A large scale screen for genes （3rd chromosome） related to Wingless signaling pathway

A wing specific F 1 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.     

Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.     

Isolation and characterization of rice lesion mimic mutants from a T-DNA tagged population

A rice ( Oryza sativa L. ssp. japonica cv. Nipponbare) T-DNA tagged population consisting of about 7000 individual lines was generated and screened for rice lesion mimic mutants in the T1 generation. Ten lines were found to develop spontaneous lesions in the absence of pathogen infection and displayed distinct lesion phenotypes. These mutants were tentatively designated as lm1 -lm10 (for lesion mimic), respectively. Lesion formation of lm mutants was developmentally regulated, and all the mutants showed stunted growth and reduced fertility. Genetic analysis demonstrated that all the mutations were recessive, and five partially fertile mutants (lm4-lm8) were derived from different loci. Mimic lesions occurring on the leaves of lm mutants resulted from cell death as revealed by trypan blue staining. Six of them ( lm3 -lm8 ) exhibited enhanced resistance to five bacterial blight isolates, indicating their wide-spectrum resistance to this pathogen. These results imply that some lesion mimic mutations of rice might be involved in disease resistance signaling pathways,and that isolation of these mutated genes may be useful for elucidating molecular mechanisms of plant disease resistance. Among the mutants, only one mutant, lm6, was preliminarily shown to cosegregate with the inserted T-DNA in its T1 generation, making it feasible to isolate the gene responsible for the phenotype of this mutant.     

Isolation and characterization of a <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> mutant that cannot utilize proline as the sole carbon and nitrogen source

Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open reading frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, namedp GXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased.The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type.     

Regulation of eight avr genes by hrpG and hrpX in Xanthomonas campestris pv. campestris and their role in pathogenicity

Eight putative avirulence genes in Xanthomonas campestris pv. campestris (Xcc) strain 8004 were characterized by Tn5gusA5 mutagenesis and gene expression analysis. The virulence test of mutants on Chinese radish showed that all mutants in individual avr genes except avrBs2 mutant were not significantly different from the wild type in virulence. The avrBs2 mutant showed reduced virulence and bacterial growth in planta. Gene expression analysis using β-glucuronidase as reporter indicated that avrBs1.1，avrBs1，avrXccB，avrXccC，avrXccE1 were regulated by hrpG, whereas avrXccA1, avrXccA2 and avrBs2 were not. RT-PCR analysis showed that all hrpG-regulated genes except avrBs1 were also regulated by hrpX. In addition, it was demonstrated that avrBs1　 was responsible for elicitation of a type III dependent hypersensitive reaction (HR) on nonhost plant pepper ECW-10R, and wild type Xcc 8004 was unable to cause HR on pepper ECW-20R.     

Genetic analysis and identification of a large leaf angles （lla） mutant in rice

Mutants are essential genetic muterials to elucidation of biological functions of genes involved.Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era.T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species.in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA inscrtion.     

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type II secretion pathway

Phosphorus is one of the major essential macronutrients for virtually metabolic processes in plant growth and de-velopment[1]. This creates a paradox with major agro-nomic implications since the phosphate form of phospho-rus is one of the least soluble mineral nutrient ions in the soil. The concentration of soluble phosphorus in soil is usually very low, normally at levels of 1 ppm or less (10 mol/L H2PO4?). Mineral forms of phosphorus are repre-sented in soil by primary minerals, such as ap…     

Reporter-based screen for <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants compromised in nonhost resistance

Plants are exposed to many potentially pathogenic microbes in the environment, but each species is only susceptible to a limited number of pathogens. The broad resistance is referred to as nonhost resistance. To date, little is known about the underlying mechanism of nonhost resistance and the signaling transduction process. Here we describe a simple method for isolating Arabidopsis nonhost resistance mutants against a nonadapted bacterial pathogen. A RAP2.6 promoter-driven LUC reporter system was developed to replace the tedious bacterial growth assay during the primary screening. The RAP2.6-LUC reporter gene is normally induced by the virulent bacterium Pseudomonas syringae pv tomato but not the nonadapted bacterium P. syringae pv phaseolicola. By using this method we iso- lated 4 mutants displaying strong reporter activity in response to P. syringae pv phaseolicola, which were characterized in some details, ebsl, ebs2, ebs3, and ebs4 （enhanced bacterial susceptibility） were compromised in resistance against P. syringae pv phaseolicola and/or P. syringae pv tomato. In addition, ebs4 showed enhanced hypersensitive response to the incompatible bacterium P. syringae pv tomato （avrB）. These results demonstrated that the method is suited for large scale screening for nonhost resistance mutants.     

