脂肪酶产生菌的选育及产酶条件的优化

A lipase-producing bacterium strain was isolated from soil and was identified as Pseudomonas sp.. Its lipase yield was improved 2.25-fold by combined beatment of UV irradiation and NTG. The lipase fermentation condition for the mutant strain was optimized with Plackett-Burman design and Response Surface Analysis(RSA), and the formula of the optimum medium suitable for industrial scale fermentation was thereby established. A maximum yield of 87.5 U/ ml was obtained.     

16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers

Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp.and Heterorhabditis sp.) were isolated and identified from infected insect cadavers(Galleria mellonella larvae) after 48-hour post infections.Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi,Schineria larvae and Ochrobactrum anthropi,respectively.The isolates O.anthropi and S.larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136,respectively,whereas O.cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany(LMG3311T) and China (X-14),while the strain SRK4 was identical to the isolates of S.larvae (L1/57,L1/58, L1/68 and L2/11) from Wohlfahrtia magnifica in Hungary.The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences.This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation.Therefore,these strains are not host-dependent, but environment-specific isolates.     

Identification and Culture Condition Study of MarineActinomycete HT-8 with Nematicidal Activity against Pine Wood Nematode

Pine wilt disease (PWD) is a devastating disease to pine trees, which was caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, and causes major losses in coniferous forests in many countries and regions in the world. In order to screen actinomycete strains with high nematicidal activity against PWN, marine actinomycetes were isolated from the marine environment and their nematicidal activity were tested.The marine actinomycetes were isolated from the samples collected from the subtidal zones near Qingdao coast using the dilution and streak plate method and their culture supernatant were assayed in vitro for nematicidal activity against PWN using immersion test. The strain with high nematicidal activity was identified on the basis of morphology, cultural characteristics and 16S rDNA sequence analysis, and its optimal culture conditions for the production of nematicidal substances were investigated through the single-factor experiments.A total of 28 marine actinomycete strains were isolated from the samples. One of these strains, designated as strain HT-8 and isolated from sea sand, exhibited stronger nematicidal activity with a 88.30% corrected mortality of PWN treated with the culture supernatant for 30 h. The strain HT-8 was identified as Streptomyces termitum on the basis of morphology, cultural characteristics, 16S rDNA sequence and phylogenetic analysis. The results of the single factor experiment demonstrated that the optimal cultivation conditions of HT-8 for the production of nematicidal substances were inoculum age was 48 h, inoculum concentration was 6%, concentration of seawater was 100%, initial pH was 7.5 and incubating at 25 ℃ for 7 days.This study provides a theoretical basis for the development of marine microorganism resources and the utilization of natural nematicidal substances.     

Foliar persistence and residual activity of methoxyfenozide against beet armyworm （Lepidoptera： Noctuidae）

A greenhouse study was conducted to investigate the persistence of methoxyfenozide in pepper （Capsicum annuum L.） foliage. An aqueous suspension of methoxyfenozide was sprayed on pepper plants at concentrations of 72 and 144 mg of active ingredient （a.i.）/L. Foliage was collected at different intervals of time （0, 3, 5, 10, 20, 30, 40, and 50 days） after the treatment, and the methoxyfenozide residue was measured by high-performance liquid chromatography. The foliage was also used in bioas- says to determine the residual toxicity on and the consumption rate of the third-instar larvae of the beet armyworm, Spodoptera exigua （Htibner） （Lepidoptera： Noctuidae）. The methoxyfenozide concentrations observed after 50 days had decreased to 19 and 69μg/g per sample, corresponding to a loss of 61% and 28% from the application concentrations of 72 and 144 mg a.i./L, respectively. When fitting a first-order kinetics degradation model, the half-life （DT50） of this compound was 76 days. Both application concentrations of methoxyfenozide caused a high mortality rate （〉97%） when the larvae were fed the pepper foliage collected at all of the time intervals. Lastly, at all of the time points, the consumption rate by the larvae was reduced to between 57% and 92% for both concentrations that were bioassayed. Our results indicate that, under the present greenhouse conditions, the degradation of methoxyfenozide was slower than that reported by other authors and that its residues were highly toxic to S. exigua larvae. The implications of these results for the management programs of this pest are discussed.     

红树林细菌Rhodococcus ruber1K降解邻苯二甲酸二丁酯的研究

A di-n-butyl phthalate (DBP)degrading bacterium Rhodococcus ruber was isolated from mangrove soil,and its degrading characteristics were studied.The results showed that the bacterium could grow well on the substrate with DBP as the sole source of carbon and energy,and the DBP of 50 mg稬^-1 could be completely degraded after 48 h.Under aerobic condition,the tentative pathway proposed for DBP degradation was through monoester initially,then phthalic acid,and finally CO₂ and H₂O.     

