AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its immunogenicity.METHOD...AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its immunogenicity.METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya1 delta Crp1 delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supematant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed.Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supematant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, bhe entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0×l0^10 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protection and care of H pylori infection.展开更多
AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was t...AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.展开更多
AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of BabA.METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression...AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of BabA.METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore,BabA immunogenicity was studied by animal test.RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank.The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA.CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of Hpyloriinfection.展开更多
The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoc...The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoculated with Potato virus X, Potato virus Y, TMV and CMV isolate RB (CMV-RB), respectively. Symptom observation showed there were no symptom differences in transgenic tobacco plants as compared with those in non-transgenie tobacco plants. ELISA also illustrated that the virus concentrations in the transgenic plants were similar to those in non-transgenic plants, indicating that no synergism is found in these plants. The transgenic tobacco plants expressing movement protein gene of ToMV or partial replicase gene of CMV-P1 were inoculated with TMV and CMV-RB, respectively. The local or systemic infected leaves were then used for elucidation of the possible virus recombination in transgenic plants with biological infectivity test, ELISA and immuno-capture RT-PCR. Within 16 months, no recombination was found between transformed genes and inoculated virus genomes. The research provides fundamental data for understanding of the possible risk of the transgenic plants expressing viral sequences.展开更多
Seventeen species of Trichoderma, isolated from soil or tree bark from Ch ina are identified based on morphological and physiological characters, and from their phylogenetic position inferred from parsimony analyses o...Seventeen species of Trichoderma, isolated from soil or tree bark from Ch ina are identified based on morphological and physiological characters, and from their phylogenetic position inferred from parsimony analyses of nucleotide sequ ences of the internal transcribed spacer regions of the rDNA cluster (ITS1 and 2) and partial sequences of translation elongation factor 1-alpha (te f1) . There were T.citrinoviride, T.longibrachiatum, T.sinensis in section Long ibrachiatum, T.atroviride, T.koningii, T.viride, T.asperellum, T.hamatum, T.e rinaceum in section Trichoderma, T.harzianum (H.lixii), T.inhamatum, T. ve lutinum, T.cerinum, T.strictipile, T.spirale, T.virens, H.nigrovirens (Trichode r ma sp.) in section Pachybasium. Among them four species: T. asperellum, T .velutinum, T.cerinum, T. spirale were reported firstly in China. In addition, two suspected new taxa (Trichoderma spp.) in Trichoderma s ec tion were proposed: Trichoderma sp.1 (ZAUT261, 4, 4A, 15A, 2C), Trich od erma sp.2 (2B, 5, 7A, 7B, 9A). Trichoderma sp.1 was similar to T.h a matum , but the temperature optimum for mycelial growth was lower than that of T.hamatum and the species tended to form hemisphaerical pustule with rela tively larger conidia (average length 4.6 μm×2.8 μm). Trichoderma sp.2 was distinguished morphologically from related species T. strigosum, T. pub escens, T. erinaceum, T. hamatum and Trichoderma sp.1 in pustules on CM D without fertile or sterile conidiophore elongation and distinctive phialide sh ape, the conidiophore branches similar to T.koningii, but the conidia sim ilar to T. viride, subglobose, conspicuously tuberculate.展开更多
By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes confe...By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes conferring blast resistance in rice. Genomic location of gene Pi25(t) conferring neck blast resistance to the Chinese isolate 92-183 (race ZC15) was verified to be located between markers A7 and RG456 on chromosome 6, with genetic distances of 1.7 cM and 1.5 cM to A7 and RG456, respectively. Leaf blast resistance of Gumei 2 to the Philippine isolate Ca89 (lineage 4) was found to be controlled by a single gene. The gene tentatively designated as Pi26(\) was located between makers B10 and R674 on chromosome 6, with genetic distances of 5.7 cM and 25.8 cM to B10 and R674 respectively. Resistant alleles at both gene loci were derived from Gumei 2, indicating an existence of resistance gene cluster in Gumei 2.展开更多
AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal D...AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.展开更多
High embryogenic calli of three cutivars of Kentucky bluegrass, Md, Bd, and Gm, were induced from mature embryos, and were proliferated on medium K3 and K5. Embryogenic calli were transformed with plamids pDM803 and p...High embryogenic calli of three cutivars of Kentucky bluegrass, Md, Bd, and Gm, were induced from mature embryos, and were proliferated on medium K3 and K5. Embryogenic calli were transformed with plamids pDM803 and pBY520 by microprojectile bombardment. Fourty-two transgenic lines had been obtained. The highest efficiency of transformation reached to 3.7% for cv. Md, 2.8% for cv. Gm, and 5 % for cv. Bd. The micro nutriment of Cupric had significant effect on transformation. The embryogenic callus cultured in dim-light condition had higher transformation efficiency than the green callus cultured in light condition for one month before transformation. The selective regime and selective pressure on the putative transgenic plants were important for obtaining the desire number of transgenic plants. It also affected the copy number of integrated genes in the genomic DNA of transgenic plants.展开更多
Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Tri...Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase (ThEn-42) was amplified by Arabidopsis leaf-PCR or genomic DNA PCR. Unfertile, dwarf and normal phenotypes appeared in the T1 generation. In addition, an enhanced resistance to S. sclerotiorum was observed. The mortality percentage (7.7% to 33.3%) in transgenic plants was significantly lower than in non-transgenic plants (86.7%) 10 days after inoculation with the pathogen.展开更多
CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably s...CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably secreted at about 180 IU/ 1e6 cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reatthch and proliferate on fresh microcarriers. This makes it very easy to scale up preduction. The microcarriers could be reused several times without affecting adhesion. proliferation and prourokinase secretion. With CMPECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1×105 IU/mg of protein. The purification factor was about 600 fold, and approxiamately 90 % of the biological activity was recovered.展开更多
The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars a...The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat P1 1 and Sat P1 2). Sat P1 1 contained 335 nucleotides, and Sat P1 2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat P1 1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat P1 2 represents the emergence of a new satellite (necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.展开更多
The physiological mechanism of maintaining the green colour of pak choy leaves (Brassicarapa var chinensis) with heat-shock treatment was studied. Chlorophyll in the outerleaves of pak choy degraded rapidly during sto...The physiological mechanism of maintaining the green colour of pak choy leaves (Brassicarapa var chinensis) with heat-shock treatment was studied. Chlorophyll in the outerleaves of pak choy degraded rapidly during storage at ambient temperature (20±2℃), aslight yellow appeared. Heat-shock treatment (46-50℃) had a mild effect on maintainingthe green colour of outer leaves. Normal chlorophyll degradation was associated with abinding of chlorophyll with chlorophyll-binding-protein preceding chlorophyll breakdown.Heat-shock treatment was found to reduce the binding-capacity between chlorophyll-binding-protein and chlorophyll. In the chlorophyll degradation pathway, pheide dioxygenasewas synthesized during leaf senescence which was considered to be a key enzyme inchlorophyll degradation. Activity of this enzyme was reduced following heat-shocktreatment, which might explain the observed reduction in chlorophyll breakdown. Twogroups of heat-shock proteins were detected in treated leaves, the first group containingproteins from 54KDa to 74Kda, and the second group contained proteins from 15KDa to29KDa. Heat-shock treatment was also found to retard the decline of glucose and fructose(the main energy substrates) of outer leaves.展开更多
Three strains of Trichoderma spp. TV112, TX003, TY009 obtained from previous experiments could inhibit the sclerotial formation of two strains of Rhizoctonia salani AG1 (-1A) isolated from the rice paddies in Hanzhou ...Three strains of Trichoderma spp. TV112, TX003, TY009 obtained from previous experiments could inhibit the sclerotial formation of two strains of Rhizoctonia salani AG1 (-1A) isolated from the rice paddies in Hanzhou of China. However, it is unclear if there are the antagonism and mycoparasitism of the Trichoderma strains tested against the mycelial growth of R. solani . The objective of this research was to evaluate the ability of the Trichoderma strains to inhibit the mycelial growth of R. solani in vitro . Dual culture testes showed all the Trichoderma strains tested inhibited the mycelial growth of R. solani. The strains also produced toxic metabolites with activity against R. solani, inhibiting the mycelial growth by 74%, 81.8%, and 53%, repectively. Electron microscopic observations revealed that all the Trichoderma strains interacted with R. solani . The strains TV112 and TX003 grew toward and coiled tightly around the host cells, penetrating and destroying the hyphae. TX009 penetrated the cell wall of R. solani by antagonist directly without formation of appressorium-like structure. Penetration of the Trichoderma strains on host cells was apparently accomplished by mechanical activity. These results demonstrated that all the three strains were effective in inhibiting the mycelial growth of R. solani .展开更多
Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3 311 unique rice transcripts (including 1 639 endosperm-derived transcripts and 1 672 mature stem-derived transcripts)...Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3 311 unique rice transcripts (including 1 639 endosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of 1-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [α-33P| labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cercal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6 %) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings while considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profiles, 84 genes were identified constantly expressed in all me four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.展开更多
AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA...AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.展开更多
Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typ...Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typical genomic organization of a begomovirus, encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virion- sense DNA and 4 ORFs (AC1—AC4) in complementary- sense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that of Okra yellow vein mosaic virus-[201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirus species, for which the name Malvastrum yellow vein virus is proposed. Satellite DNA molecule (Y47b) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAb. Y47b consists of 1348 nucleotides, with a functional ORF (C1) in complementary-sense DNA. Y47b has 62%—67% sequence identity with DNAb molecule associated with Cotton leaf curl Multan virus or Cotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNAb molecules. Relationship dendrograms show that DNAb molecules are co-evolved with their help begomoviruses.展开更多
Increased expression of Fas by hematopoietic progenitors in aplastic anemia(AA)suggests that Fas/Fas ligand (FasL)system plays a key role in the formation of severe pancytopenia.To further confirm the above hypo- thes...Increased expression of Fas by hematopoietic progenitors in aplastic anemia(AA)suggests that Fas/Fas ligand (FasL)system plays a key role in the formation of severe pancytopenia.To further confirm the above hypo- thesis,T cells from 8 patients with AA were systematically studied for their FasL's distribution pattern, releasing manner and proapoptotic activity,compared with normal resting T cells and artificially activated T cell blasts.The results demonstrated that AA T cells abnormally expressed low levels of membrane-bound FasL and contained high levels of intracellular FasL which could be triggered to release by high-dose phyto- hemagglutinin(PHA)pulse-stimulation.The supernatants from the PHA-stimulated AA T cells had apparent cytotoxicity against FasL-sensitive Jurkat cells,which could be significantly inhibited by monocional antibody against FasL in a dose-dependent manner,or nearly completely abrogated by ultracentrifugation.The above phenomena also appeared on artificially activated T cell blasts,but this was not the case on normal resting T cells.These results indicate that AA T cell is a type of“preactivated”T lymphocyte,characterized by over- expression of FasL,especially intracellular FasL which can be stimulated to release in bioavtive exosomes- bound form.Taken together,our data provide further and direct evidence for the hypothesis that T cells might mediate the destruction of hematopietic progenitor in AA through Fas/FasL system.Cellular & Molecular Immunology.2004;1(2):142-147.展开更多
Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In con- trast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35...Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In con- trast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP si- lencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silenc- ing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silenc- ing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA si- lencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. There- fore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.展开更多
