二乔玉兰花粉贮存条件的比较研究

探讨了二乔玉兰(MagnoliasoulangeanaSoul.-Bod.)花粉在不同温度和贮存条件下的活力。结果表明:二乔玉兰花粉在5%蔗糖 0.01%硼酸及5%蔗糖 0.1%硼酸两种培养液上萌发较好,萌发率均达到70%以上。随着保存时间的延长,花粉萌发率不断降低,降低速度从快到慢依次为室温、5℃和-20℃。超低温(-196℃)保存花粉的萌发率并没有随着保存时间的延长而降低,液氮保存2a的花粉萌发率达到79.3%,与新鲜花粉的萌发率差异不显著。超低温反复冻存6次的花粉萌发率与新鲜花粉没有显著变化。     

不同形态的磷源对球形棕囊藻生长及碱性磷酸酶的影响

研究了不同浓度和形态的无机磷(DIP)和有机磷(DOP)对球形棕囊藻细胞生长、培养基中磷浓度的变化以及碱性磷酸酶活性的变化的影响。在缺磷和大分子DOP(卵磷脂)作为磷源的情况下,碱性磷酸酶活性迅速提高,在第9天达到了最大值,其活性分别为19.11U和18.28U,显然在缺磷胁迫下和大分子DOP的利用过程中碱性磷酸酶起着重要作用。DIP(磷酸二氢钾)和小分子DOP(β-甘油磷酸钠)都容易直接被球形棕囊藻吸收利用,碱性磷酸酶活性变化不明显。     

海蚕新型抗菌肽Perinerin在毕赤酵母中的表达及活性测定

摘要：利用巴斯德毕赤酵母表达系统,对海蚕抗菌肽 Perinerin 进行分泌性表达,并进行活性检测。首先通过 SOE 法（Gene splicing by over lap extension）设计三对互补引物来合成完整的Perinerin 的基因,将该基因片断插入到含有AOX1启动子和α分泌信号肽序列载体pPICZαA中,构建了重组表达质粒 pPICZαA-PEN ,转化Pichia pastoris GS115 宿主菌,利用甲醇来诱导外源基因在阳性菌株中的表达,表达产物经 Tricine-SDS-PAGE 电泳验证。生物学活性检测证实重组蛋白对部分革兰阳性及革兰阴性细菌有明显抑制作用,尤其对绿脓杆菌有强烈的抑制作用。     

重组人IL-4大肠杆菌表达与纯化

根据大肠杆菌密码子偏爱性优化并合成人白细胞介素4基因,以pET30a( )为载体构建了重组表达质粒pET30a( )/rhIL-4,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,诱导表达并超声破菌检测重组蛋白的表达形式。采用5L发酵罐培养工程菌,发酵液OD600为0.6时诱导3.5h收集菌体,检测目的蛋白的表达量。收集的菌体经压榨破菌获得包涵体,通过包涵体变性、层析、透析复性等方法对rhIL-4进行纯化。采用人红细胞白血病细胞(TF-1)测定纯化的rhIL-4的生物活性。测序表明目的基因已插入载体pET30a( )中,重组蛋白以包涵体形式表达,单位体积重组蛋白的表达量达200mg/L发酵液,建立了对包涵体形式表达的rhIL-4纯化方法,最终得率为40mg/L发酵液,纯度大于98%,回收率为20%以上。免疫印迹法检测诱导表达的重组蛋白和纯化的蛋白为IL-4,N端氨基酸序列测定结果与理论相符,生物活性检测纯化的蛋白比活性达2.5×106AU/mg。这为rhIL-4进一步产业化研究建立了基础。     

表达流感病毒神经氨酸酶基因的重组腺病毒的构建

通过RT-PCR方法扩增流感病毒神经氨酸酶基因,将其克隆到腺病毒穿梭载体pTrackCMV,此重组质粒与腺病毒DNA共转化大肠杆菌BJ5183,通过细菌内同源重组获得重组腺病毒DNA,将其转染293细胞获得重组腺病毒.经PCR证实目的基因已整合至腺病毒基因组中,western blot检测到神经氨酸酶的表达.重组病毒经滴鼻和灌胃两种途径免疫小鼠,结果表明2次免疫后滴鼻组和灌胃组均产生明显的免疫应答反应,滴鼻组的免疫效果优于灌胃组.     

Morphologies and phylogenetic classification of cellulolytic myxobacteria

The evolutionary distances of the 16S rDNA sequences in cellulolytic myxobacteria are less than 3%, which units all the strains into a single genus, Sorangium. The size of myxospores and the shape of sporangioles, rather than fruiting body colors or swarm morphologies are consistent with the changes of the 16S rDNA sequences. It is suggested that there are at least two species in the genus Sorangium: one includes strains with small myxospores and spherical sporangioles, and the color of the fruiting bodies is normally orange or brown, though sometimes yellow or black. The second species has large myxospores, polyhedral sporangioles with many inter-cystic substrates, and normally deep brown to black color.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

电击法介导的紫孢侧耳原生质体转化

使用基因脉冲导入仪成功地将糙皮侧耳DNA导入紫孢侧耳单核原生质体内,获得了具有"锁状联合”特征的双核转化菌株T₁,和T₂。转化率为8．2×10^-5,转化比为3．6％。酯酶同I酶分析结果表明,转化菌株除具有受体菌的酶带外,还存在供体菌的酶带,由此证明转化菌株确为紫孢侧耳和糙皮侧耳DNA重组的产物。转化菌株子实体形态也发生了变化。两菌株子实体均不释放孢子;T₁。菌柄中生,T₂成熟子实体菌盖中部易长出菌丝。