Biology and life table studies of the oriental tobacco budworm,Helicoverpa assulta （Lepidoptera： Noctuidae）, influenced by different larval diets

Development, survivorship, pupal weight, oviposition, and life table parameters of the oriental tobacco budworm, Helicoverpa assulta Guenée, were evaluated in the laboratory on an artificial diet, pepper （Capsicumfrutescens L.）, and tobacco （Nicotiana tobacum L.）. We found that the average developmental time of immature stages was longest on tobacco （36.2 d）, intermediate on pepper （34.4 d）, and shortest on artificial diet （33.5 d）. Immature survival from egg to pupa varied from 31% on tobacco, 43% on pepper, and 74% on artificial diet. Pupal weight ranged from 197.4 mg/pupa on tobacco, 233.1 mg/pupa on pepper and 253.4 mg/pupa on artificial diet. The average numbers of eggs laid by adults reared as larvae on the artificial diet, pepper, or tobacco were 614, 421 and 334 eggs/female, respectively. Numbers of remaining eggs in ovaries of the adult females reared as larvae on the artificial diet, pepper, or tobacco were 16, 26, and 42 eggs/female, respectively. The longevity of adult females developed from larvae reared on the three diets was not significantly different, whereas the longevity of male adults from the larvae reared on artificial diet was longer （16.8 d） than that for males reared on tobacco （13.8 d） and pepper （13.3 d）. The intrinsic, finite, gross, and net rates of increase were highest for females reared as larvae on artificial diet, lowest for females emerging from larvae reared on tobacco, and intermediate for females emerging from larvae reared on pepper. Generation times and doubling time of H. assulta were shortest for larvae fed artificial diet, intermediate from larvae reared on pepper, and longest from larvae reared on tobacco. We concluded that the artificial diet was the most suitable larval diet ofH. assulta followed by pepper, and tobacco.     

CHANGES OF GLYCEROL CONTENT IN DIAPAUSE LARVAEOF THE ORANGE WHEAT BLOSSOM MIDGE, SITODIPLOSIS MOSELLANA （GEHIN） IN VARIOUS SEASONS

The glycerol contents in diapause larvae of the orange wheat blossom midge, Sitodiplosis mosellana (Gehin), collected from various seasons, were measured. The results showed that there was less glycerol content in larvae during living on the wheat head. Content of glycerol began to increase significantly when the larvae left the wheat head and entered the soil. A change trend of upper- lower- upper- lower in larvae glycerol contents during diapause in soil was observed from June to April of next year. More glycerol could be examined in larvae collected in summer and winter than in spring and autumn. There was not more glycerol in cocooned larvae than that in non-cocooned larvae during various seasons from the point of statistics. Comparing the glycerol content of larvae being diapause in the first year with that of larvae in the second year, there was yet no obvious difference when larvae were collected in the same season belonged to different years. Therefore, it is shown that the content of glycerol in larvae of the wheat midge in diapause is affected mainly by the seasons or diapause intensity.     

Characterization and catabolic genes analyses of oil-degrading Pseudomonas aeruginosa 1217

One oil-degrading bacterial strain 1217 isolated from oil contaminated soil could degrade crude oil. It was identified and designated as Pseudomonas aeruginosa by morphology, physiology, biochemical and 16S rDNA sequence analyses. The strain grew well at 5-65 ℃ with the initial pH of 2-10 and NaCl concentrations of 0%-9%. It grew well in the medium containing different organic substrates as sole carbon sources, such as n-dodecane, n-octadecane, benzene, toluene, xylene and naphthaline. The degrading rates for hydrocarbons were 21.57% and 15.15% when it was cultured in minimal medium containing crude oil and different chain alkanes at 30 ℃ and 10 ℃ for 7 days. The bacterium produced biosurfactants with the surface tension reduction from 72.20 mN/m to 35.14 mN/m. The oil degrading related genes such as alkane monooxygenase, toluene dioxygenase, biphenyl dioxygenase, ring hydroxylating dioxygenase and oxidoreductase genes were detected in Pseudomonas aeruginosa 1217. The alkane monooxygenase and ring hydroxylating dioxygenase genes were further cloned and analyzed. The sequence similarities of the two genes with those of Pseudomonas aeruginosa PAO1 were 99.91% and 99.22% respectively. The isolated strain exhibits great potential for the bioremediation of the hydrocarbons contaminated environments.     

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.     

Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco

A strain of canine parvovirus (CPV) was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.     

