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Construction of biological control strain of Trichoderma viride and study of their ability to induce plant disease resistance

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浙江大学学报（农业与生命科学版） 2004年第4期30卷 416-417页

作者： LIUShi-wang GUOZe-jian biotechnologyinstitute ZhejiangUniversityHangzhou310029China

Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow sec

关键词：生物防治菌株真菌木霉属植物疾病抗性

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Cloning,Structural Organization and Chromosomal Mapping of Rat Costimulatory Molecule 4-1BBL

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 694-701页

作者： Qiu-Ming DONG~1 Li-Jie MA~2 Guang-Bo ZHANG~1 Ya-Fang WU~3 Jia-Yao SHEN~1 Ying CHEN~1 Yong-Jing CHEN~1 Xiang-Ke PU~1 Sai-Yu HANG~1 Xue-Guang ZHANG~(1*) 1 Biotechnology Institute,Soochow University,Suzhou 215007,China 2 Genetics Institute,Fudan University,Shanghai 200433,China 3 First Affiliated Hospital of Soochow University,Suzhou 215006,China biotechnologyinstitute SoochowUniversitySuzhou215007China

4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily,whichis expressed on some activated antigen presenting cells and B *** isolated a rat cDNA clone encodingthe rat homologue of the human 4-1BBL (GenBank accession ***259541).The deduced rat 4-IBBLprotein,consisting of 308 amino acids with a molecular weight of 33,469 Da,was a typical type Ⅱtransmembrane glycoprotein,the same as human and routine 4-IBBL.“SDAA” in the cytoplasmic domain ofrat 4-1BBL was deduced to act as the phosphorylation site for casein kinase Ⅰ(“SXXS” motif),which ispresent in the cytoplasmic domains of human and murine 4-1BBL,and all other TNF ligand family membersknown to utilize reverse *** two introns of 4-1BBL were also cloned (GenBank accession ***332409).Rat 4-IBBL is much more homologous with murine 4-1BBL than with human 4-1BBL,in bothnucleotide and amino acid *** 4-1BBL was expressed in all tested tissues:brain,lung,colon,liver,thymus,testicle,kidney,adrenal,stomach,spleen and *** chromosomal location of rat 4-1BBL wasfirst identified by bioinformatics,then by fluorescence in situ hybridization at 9q11(GenBank accessionUni Gene ***.46783).Rat,murine and human 4-1BBL genes are evolved from a common *** and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biologicalfunction of this molecule.

关键词： TNF superfamily rat 4-1BBL cloning mapping

Expression of Cryptogein in tobacco plants exhibits enhanced disease resistance and tolerance to salt stress

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Chinese Science Bulletin 2004年第8期49卷 803-809页

作者： JIANGDonghua CHENXujunt WUKunlu GUOZejian CollegeofAgronomyandBiotechnology ChinaAgriculturalUniversityBeijing100094China biotechnologyinstitute ZhejiangUniversityHangzhou310029China

Cryptogein (Crypt), an elicitin secreted from Phytophthora cryptogea, was used for genetic engineering of biotic and abiotic resistance plants. We generated trans-genic tobacco plants harboring a rice phenylalanine ammo-nia-lyase (PAL) promoter and Crypt fusion gene (PAL::Crypt) or the mutated Crypt (mutation of the lysine at the position 13 to valine) under the control CaMV35S promoter (CaMV35S::CryK13V). T2 progeny of the transgenic plants showed significantly enhanced disease resistance to patho-gens of fungal Phytophthora parasitica var nicotiana (Ppn) and Alternaria alternata, and bacterial Pseudomonas syringae pv tabaci. The amount of mRNA accumulation of Crypt and CryK13V was quite low in the transgenic lines analyzed by Northern blot, and was detected by a reverse transcription PCR method. Plants harboring PAL::Crypt construct showed faster and stronger induction of PR-1a gene after Ppn inoculation than that in the wild-type plants. The re-sults suggested that the inducible PAL promoter could rap-idly respond to pathogen attack and efficiently suppress the pathogen infection. Furthermore, the enhanced tolerance to salt stress in both of the Crypt and CryK13V expressing tobacco plants was also observed compared with that in the control plants. The constitutive expression of PR and tran-scription factor genes in the transformants was probably associated with the salt tolerance. The above observations suggested that a cross-talk between biotic and abiotic stresses existed in tobacco plants.

关键词：隐性基因耐盐性疾病抵抗力基因工程烟草

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cvhA Gene of Streptomyces hygroscopicus 10-22 Encodes a Negative Regulator for Mycelia Development

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Acta Biochimica et Biophysica Sinica 2006年第4期38卷 271-280页

作者： Heng-An WANG~(1,2#) Lei QIN~(2#) Ping LU~(1*) Zhi-Xuan PANG~2 Zi-Xin DENG~(3*) Guo-Ping ZHAO~(2*) 1 Biotechnology Institute,School of Agriculture and Biology,Shanghai Jiaotong University,Shanghai 201101,China 2 Laboratory of Molecular Microbiology,Institute of Plant Physiology and Ecology,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China 3 Bio-X Life Science Research Center,Shanghai Jiaotong University,Shanghai 200030,China. biotechnologyinstitute SchoolofAgricultureandBiologyShanghaiJiaotongUniversityShanghai201101China LaboratoryofMolecularMicrobiology InstituteofPlantPhysiologyandEcologyShanghaiInstituteforBiologicalSciencesChinese

A five-gene cluster cvhABCDE was identified from Streptomyces hygroscopicus *** thefirst gene of this cluster,cvhA encoded a putative sensor histidine kinase with a predicted sensor domainconsisting of two trans-membrane segments at the N-terminus and a conserved HATPasecdomain at the *** C-terminus polypeptide of CvhA expressed in Escherichia coli was purified and shown to beautophosphorylated with [γ-32P]ATP in *** phosphoryl group was acid-labile and basic-stable,whichsupported histidine as the phosphorylation *** obvious difference of mycelia development was ob-served between the null mutant of cvhA generated by targeted gene replacement and the wild-type parentalstrain 10-22 grown on solid soya flour medium with 2%-8% glucose or sucrose,but the cvhA mutant couldform much more abundant aerial mycelia and spores than the wild-type strain on solid soya flour mediumsupplemented with 6%-8% mannitol,6%-8% sorbitol,4%-6% mannose,or 4%-6% *** pheno-type was complemented by the cloned wild-type cvhA gene,and no difference was observed for growthcurves of the cvhA mutant and the wild strain in liquid minimal medium with the tested sugars at a concen-tration of 4%,6% and 8%.We thus propose that CvhA is likely a sensor histidine kinase and negativelyregulates the morphological differentiation in a sugar-dependent manner in *** 10-22.

关键词： sensor histidine kinase aerial mycelia sugar metabolism