基于转座子策略提高链霉菌中landomycin E产量的研究

【目的】对影响放线菌链霉菌Streptomyces globisporus产landomycin E(laE)的代谢网络进行研究,以提高次生代谢物的产量。【方法】通过构建含强启动子和抗性标记的转座子Tn7为基础的转座子,整合至S.globisporus的染色体产生突变库,筛选高产量的突变株并对其代谢网络进行研究分析。【结果】利用构建好的Tn7-转座子连续转化链霉菌S.globisporus,经过数轮的突变和筛选,得到6株产量有较大改变的突变株,对整合位点的亚克隆和测序结果表明,该位点整合导致编码类似细菌的某些调节因子如TetR和GntR家族的蛋白的基因失活。【结论】所构建的经过修饰的微型Tn7-转座子不仅带有抗性标记且有启动子,可插入链霉菌染色体产生突变,进而提高次生代谢物laE的产量,同时也证明,转座子基载体可应用于非模式菌链霉菌。     

The effect of pmpR on the type III secretion system in Pseudomonas aeruginosa

The type III secretion system(T3SS) plays important roles in Pseudomonas aeruginosa pathogenicity.Previously,we reported that the uncharacterized protein PmpR could regulate pqsR,an important regulator in the quorum-sensing system,by directly binding to its promoter region.As the T3SS is controlled by the quorum-sensing system,here,we investigated the relationship between PmpR and the T3SS.Our data showed that expression of the T3SS genes exoS,exoY,exoT,and exsD was dramatically increased in a pmpR-deletion mutant compared with that in the wild-type P.aeruginosa strain PAO1.Data from DNA mobility assays indicated that PmpR affects the T3SS indirectly.It is unlikely that PmpR controls the T3SS via the Pseudomonas quinolone signal(PQS) because the PQS negatively regulates the T3SS,while pmpR negatively regulates the PQS.The effect of PmpR on the T3SS seems to be independent of the PQS;further investigation is required to uncover the underlying regulatory pathways.     

长针科线虫2种传毒种类和3种非传毒种类分子鉴定和亲缘分析

 裂尾剑线虫Xiphinema diversicaudatum和逸去长针线虫Longidorus elongatus是植物病毒的重要传毒线虫,具有重要的经济意义,被我国列入进境植物检疫性有害生物名单.通过对上述2种检疫性线虫和中国云南不同地区3种长针科线虫共7个种群rDNA的内转录间隔区(ITS)进行PCR扩增和测序,分别获得1 400～2 100 bp PCR产物片段,通过与Genbank上5种8种群的ITS区序列进行分析,结果发现裂尾剑线虫与包括逸去长针线虫等在内的其他4种线虫的同源性仅有55%,逸去长针线虫与3种长针科非传毒线虫的同源性仅为56%.同种不同种群间的rDNA-ITS的基因变异分别从0～10%不等.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Induction of nicotine in tobacco by herbivory and its relation to glucose oxidase activity in the labial gland of three noctuid caterpillars

Tobacco Nicotiana tabacum L. is a host plant ofHelicoverpa armigera (Huibner), Helicoverpa assulta Guenee and Spodoptera litura (Fabricius) (Lepidoptera, Noctuidae). The difference in leaf nicotine response to the feeding by these three larvae and the mechanical simulation of their feeding was examined by HPLC. Results indicated that nicotine induction was suppressed by H. armigera and H. assulta larvae feeding or by simulated damage treated with their labial glands extracts. The production of nicotine was also suppressed by the glucose oxidase from Aspergillus niger when it was treated on mechanically wounded leaf area. On the contrary, the nicotine production was stimulated by S. litura larva feeding or by simulated damage treated with its labial gland extract. Heat denature can not counteract the stimulation effect of the S. litura labial gland extracts to tobacco nicotine production. The glucose oxidase activity was detected in labial gland extracts of both H. armigera and H. assulta, but the activity in H. armigera was significantly higher than that in H. assulta. No glucose oxidase activity was detected in labial gland extracts of S. litura. It is shown that the glucose oxidase activity in labial glands of caterpillars plays an important role in the nicotine response to herbivory. The glucose oxidase was mainly contained in the labial gland of H. armigera larva, and had the highest activity at pH 7.0. D-Glucose was the optimal substrate of the glucose oxidase. Labial gland glucose oxidase activities varied daily during larval development with high activities found when larvae were actively feeding.     

氰戊菊酯对Tn细胞系P450及蛋白质谱表达的影响

研究氰戊菊酯对粉蚊夜蛾(Trichoplusia ni, Tn)细胞系细胞色素P450(P450s)的诱导作用及蛋白质谱表达的影响.用MTT法和台盼兰法观察不同浓度氰戊菊酯处理Tn细胞12 h后细胞的存活率；光谱测定法测定P450s含量；以12.5 μmol/L氰戊菊酯处理Tn细胞12 h，通过二维液相色谱分离技术分析处理前后Tn细胞系蛋白质谱的变化.结果显示：当氰戊菊酯处理浓度高于17.5 μmol/L时，Tn细胞形态发生明显变化，且细胞死亡率显著高于对照组(P<0.01).P450s的含量有随氰戊菊酯浓度增加而升高的趋势，并在氰戊菊酯浓度为12.5 μmol/L时达到最高(8.562 nmol/mg protein)；P450s的含量亦有随处理时间延长而升高的趋势，且在12 h时达到最高(5.28±1.14 nmol/mg protein).通过二维液相色谱分离技术得到124个差异组分，其中42个组分上调，82个组分下调.结果表明：氰戊菊酯对P450s确有诱导作用，且氰戊菊酯诱导前后Tn细胞中蛋白的变化可能与P450s诱导途径中某些受体的变化有关.     

北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.