Luo Yunbo

Cloning and DNA-binding properties of ethylene response factor, LeERF1 and LeERF2, in tomato.

Two new genes, LeERF1 andLeERF2, were isolated from a tomato (Lycopersicon esculentum cv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of the NP24 gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their binding in vitro.

Ochratoxin A and ochratoxin-producing fungi on cereal grain in China: a review

The mycotoxin ochratoxin A (OTA) is known to be the main contaminant of cereal grain and has become increasingly important in recent years. Therefore, a survey of ochratoxigenic fungi and OTA contamination in China is a special challenge. This paper summarises data on cereals and moulds (Aspergillus niger, Penicillium verrucosum, Aspergillus ochraceus, for example) and on grain and OTA from 1973 by searching Chinese information databases (NCKI, VIP, DuXiu etc.), calculating the OTA-producing mould detection rate, referring to sampling locations, latitude and temperature, and also combining six grain-producing areas of ochratoxigenic fungi and OTA positive rate through a comprehensive analysis. It is concluded that in China rice (excluding shell rice) has less OTA contamination than wheat or maize. The contamination of cereal grains with Aspergillus section Nigri (formerly of the A. niger group) is a serious problem in China, and these fungi may be the main ochratoxigenic fungi on cereals.

Transgenic cotton could safely be grown since CpTI toxin rapidly degrades in the rhizosphere soil

Abstract Cultivation of transgenic plants is debated worldwide. Potential environmental risks have to be considered, before acceptance of expanding cultivation, despite the advantages of the use of fewer pesticides. Here, the potential effects on soil ecosystems of transgenic plants have been studied. As a model, genetically engineered cotton producing cowpea trypsin inhibitor (CpTI) has been used. The degradation of CpTI in the rhizosphere of the transgenic CpTI+Bt (Bacillus thuringiensis) cotton cultivar SGK321 was assessed. During plant development, concentrations of CpTI toxin in the rhizosphere were measured using an enzyme-linked immunosorbent assay (ELISA). As the plants developed, the residue of CpTI in the rhizosphere increased, and reached a peak at topping stage (100 days after planting). After this stage, the residue began to decrease, and was nil the following year (258 days after planting). The conclusion is that genetically engineered cotton can safely be cultivated since no accumulation of substances released from the transgenic plants was persistent in the soil.

Real-time quantitative PCR detection of Escherichia coli O157:H7

A rapid and accurate real-time quantitative polymerase chain reaction (Real-time PCR) method with SYBR GreenⅠfor detecting Escherichia coli O157:H7 was established. A pair of amplify primers were designed to amplify eae gene. The dissociation curves showed that the amplified product was very specific. The optimal reaction program and standard curve were founded. The result indicated that Real-time PCR was sensitive 1000 times more than ordinarily PCR.

Relationship between calcium and ABA in ethylene synthesis in tomato fruit

The influence of calcium and ABA on ethylene biosynthesis was investigated at different stages and patterns of tomato ( Lycopersicon esculentum cv. Lichun) fruit to analyse the relationship between calcium and ABA in relation to the ethylene biosynthesis system. The ethylene production of discs excised from mature green tomato fruits increased rapidly with 100 μmol/l ABA or 100 mmol/l calcium treatment. The increase with the two treatments combined was more than that after application of a single chemical, which suggests that these two chemicals could play synergistic roles in the ethylene synthesis of mature green tomato fruits. It was also found that application of the calcium chelator EGTA inhibited ethylene synthesis, and the production of ethylene after application of EGTA plus ABA was higher than that after treatment with EGTA alone. However, the effect changed when immature fruits were treated with these chemicals. ABA inhibited ethylene synthesis obviously and calcium still promoted it. When we treated immature fruit discs with both calcium and ABA simultaneously, ethylene production was more than that of ABA and less than that of calcium, which indicates that ABA and calcium play agonistic roles in ethylene biosynthesis of the immature tomato fruit discs. Furthermore, ethylene release from transgenic antisense ACS immature tomato fruit discs with application of both calcium and ABA was inhibited markedly, but was stimulated when the mature fruits were treated with ABA and calcium. All of these data suggest that there are different relationships between calcium and ABA in the ethylene synthesis system in different patterns and stages of tomato fruit ripening.

Enzyme linked immunosorbent assay for PAT protein detection in genetically modified rape

The immunoassay method to detect genetically modified (GM) rape(Brassica campestris ) containing the phosphinothricin acetyltransferase (PAT) were developed and applied. The purified PAT was identified by Western blot and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both saturated ammonium sulfate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of polyclonal antibody has been demonstrated in ELISA test. The result of the ELISA for antiserum sensitivity was about 2×10-5 mg/mL and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1 %. The detections of transgenic plants were evaluated with two transgenic rapes (MS1/RF1 and MS8/RF3) and the transgenic rapes (MS1/RF1 and MS8/RF3) could be easily distinguished by ELISA.

Influence of salt stress on expression of some genes involved in the ethylene signalling pathway in tomato seedlings

Differences in immunostimulation activity of CpG oligodeoxynucleotides (ODNs) between pigs and mice were studied. In accordance with current research data, three kinds of CpG ODN – 2006, D19 and 1826, which were identified as optimally active in humans, pigs and mice, respectively – and control ODN D48, a non CpG-containing ODN, were selected and synthesized. Results indicated that CpG ODN D19 strongly stimulated the proliferation of porcine peripheral blood mononuclear cells (PBMCs), and the amount of γ-interferon in the supernatant of activated cells increased, exceeding that of all other ODNs tested, while mouse splenocytes did not respond. CpG ODN 1826 strongly stimulated the proliferation of mouse splenocytes, but porcine PBMCs remained unresponsive. CpG ODN 2006 effectively stimulated proliferation of both porcine PBMCs and mouse splenocytes. Therefore some specific CpG ODNs, D19 and 1826, selectively stimulated animal species, while CpG ODN 2006 possessed a common stimulation effect. In addition, CpG ODN D19 predominantly induced the response of type 1 T-helper cells in vivo .

PCR for the detection of the anti-herbicide genes in genetically modified organisms

Genetically modified organisms (GMOs) containing anti-herbicide genes account for more than 75% of all GMO and the proportion is increasing. The polymerase chain reaction (PCR) method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material in foodstuffs and PCR screening for the presence of transgenic components in food is becoming a routine method in modern food analysis. In this study, the five kinds of the anti-herbicide genes (bar, pat, cp4-epsps1, cp4epsps2 and gox) were examined by PCR in four kinds of genetically modified crops (soy, maize, cotton and rape). Results indicate that the method was sensitive, specific and credible, and that all genetically modified and approved crops containing the anti-herbicide genes can be examined by this method.