特异性杀虫基因Bt cry3A在桑粒肩天牛幼虫两种肠道常驻内生菌中的转化和表达

[目的]将特异性杀虫毒蛋白基因Bt cry3A转入桑粒肩天牛(Apriona germari Hope,Ag)幼虫肠道常驻内生菌中,构建能在天牛幼虫肠道中定殖并表达特异性杀虫基因Bt cry3A的工程菌.[方法]以传统方法和16S rDNA分子生物学分析等方法分离、鉴定Ag幼虫肠道优势的常驻内生菌,从中筛选出适合转化的候选菌株.利用电转化技术将含有对鞘翅目昆虫具专一性毒力Bt cry3A基因的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911分别转入Ag幼虫肠道常驻内生菌短短芽孢杆菌(Brevibacillus brevis Ag12,Ag12)和苏云金芽孢杆菌(Bacillus thuringiensis Ag13,Ag13)中.[结果]从Ag幼虫肠道共分离获得18个不同种的可培养细菌菌株,并从中选取菌株Ag12和Ag13作为出发菌株转入Bt cry3A基因.经质粒稳定性试验、转化子生长特性测试、伴胞晶体电镜检测、毒蛋白SDS-PAGE分析、工程菌定殖性分析以及生物毒力测试,结果显示cry3A基因已经成功转入Ag幼虫的常驻内生菌短短芽孢杆菌和苏云金芽孢杆菌中,并且工程菌Ag12-305a、Ag13-305a、Ag 12-7911和Ag13-7911都能在天牛幼虫肠道内稳定生长、繁殖并表达分子量约65kDa的伴孢晶体杀虫蛋白Cry3A.[结论]Bt cry3A基因已成功转入桑粒肩天牛幼虫肠道优势常驻内生菌中,获得了四株能在桑粒肩天牛幼虫肠道内定殖,并能表达目的杀虫基因Btcry3A的转基因工程菌.     

Bt杀虫基因在桑粒肩天牛幼虫肠道内生优势菌中的转化研究

桑粒肩天牛(Apriona germariHope,Ag)幼虫是一种营钻蛀性生活的重要林业害虫,通过传统纯培养、生理生化鉴定和16SrDNA分子生物学分析等方法分离、鉴定出其肠道优势内生菌溶血葡萄球菌(Staphylococcus haem olyticus,S.haem olytic-us)Ag06菌株和人葡萄球菌(Staphylococcus hom is)Ag08菌株。从中筛选菌株S.haem olyticusAg06进行质粒消除后作为出发菌株,利用电转化技术将含有对鞘翅目昆虫具专一性毒力B t杀虫基因cry3A的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305 a和pHT7911分别转入其中。经质粒稳定性试验、转化子生长特性测试等分析,结果显示cry3A基因已经成功转入Ag幼虫的优势内生菌溶血葡萄球菌中。     

桑粒肩天牛肠道纤维素分解细菌的分离和鉴定*

为研究天牛消化利用纤维素的机理,利用纤维素-刚果红琼脂培养基,从桑粒肩天牛(A.germari)幼虫中肠分离到一种兼性厌氧纤维素分解菌。菌落为白色圆形,边缘比较规则,周围溶纤维素透明圈直径一般可达10mm～20mm,细菌大小为0.5～0.8μm×1～3μm,革兰氏染色阳性,杆状,极生鞭毛,无芽孢。结合生化实验结果,初步鉴定该菌株为纤维单孢菌属(Cellulomonas)。     

蜜蜂幼虫粪肠球菌的分离与鉴定

在对患有欧洲幼虫腐臭病的蜜蜂幼虫进行细菌分离与培养时,获得1株未知菌株。从分离培养特性、形态学、生理生化特性和分子生物学等方面对该菌株进行了鉴定,得知该菌株为欧洲幼虫腐臭病的次生菌——粪肠球菌,属于肠球菌属,并暂定名为Enterococcus faecalis FB102。同时,得知根据该菌的分离培养特性可与欧洲幼虫腐臭病病原区分,并且将其单独接种蜜蜂幼虫后未能使幼虫患病。根据粪肠球菌较欧洲幼虫腐臭病病原易于分离培养的特性,得知通过对粪肠球菌的鉴定可以简化欧洲幼虫腐臭病的诊断过程,而且推测该菌株可能对人体健康有一定的影响。     

长期留置管细菌分布及生物膜形成能力调查

目的了解长期留置管细菌分布及生物膜形成能力。方法10％血清肉汤振荡培养留置管中细菌,常规生化和分子生物学方法鉴定分离菌,结晶紫半定量法检测分离菌生物膜形成能力。结果留置管细菌检出率为49．1％（53／108）;共分离60株菌,以葡萄球菌分离率最高（70．0％）,其次为肠球菌（15．O％）和大肠埃希菌（6．7％）;所有分离菌均具有生物膜形成能力,其中38株（63．3％）具有较强的生物膜形成能力。结论长期留置管细菌检出率较高,大部分分离菌具有较强的生物膜形成能力。     

