巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings express-ing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca^2 ]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H202. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca^2 ]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.     

褐飞虱、白背飞虱种间、种内密度制约效应研究

以高感品种TN1为材料．研究了褐飞虱、白背飞虱成、若虫密度对种群的制约效应。实验表明,单虫种一龄善虫密度15头／株和30头／株相比．褐飞虱成虫的羽化率、短翘率、体长和若虫历期均无明显差异,但成虫雌性比率前高;白背飞虱成虫羽化率前比后高21．22％．短翘率高15.25％。在两种飞虱一龄若虫密度30、45、60头／株等虫量混合组配下,褐飞虱成虫羽化率60头／株比30头／株下降28％,白背飞虱若虫历期以30头／株最长。在总虫量为30头／株时,种内(30头／株)和种间(15：15头／株)比．白背飞虱种内处理成虫羽化率高36．83％．昔虫历期短;褐飞虱以种内处理成虫短翘率高。从产卵位点来看．褐飞虱喜产在稻株叶鞘中下部厚肉组织上,白背飞虱以叶脉基部产卵为主．成虫密度2、4、8对／株3处理,其叶鞘、叶脉上的平均卵块变小;10天内的平均卵量亦减步。同密度下,种间组合比种内组合的产卵量多。从各指标对密度反应来看,褐飞虱比白背飞虱更耐拥挤。     

培养时间对蛹拟青霉(Paecilomyces militaris)菌丝体中代谢产物的影响

[目的]通过测定不同培养时间蛹拟青霉(Paecilomyces militaris)菌丝体中次级代谢产物的变化,分析蛹拟青霉次级代谢产物与培养时间之间的关系.[方法]液体种子接入SDAY固体培养基 ;培养温度为25℃,培养周期为9d,从第2天开始每天取样 ;用甲醇与乙酸乙酯分别提取菌丝体中次级代谢产物,离心、过滤后合并提取液,用液质联用仪进行分析,用MetaboAnalyst software软件进行数据采集分析.[结果]主成分分析结果表明供试菌株在不同培养时间内其菌丝中次级代谢产物差异显著.聚类分析结果显示,供试拟青霉对生物碱、肽类和核苷等易形成阳离子类物质的代谢物可分为前中后三个阶段.供试拟青霉对糖类和有机酸等易形成阴离子类物质的代谢主要分为前后两个阶段.差异代谢物及热图分析结果表明,在培养的第2和第3天含量显著增加的代谢产物种类较多,主要有酯类及其水解产物、细胞破坏素B和拟青霉素,以及多种尚未鉴定的含氮化合物等 ;在培养第4和第5天含量显著增加的差异代谢物质种类较少,主要有细胞破坏素A和团囊虫草素等肽类抗菌杀虫物质 ;在培养第6天至第9天含量显著增加的代谢物种类较多,除多种白僵菌交酯和细胞破坏素等肽类抗生素外,显著增加的还有多种脂肪酸、氨基酸、鼠李糖、海藻糖、脑苷脂类化合物和核黄素等物质.[结论]培养时间对蛹拟青霉菌丝中次级代谢产物的产生有显著影响.在培养初期,菌体中酯类及细胞破坏素B和拟青霉素等含氮化合物的合成旺盛.在培养中期,次生代谢物明显减少,但细胞破坏素A2和团囊虫草素等肽类抗生素的合成却仍显著增多.在培养后期,供试蛹拟青霉除代谢出多种白僵菌交酯等肽类抗生素外,还大量产生有机酸、氨基酸和海藻糖等物质.虽然在整个培养过程中都有肽类抗生素产生,但这些抗生素并不相同,同时后一阶段新的抗生素的产生常常伴随着前一阶段的抗生素减少,因此前期的抗生素可能是后期产生的抗生素的前体.     

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5′-untranslated region (5′UTR), a 194 bp 3′-untranslated region (3′UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.     

真菌降解木质素研究进展及在好氧堆肥中的研究展望

综述了近十年来真菌降解木质素的研究进展,包括木质素的存在与结构,真菌降解木质素生物学、酶系及作用机理、生理学以及在环境工程中应用方面的研究进展,并对好氧堆肥处理城市垃圾中木质素生物降解的研究作了展望 。     

转瓶内部结构对无血清悬浮培养昆虫细胞的影响

以昆虫细胞为宿主进行基因工程产品的开发是动物细胞培养领域十分有吸引力的研究方向[1] 。由于昆虫细胞对营养要求极高 ,且对培养环境非常敏感 ,所以一般是在含有兼具营养及保护功能的胎牛血清的培养基中进行培养。血清一方面因其高额成本而限制了昆虫细胞大规模培养技术的发展 ,另一方面又因其成分复杂、富含蛋白而给外源基因表达产物的后处理带来困难。因此 ,昆虫细胞无血清培养技术的开发一直是细胞培养工程领域的研究热点 ,采用无血清培养技术取代传统的有血清培养技术已成为昆虫细胞 杆状病毒表达系统的发展趋势[2 ] 。然而 ,昆虫细…     

Novel Approach for Improving the Productivity of Antibiotic-Producing Strains by Inducing Combined Resistant Mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str^r), gentamicin (Gen^r), or rifampin (Rif^r), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the β-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees.

Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.     

温度对茶尺蠖核型多角体病毒增殖动态的影响

多角体计数、对流免疫电泳、单向免疫扩散及火箭免疫电泳测定的结果表明:26℃适于茶尺蠖核型多角体病毒(EoNPV)的增殖。多角体含量或其相对值随着时间的推移而增长,并渐趋于平稳,两者间呈Logistic曲线关系。单位体重或单头幼虫所含的多角体数量(y_1或y_2)、扩散环直径(y_3)和火箭峰值(y_4)与时间(t)的关系式分别为:y_1=(8.1481)/(1 EXP(9.4210-0.0608t))×10~9PIB/克;y_2=(6.1596)/(1 EXP(5.4809-0.0376t))×10~8PIB/头,y_3=(1.4)/(1 EXP(2.710-0.015t))cm;y_4=(3.52)/(1 EXP(4.580-0.040t))cm。但30℃下,EoNPV增殖严重受抑制,饲毒后24～168小时内难以测出多角体,其后多角体含量也极显著低于26℃,乃至难以被三种免疫测定法测出。