A novel type of circular single-stranded satellite DNA, known as DNAb, was recently characterized and demonstrated to be associated with monopartite begomovi-ruses. DNAb was essential for induction of characteristic s...A novel type of circular single-stranded satellite DNA, known as DNAb, was recently characterized and demonstrated to be associated with monopartite begomovi-ruses. DNAb was essential for induction of characteristic symptoms in plants. DNAb has three structural features: an 115 bp highly conserved region, bC1 gene and A-Rich region. The in-frame ATG mutation of bC1 gene of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-TY10) DNAb demonstrated that bC1 gene is required for leaf curl symptom. Here, the function of A-Rich region in TYLCCNV-Y10 DNAb was identified. When A-Rich region was deleted, the A-Rich deleted mutant was still capable of replication and systemic infection in plant, indicating that A-Rich region is not required for trans-replication of DNAb. The immunotrapping-PCR demonstrated that A-Rich de-leted mutant could be encapsidated in the coat protein en-coded by TYLCCNV-Y10 DNA-A, suggesting that A-Rich region is not related with DNAb encapsidation. However, the A-Rich region deleted mutant caused milder symptom.展开更多
Organisms have variable genome sizes and contain different numbers of genes. This difference demon-strates that new gene origination is a fundamental process in evolutionary biology. Though the study of the originatio...Organisms have variable genome sizes and contain different numbers of genes. This difference demon-strates that new gene origination is a fundamental process in evolutionary biology. Though the study of the origination of new genes dated back more than half a century ago, it is not until the 1990s when the first young gene jingwei was found that empirical investigation of the molecular mechanisms of origination of new genes became possible. In the recent years, several young genes were identified and the studies on these genes have greatly enriched the knowledge of this field. Yet more details in a general picture of new genes origination are to be clarified. We have developed a systematic approach to searching for young genes at the genomic level, in the hope to summarize a general pattern of the origination and evolution of new genes, such as the rate of new gene appearance, im-pact of new genes on their host genomes, etc.展开更多
基金Supported by the National Natural Science Foundation of China,No.30270078
文摘AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its immunogenicity.METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya1 delta Crp1 delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supematant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed.Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supematant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, bhe entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0×l0^10 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protection and care of H pylori infection.
基金the National Natural Science Foundation of China,No.30270078
文摘AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity.METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+), which was transformed into BL21 (DE3) E. coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments.RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2 % of the total bacterial protein,and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself.CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.
基金Supported by the National Natural Science Foundation of China,No.30270078
文摘AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of BabA.METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore,BabA immunogenicity was studied by animal test.RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank.The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA.CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of Hpyloriinfection.
基金supported by the National Natural Science Foundation of China(39870499)funded by the Nationa1 Outstanding Youth Foundations from National Natural Science Foundation of China(30125032).
文摘The transgenic tobacco plants transformed with movement protein gene of Tomato mosaic virus (ToMV) or Tobacco mosaic virus (TMV) and partial replicase gene of Cucumber mosaic virus (CMV) P1 isolate (CMV-P1), were inoculated with Potato virus X, Potato virus Y, TMV and CMV isolate RB (CMV-RB), respectively. Symptom observation showed there were no symptom differences in transgenic tobacco plants as compared with those in non-transgenie tobacco plants. ELISA also illustrated that the virus concentrations in the transgenic plants were similar to those in non-transgenic plants, indicating that no synergism is found in these plants. The transgenic tobacco plants expressing movement protein gene of ToMV or partial replicase gene of CMV-P1 were inoculated with TMV and CMV-RB, respectively. The local or systemic infected leaves were then used for elucidation of the possible virus recombination in transgenic plants with biological infectivity test, ELISA and immuno-capture RT-PCR. Within 16 months, no recombination was found between transformed genes and inoculated virus genomes. The research provides fundamental data for understanding of the possible risk of the transgenic plants expressing viral sequences.