Effects of Serratia marcescens on the F1 generation of laboratory-reared Heliothis virescens (Lepidoptera: Noctuidae)

The effects of the bacterium Serratia marcescens (Bizio) was investigated on the F1 generation of laboratory-reared Heliothis virescens (F.). There was no difference in adult male or female longevity (i.e., parental generation) for individuals inoculated with S. marcescens as larvae (Serratia treatment) and those that were free of the bacterium (control treatment). However, the number of eggs laid and the prevalence of eclosion of eggs from Serratia treatment adults were reduced relative to control treatment adults. A very low number of F1 Serratia treatment eggs exhibited signs of infection, but a higher prevalence of mortality was observed for F1 larvae (n = 2,888) for the Serratia (3.5-4.6%) than for the control (1.1-1.5%) treatment. No S. marcescens was isolated from dead control larvae; whereas, 48 -54% of dead F1 larvae for the Serratia treatment were positive for the bacterium. However, there was no significant difference in larval weights between treatments. There were also no differences in either mortality or weight of F1 male pupae between treatments, but F1 female pupae were significantly smaller and prevalence of mortality was higher for the Serratia treatment. Serratia marcescens was not isolated from any of the control F1 pupae, but 6% of pupal cadavers for the Serratia treatment were positive for the bacterium. No S. marcescens was recovered from the meconia of any of the F1 adults (n = 2,600) regardless of treatment, and there were no differences in adult weights between treatments. Although sublethal effects of S. marcescens were detected, the impact and prevalence of the bacterium were tremendously reduced over the F1 generation in the absence of all but the most basic management strategies.     

Draft genome sequence of Bacillus thuringiensis 147, a Brazilian strain with high insecticidal activity

Bacillus thuringiensis is a ubiquitous Gram-positive and sporulating bacterium. Its crystals and secreted toxins are useful tools against larvae of diverse insect orders and, as a consequence, an alternative to recalcitrant chemical insecticides. We report here the draft genome sequence ofB. thuringiensis 147, a strain isolated from Brazil and with high insecticidal activity. The assembled genome contained 6,167,994 bp and was distributed in seven replicons (a chromosome and 6 plasmids). We identified 12 coding regions, located in two plasmids, which encode insecticidal proteins.     

Catabolism of sulfamate by Mycobacterium sp. CF1

A bacterium able to utilize sulfamate as N-source for growth was isolated from soil and identified as a Mycobacterium sp. An apparently previously unrecorded enzyme, sulfamate hydrolase (EC 3.10.1.-), converts sulfamate to equimolar amounts of ammonia and sulfate. This enzyme was purified to homogeneity and had a Km for sulfamate of 26.36 +/- 4.01 mM. Its Specificity Constant value, 74 M(-1) s(-1), was low, indicating that it was not a particularly good catalyst for this reaction and it may be a hydrolase recruited to this role from some other reaction sequence. However, under equivalent conditions it showed no detectable action on the other sulfamates, cyclamate and sulfamoylbenzoate, or on urea or methylamine.     

Cryopreservation of common carp (Cyprinus carpio) sperm in 1.2 and 5 ml straws and occurrence of haploids among larvae produced with cryopreserved sperm

Experiments were carried out on the cryopreservation of common carp (Cyprinus carpio) sperm in order to test the suitability of using 1.2 and 5 ml straws and to investigate the ploidy of malformed larvae found among the hatched progeny. In the first set of experiments, the effect of freezing time was investigated on the hatch rate of embryos. The highest hatch rate for 1.2 ml straws was 69+/-16% at the freezing time of 4 min, and 39+/-27% for 5 ml straws at 5 min. In the second set, the effect different egg volumes fertilized with one straw of sperm on the hatch rate and the rate of malformed larvae was investigated. The highest hatch rate with 1.2 ml straws (86+/-12%) was observed when 10 g of eggs were fertilized with one straw, whereas with 5 ml straws the hatch rate was highest (65+/-18%) when 40 g of eggs were fertilized. The highest rate of malformed larvae (15+/-9%) was found in the control, whereas the highest rate of malformed larvae among the groups fertilized with cryopreserved sperm (13+/-7%) was found in the 1x dose group fertilized with 5 ml straw. The chromosome numbers of malformed larvae were investigated and haploids were found among those hatched from eggs fertilized with cryopreserved sperm whereas only diploids were found in the controls.     

杨树粒肩天牛幼虫林间化学防治试验

选用3种化学药剂应用不同的施药方法对杨树粒肩天牛幼虫进行林间药效试验,结果表明,采用虫道注射与毒签两种施药方法林间防治粒肩天牛幼虫都具有良好的防治效果,15d后防治效果达71.44～97.54%,可在生产中推广应用。