoted in the experimental group compared with that of the control group. The proliferative effect was obviously inhibited in the intervention group. The mRNA expressions of PCNA, α-SMA, and VEGF, and protein expressions of α-SMA and VEGF were all dramatically up-regulated in each group, and were strongly inhibited by SB-431512. TAFs promote the proliferation of OCCs via paracrine and up-regulated expression of angiogenic genes and proteins, which can be effectively inhibited by inhibiting the TGF-ß signaling pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/metabolismo , Neoplasias Ovarianas/metabolismo , Actinas/genética , Actinas/metabolismo , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos/fisiologia , Humanos , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/patologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The blood clam, Tegillarca granosa, is widely cultivated in China. We isolated 6 microsatellite loci from T. granosa and used them to investigate genetic diversity and population structure of 5 widely distributed populations of blood clam collected from eastern and southeastern China. The allele number per locus varied from 4 to 9, and the polymorphism information content value was 0.301 to 0.830. The mean observed and expected heterozygosities varied from 0.304 to 0.460 and 0.556 to 0.621, respectively; the population from Yueqing had the smallest observed heterozygosity. In the neighbor-joining tree, Shandong, Fenghua and Yueqing populations clustered together, and there was geographic divergence between Shandong and Guangxi populations. Some microsatellite loci that were isolated from these mainland China samples were not found in blood clams collected from Malaysia.