文摘Seventeen species of Trichoderma, isolated from soil or tree bark from Ch ina are identified based on morphological and physiological characters, and from their phylogenetic position inferred from parsimony analyses of nucleotide sequ ences of the internal transcribed spacer regions of the rDNA cluster (ITS1 and 2) and partial sequences of translation elongation factor 1-alpha (te f1) . There were T.citrinoviride, T.longibrachiatum, T.sinensis in section Long ibrachiatum, T.atroviride, T.koningii, T.viride, T.asperellum, T.hamatum, T.e rinaceum in section Trichoderma, T.harzianum (H.lixii), T.inhamatum, T. ve lutinum, T.cerinum, T.strictipile, T.spirale, T.virens, H.nigrovirens (Trichode r ma sp.) in section Pachybasium. Among them four species: T. asperellum, T .velutinum, T.cerinum, T. spirale were reported firstly in China. In addition, two suspected new taxa (Trichoderma spp.) in Trichoderma s ec tion were proposed: Trichoderma sp.1 (ZAUT261, 4, 4A, 15A, 2C), Trich od erma sp.2 (2B, 5, 7A, 7B, 9A). Trichoderma sp.1 was similar to T.h a matum , but the temperature optimum for mycelial growth was lower than that of T.hamatum and the species tended to form hemisphaerical pustule with rela tively larger conidia (average length 4.6 μm×2.8 μm). Trichoderma sp.2 was distinguished morphologically from related species T. strigosum, T. pub escens, T. erinaceum, T. hamatum and Trichoderma sp.1 in pustules on CM D without fertile or sterile conidiophore elongation and distinctive phialide sh ape, the conidiophore branches similar to T.koningii, but the conidia sim ilar to T. viride, subglobose, conspicuously tuberculate.
文摘By using 304 recombinant inbred lines derived from indica rice cross Zhong 156/Gumei 2, a linkage map consisting of 177 marker loci and covering 12 rice chromosomes was constructed and employed for mapping genes conferring blast resistance in rice. Genomic location of gene Pi25(t) conferring neck blast resistance to the Chinese isolate 92-183 (race ZC15) was verified to be located between markers A7 and RG456 on chromosome 6, with genetic distances of 1.7 cM and 1.5 cM to A7 and RG456, respectively. Leaf blast resistance of Gumei 2 to the Philippine isolate Ca89 (lineage 4) was found to be controlled by a single gene. The gene tentatively designated as Pi26(\) was located between makers B10 and R674 on chromosome 6, with genetic distances of 5.7 cM and 25.8 cM to B10 and R674 respectively. Resistant alleles at both gene loci were derived from Gumei 2, indicating an existence of resistance gene cluster in Gumei 2.
基金the National Natural Science Foundation of China, No.30270078
文摘AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.
基金This project is supported by the Foundation of State Key Laboratory of Freshwater Ecology and Biotechnology,China(2001FB10)Young Foundation of Shanxi,China(20001038)+2 种基金Shanxi Scholarship Foundation,Chinathe Hational Natural Science Foundation of China(30270204)DLF-Trifolium,Research Division,Denmark.
文摘High embryogenic calli of three cutivars of Kentucky bluegrass, Md, Bd, and Gm, were induced from mature embryos, and were proliferated on medium K3 and K5. Embryogenic calli were transformed with plamids pDM803 and pBY520 by microprojectile bombardment. Fourty-two transgenic lines had been obtained. The highest efficiency of transformation reached to 3.7% for cv. Md, 2.8% for cv. Gm, and 5 % for cv. Bd. The micro nutriment of Cupric had significant effect on transformation. The embryogenic callus cultured in dim-light condition had higher transformation efficiency than the green callus cultured in light condition for one month before transformation. The selective regime and selective pressure on the putative transgenic plants were important for obtaining the desire number of transgenic plants. It also affected the copy number of integrated genes in the genomic DNA of transgenic plants.
文摘Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase (ThEn-42) was amplified by Arabidopsis leaf-PCR or genomic DNA PCR. Unfertile, dwarf and normal phenotypes appeared in the T1 generation. In addition, an enhanced resistance to S. sclerotiorum was observed. The mortality percentage (7.7% to 33.3%) in transgenic plants was significantly lower than in non-transgenic plants (86.7%) 10 days after inoculation with the pathogen.
文摘CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably secreted at about 180 IU/ 1e6 cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reatthch and proliferate on fresh microcarriers. This makes it very easy to scale up preduction. The microcarriers could be reused several times without affecting adhesion. proliferation and prourokinase secretion. With CMPECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1×105 IU/mg of protein. The purification factor was about 600 fold, and approxiamately 90 % of the biological activity was recovered.
