Journal of Zhejiang University - Science A

Ferguson, Ian

doi: 10.1007/BF02840912pmid: 14674021

Mei, Liu; Zong-xiu, Sun; Jie, Zhu; Tong, Xu; Harman, Gary; Matteo, Lorito

doi: 10.1007/BF02840913pmid: 14674022

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and glu 78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech 42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani , while the exochitinase-encoding gene, nag 70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc 78. A few lines expressing either ech 42 or nag 70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Chang-jie, Xu; Kun-song, Chen; Ferguson, Ian

doi: 10.1007/BF02840914pmid: 14674023

Suspension-cultured apple fruit cells ( Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

Jing, Jin; Cheng, Zhu; Hong-xin, Zhang; Zong-xiu, Sun

doi: 10.1007/BF02840915pmid: 14674024

A gravity-insensitive mutant was isolated from rice ( Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens . The mutant’s shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

Guo-qing, He; Qi-he, Chen; Xiao-jie, Ju; Nai-dong, Shi

doi: 10.1007/BF02840916pmid: 14674025

An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface methodology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K 2 HPO 4 , MgSO 4 ·7H 2 O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K 2 HPO 4 0.206 g/100 ml and MgSO 4 ·7H 2 O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth.

Jin-fu, Wang; Yi-fan, Wu; Jenny, Harrintong; McNiece, Ian

doi: 10.1007/BF02840917pmid: 14674026

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34 + /c-kit + cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34 + /c-kit + cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34 + /c-kit + cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34 + cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34 + /c-kit + cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients.

Xing-guo, Gong; Wen-tao, Zhong; Wen-ying, Wu

doi: 10.1007/BF02840918pmid: 14674027

β-1,4-galactosyltransferase (β4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of β4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse β4Gal-T gene in Escherichia coli (E. coli) . The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E.coli with isopropyl-β-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse β4Gal-T. The transcriptional product of β4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.

Yong-quan, Li; Pei-lin, Cen; Shi-fei, Chen; Dan, Wu; Jing, Zheng

doi: 10.1007/BF02840919pmid: 14674028

Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor . DNA microarray analysis revealed that ecr A1/A2, which mapped at distant sites from red lucus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway. ecr A1 and ecr A2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecr A1/A2 was positive regulatory element for red gene cluster.

Yan-qing, Cong; Zu-cheng, Wu; Qian, Ye; Tian-en, Tan

doi: 10.1007/BF02840920pmid: N/A

A novel in-situ electrochemical oxidation method was applied to the degradation of wastewater containing chlorophenol. Under oxygen sparging, the strong oxidant, hydrogen dioxide, could be in-situ generated through the reduction of oxygen on the surface of the cathode. The removal rate of chlorophenol could be increased 149% when oxygen was induced in the electrochemical cell. The promotion factor was estimated to be about 82.63% according to the pseudo-first-order reaction rate constant (min −1 ). Important operating parameters such as current density, sparged oxygen rate were investigated. Higher sparged oxygen rate could improve the degradation of chlorophenol. To make full use of oxygen, however, sparged oxygen rate of 0.05 m 3 /h was adopted in this work. Oxidation-reduction potential could remarkably affect the generation of hydrogen peroxide. It was found that the removal rate of chlorophenol was not in direct proportion to the applied current density. The optimum current density was 3.5 mA/cm 2 when initial chlorophenol concentration was 100 mg/L and sparged oxygen rate was 0.05 m 3 /h.

Xin-da, Lin; Xin-hua, Lin; Jia-an, Cheng

doi: 10.1007/BF02840921pmid: 14674030

A wing specific F1 genetic screen was carried out using the powerful Drosophila genetic system, combined with yeast FRT/FLP and GAL4/UAS system. Form the wing phenotypes and germline clone embryonic cuticle phenotypes observed in these mutant alleles, a number of mutant alleles of known or unknown genes were isolated. Among them, fifteen mutant alleles related to Wingless signal transduction were further isolated; the arm of these mutations located were determined, and their location in the chromosome were roughly mapped.