Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

抗汉坦病毒核蛋白细胞内抗体抗病毒作用的研究

本文报道在内质网和细胞浆内稳定表达抗汉坦病毒核蛋白细胞内抗体,研究抗汉坦病毒核蛋白细胞内抗体抗病毒作用及其抗病毒作用机理。在获得稳定表达抗汉坦病毒核蛋白细胞内抗体的细胞系的基础上,用MTT法检测细胞内表达抗汉坦病毒单链抗体对细胞增殖的影响,证实细胞内抗体的表达不会影响到细胞的生长。不同时间收集病毒感染毒后细胞培养培养上清和细胞裂解物,用ELISA方法检测细胞内外病毒结构蛋白含量的变化,结果表明定位于细胞内质肉和细胞浆内的抗汉坦病毒核蛋白细胞内抗体均能不同程度降低感染细胞上清中的核蛋白含量,但是不能或轻微抑制细胞内汉坦病毒核蛋白的合成。利用病毒微量滴定免疫荧光方法,对细胞内抗体与病毒的增殖关系进行了研究,证实无论在内质网还是在胞质,抗核蛋白细胞内抗体都能够抑制病毒的增殖,具有抗病毒活性。抗汉坦病毒核蛋白细胞内抗体不能抑制病毒结构蛋白的合成,其抗病毒效果是通过抑制病毒的包装而实现。     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.     

微生物1,3-1,4-β-葡聚糖酶蛋白质改造及工业应用研究进展

1,3-1,4-β-葡聚糖酶(E.C.3.2.1.73)是一种重要的工业用酶,其可以通过特异性切割毗邻β-1,3-糖苷键的β-1,4-糖苷键将β-葡聚糖或地衣多糖降解为纤维三糖和纤维四糖。微生物β-葡聚糖酶属于糖苷水解酶家族16,其三维结构为卷心蛋糕状的逆向β-片层结构。文中综述了近些年来β-葡聚糖酶在工业上的应用情况及酶蛋白质工程改造的研究进展,并对其研究前景进行了展望。     

悬钩子属种质的评价

在进行了７省悬钩子资源调查的基础上,在南京建立了悬钩子属田间基因库。３年来对田间基因库内保存并开花结果的３０个种进行了开花结果性状的记栽和评价。内容包括果实特征,糖、酸、维生素类、氨基酸、矿质元素含量分析,染色体计数等。分析了种间和种内多样性物存在及在良种选育中利用的可能性。     

Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus.     

Effect of ubiquitin carboxy-terminal hydrolase 37 on apoptotic in A549 cells

Proteins destined for degradation by the ubiquitin-proteasome system are labelled with a 76-amino acid peptide, ubiquitin, through a series of conjugation steps by the E1, E2 and E3 enzymes respectively. Ubiquitin carboxy-terminal hydrolase 37 (UCH37) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. However, it is few reports about the relationship between UCH37 and apoptosis. In order to clarify the role of UCH37 on apoptosis, the A549 cells were chosen for this study. We transfected UCH37 siRNA and pcDNA3.1-UCH37 plasmid into A549 cells, respectively. Using MTT assay, Western blot, Hoechst 33342 staining assay and flow cytometry, we found that silencing of UCH37 in A549 cells induced apoptosis. The ratio of Bax/Bcl-2 was higher in silencing of UCH37 than that in control group after silencing of UCH37 in A549 cells. Meanwhile, experiments with the A549 cell line disclose that silencing of UCH37 could induce efficiently A549 cell apoptosis through activation of caspase-9 and caspase-3. On the other hand, over-expression of UCH37 led to the opposite effect. Hence, UCH37 might play an important role in apoptotic through altering Bax/Bcl-2 ratio and enzymatic activities of caspase-9 and caspase-3.