文摘The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat P1 1 and Sat P1 2). Sat P1 1 contained 335 nucleotides, and Sat P1 2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat P1 1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat P1 2 represents the emergence of a new satellite (necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.
文摘The physiological mechanism of maintaining the green colour of pak choy leaves (Brassicarapa var chinensis) with heat-shock treatment was studied. Chlorophyll in the outerleaves of pak choy degraded rapidly during storage at ambient temperature (20±2℃), aslight yellow appeared. Heat-shock treatment (46-50℃) had a mild effect on maintainingthe green colour of outer leaves. Normal chlorophyll degradation was associated with abinding of chlorophyll with chlorophyll-binding-protein preceding chlorophyll breakdown.Heat-shock treatment was found to reduce the binding-capacity between chlorophyll-binding-protein and chlorophyll. In the chlorophyll degradation pathway, pheide dioxygenasewas synthesized during leaf senescence which was considered to be a key enzyme inchlorophyll degradation. Activity of this enzyme was reduced following heat-shocktreatment, which might explain the observed reduction in chlorophyll breakdown. Twogroups of heat-shock proteins were detected in treated leaves, the first group containingproteins from 54KDa to 74Kda, and the second group contained proteins from 15KDa to29KDa. Heat-shock treatment was also found to retard the decline of glucose and fructose(the main energy substrates) of outer leaves.
文摘Three strains of Trichoderma spp. TV112, TX003, TY009 obtained from previous experiments could inhibit the sclerotial formation of two strains of Rhizoctonia salani AG1 (-1A) isolated from the rice paddies in Hanzhou of China. However, it is unclear if there are the antagonism and mycoparasitism of the Trichoderma strains tested against the mycelial growth of R. solani . The objective of this research was to evaluate the ability of the Trichoderma strains to inhibit the mycelial growth of R. solani in vitro . Dual culture testes showed all the Trichoderma strains tested inhibited the mycelial growth of R. solani. The strains also produced toxic metabolites with activity against R. solani, inhibiting the mycelial growth by 74%, 81.8%, and 53%, repectively. Electron microscopic observations revealed that all the Trichoderma strains interacted with R. solani . The strains TV112 and TX003 grew toward and coiled tightly around the host cells, penetrating and destroying the hyphae. TX009 penetrated the cell wall of R. solani by antagonist directly without formation of appressorium-like structure. Penetration of the Trichoderma strains on host cells was apparently accomplished by mechanical activity. These results demonstrated that all the three strains were effective in inhibiting the mycelial growth of R. solani .
文摘Through exploiting the high homology of cereal crop genes, membranous cDNA microarrays containing 3 311 unique rice transcripts (including 1 639 endosperm-derived transcripts and 1 672 mature stem-derived transcripts) were used for monitoring the expression profiles of 1-leaf stage seedlings of 4 cereal crop species: rice, maize, sorghum and barley. After hybridizing with [α-33P| labeled probes, 73.6 % of the arrayed genes generated reliable signals in all of the four cercal crops. Further analysis revealed that among the arrayed genes, a higher percentage of the endosperm-derived transcripts (86.6 %) expressed than that of the mature stem-derived genes (60.9 %), indicating that most of the endosperm expressed genes functioned in young seedlings while considerable amount of mature stem tissue expressed genes did not express. These results also inferred that some genes might function only at certain developmental stages. By comparing the obtained profiles, 84 genes were identified constantly expressed in all me four cereal crops. Many housekeeping genes, such as polyubiquitin, ubiquitin conjugating enzyme and ribosomal proteins were included in this catalogue. The experiment also identified 14 rice seedling specifically expressed genes, including 3 biotic and abiotic stress induced genes and 1 apoptosis suppressor encoding gene Bax inhibitor-1. This investigation provided invaluable information for comparative genomics of gramineae members.
基金Supported by the National Natural Science Foundation of China,No. 30270078
文摘AIM:To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.
基金supported by the National Outstanding Youth Foundation from the Natural Science Foundation of China(Grant No.30125032)the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of MOE.
文摘Virus isolate Y47 was obtained from Malvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typical genomic organization of a begomovirus, encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virion- sense DNA and 4 ORFs (AC1—AC4) in complementary- sense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that of Okra yellow vein mosaic virus-[201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirus species, for which the name Malvastrum yellow vein virus is proposed. Satellite DNA molecule (Y47b) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAb. Y47b consists of 1348 nucleotides, with a functional ORF (C1) in complementary-sense DNA. Y47b has 62%—67% sequence identity with DNAb molecule associated with Cotton leaf curl Multan virus or Cotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNAb molecules. Relationship dendrograms show that DNAb molecules are co-evolved with their help begomoviruses.
基金supported by grants from National Key Basic Research Program of China("973"project)(No.2001CB510003)Basic scientific research program foundation of the Commission of Science Technology an d Industry for National Defence(2003-44)Key Medical Elite Foundation of Jiangsu Provincial Govemment(No.RC2002021)
文摘Increased expression of Fas by hematopoietic progenitors in aplastic anemia(AA)suggests that Fas/Fas ligand (FasL)system plays a key role in the formation of severe pancytopenia.To further confirm the above hypo- thesis,T cells from 8 patients with AA were systematically studied for their FasL's distribution pattern, releasing manner and proapoptotic activity,compared with normal resting T cells and artificially activated T cell blasts.The results demonstrated that AA T cells abnormally expressed low levels of membrane-bound FasL and contained high levels of intracellular FasL which could be triggered to release by high-dose phyto- hemagglutinin(PHA)pulse-stimulation.The supernatants from the PHA-stimulated AA T cells had apparent cytotoxicity against FasL-sensitive Jurkat cells,which could be significantly inhibited by monocional antibody against FasL in a dose-dependent manner,or nearly completely abrogated by ultracentrifugation.The above phenomena also appeared on artificially activated T cell blasts,but this was not the case on normal resting T cells.These results indicate that AA T cell is a type of“preactivated”T lymphocyte,characterized by over- expression of FasL,especially intracellular FasL which can be stimulated to release in bioavtive exosomes- bound form.Taken together,our data provide further and direct evidence for the hypothesis that T cells might mediate the destruction of hematopietic progenitor in AA through Fas/FasL system.Cellular & Molecular Immunology.2004;1(2):142-147.
文摘Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In con- trast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP si- lencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silenc- ing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silenc- ing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA si- lencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. There- fore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.
文摘A novel type of circular single-stranded satellite DNA, known as DNAb, was recently characterized and demonstrated to be associated with monopartite begomovi-ruses. DNAb was essential for induction of characteristic symptoms in plants. DNAb has three structural features: an 115 bp highly conserved region, bC1 gene and A-Rich region. The in-frame ATG mutation of bC1 gene of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-TY10) DNAb demonstrated that bC1 gene is required for leaf curl symptom. Here, the function of A-Rich region in TYLCCNV-Y10 DNAb was identified. When A-Rich region was deleted, the A-Rich deleted mutant was still capable of replication and systemic infection in plant, indicating that A-Rich region is not required for trans-replication of DNAb. The immunotrapping-PCR demonstrated that A-Rich de-leted mutant could be encapsidated in the coat protein en-coded by TYLCCNV-Y10 DNA-A, suggesting that A-Rich region is not related with DNAb encapsidation. However, the A-Rich region deleted mutant caused milder symptom.
文摘Organisms have variable genome sizes and contain different numbers of genes. This difference demon-strates that new gene origination is a fundamental process in evolutionary biology. Though the study of the origination of new genes dated back more than half a century ago, it is not until the 1990s when the first young gene jingwei was found that empirical investigation of the molecular mechanisms of origination of new genes became possible. In the recent years, several young genes were identified and the studies on these genes have greatly enriched the knowledge of this field. Yet more details in a general picture of new genes origination are to be clarified. We have developed a systematic approach to searching for young genes at the genomic level, in the hope to summarize a general pattern of the origination and evolution of new genes, such as the rate of new gene appearance, im-pact of new genes on their host genomes, etc.
