Finite element simulation of the physical gelation of rennet casein(酶凝干酪素物理凝胶化过程的有限元模拟)

以酶凝干酪素的凝胶化过程为对象,利用有限元方法数值分析了在凝胶化过程中温度场的空间分布和时间演变规律.在此基础上,基于一阶的凝胶化动力学方程,数值模拟了凝胶体系的复剪切模量场,进而分析了材料配方、体系尺寸与冷却方案对复剪切模量场的影响规律.模拟结果表明,由于热阻的差异,体系表面的冷却速率大于内部,表面首先发生凝胶化;而由于预凝胶化阶段的平均冷却速率决定了无穷复剪切模量的值,最终体系内部的复剪切模量超过表面的.

水稻I型酪蛋白激酶的分离及生理功能研究

（第二部分的摘要） 酪蛋白激酶在许多物种的细胞分裂及分化过程中都有重要作用。在水稻中，以经过油菜素内酯处理的水稻幼苗为材料，通过cDNA微矩阵的方法得到了一个全长1939bp的基因OsCKI1（Accession number AJ487966）。该基因编码的蛋白产物属I型酪蛋白激酶（CKIs），含463个氨基酸。RT-PCR及Northern blot结果显示，基因OsCKI1在水稻各组织中表现为组成型表达，并且其表达受油菜素内酯（BR）及脱落酸（ABA）的诱导。在大肠杆菌中对该基因进行原核表达，并用表达后的蛋白粗提物进行酶活测定，显示该蛋白产物可磷酸化CKIs的特异性底物酪蛋白。通过构建OsCKI1的反义载体并转化水稻，对该基因的生理功能进行了研究。对转基因植株的纯系表型进行了观察，显示其根部发育异常，表现为具有较短的初生根、侧根及不定根数目少于对照。进一步研究显示初生根的变短是由于细胞延伸受抑制引起的。以CKI的特异性抑制剂，CKI-7处理野生型植株，也对OsCKI1缺失引起的表型进行了确认。值得注意的是，以外源生长素（IAA）处理转基因及经CKI-7处理过的野生型植株，都能恢复根部表型，使其生长正常。对反义植株初生根及次生根的游离生长素含量测定结果显示，OsCKI1可能在IAA的代谢途径中发挥作用。转基因植株的种子在萌发时对ABA及BR的处理都表现为不敏感，暗示该基因可能在各种激素信号转导途径中都有作用。OsCKI1-GFP双元表达载体的亚细胞定位的研究显示该基因主要定位于核中，可能参与了基因表达的调节。同时，以该反义转基因植株为材料，通过cDNA芯片的技术研究了受OsCKI1调节的基因的表达谱，结果显示该基因的缺失的确影响了参与信号转导及激素代谢途径的许多基因的表达。 （第四部分的摘要） 以OsCKI1反义转基因植株对照植株为材料，研究它们处于4℃低温胁迫下的反应情况。植株种子在室温下萌发并生长一段时间后，移入4℃低温下进一步生长。取对照及低温处理后的材料，对其表型进行观察，显示低温下转基因植株初生根生长受抑制程度小于对照，其生长的延缓程度低;相对电导率测定结果显示，经低温处理后，转基因植株相对电导率变化较小，质膜受害程度小;微管观察结果也显示在短期低温处理下对照根部延伸区细胞的皮层微管解聚，而转基因植株其根部延伸区细胞的皮层微管仍能保持正常状态。基因OsCKI1在低温下的表达模式表现为先升高之后又降低，推测其在低温信号的转导途径中发挥作用。通过总结以上结果，我们认为基因OsCKI1的反义转基因植株虽然在短期冷害下具有一定的抗冷能力，但其不具备形成长期稳定的冷适应的能力。

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.

川牛膝多糖的免疫活性及其水解活性片段分离研究

川牛膝多糖（CP）是从传统中药川牛膝（Cyathula officinalis Kuan）中提取的一种活性多糖，现代药理研究表明川牛膝多糖是川牛膝许多生物活性的物质基础。本实验室前期进行了川牛膝多糖的提取、分离、结构鉴定及其部分活性研究，发现川牛膝中多糖含量非常高，在对川牛膝多糖活性的初步研究中也证实了其具有免疫调节作用。我们为了进一步了解其免疫调节活性，并为构效关系的研究奠定基础，对其进行了如下研究： 1． 通过体外毒性检测、淋巴细胞增殖实验、NK细胞杀伤活性和腹腔巨噬细胞吞噬中性红活性测定，发现川牛膝多糖在10～300μg/mL浓度范围内，对细胞无毒性作用；能够促进LPS诱导的B淋巴细胞增殖(P<0.01)、增强NK细胞杀伤活性(P<0.05)和PMΦ吞噬中性红活性(P<0.01)，且随多糖浓度增高而增强；但其对ConA诱导的T淋巴细胞的增殖无促进作用(P>0.05)。 2． 通过正常小鼠体内淋巴细胞转化实验、迟发型变态反应分析、抗体生成细胞检测、碳粒廓清检测、腹腔巨噬细胞吞噬鸡红细胞活性和NK细胞活性测定，发现川牛膝多糖在适应性免疫方面能够促进SRBC免疫小鼠体内的抗体生成细胞的生成（P<0.01）和增强DNFB诱导的DTH（P<0.05），但对ConA诱导的脾淋巴细胞增殖无促进作用（P>0.05）；在固有免疫方面能够提高小鼠碳粒廓清速率（P<0.05），PMΦ吞噬 CRBC 活性（P<0.01）和NK细胞杀伤活性（P<0.05）。同时还发现其对由环磷酰胺(Cy)引起的白细胞数下降具有很好的抑制作用（P<0.01）。 3． 为了获得结构明确、均一的保留活性的川牛膝多糖片段，为其作用机制、构效关系研究提供关键研究材料，我们开展了“保留免疫活性的最小片段”的分离制备的初步研究。建立并优化了川牛膝多糖的酸水解条件，发现在6%的样品浓度，0.025mol/L的硫酸浓度，65℃的水解温度，水解时间为8min的条件下可以得到一系列连续的多糖片段；采用Bio-Gel P2 分子筛柱层析分离得到5个级分，通过体外淋巴细胞增殖实验、NK细胞活性测定、腹腔巨噬细胞吞噬中性红实验发现其中的一个片段仍保留较强的免疫活性，并测得其分子量约为2057Da，为保留免疫活性的最小片段的进一步分离奠定了基础。 Cyathula officinalis Kuan is a commonly-used Traditional Chinese Medicine (TCM) with a wide range of pharmacological activities. Modern pharmacological researches showed the polysaccharide extracted from it (CP) is an important component for many bioactivities of this TCM. In the previous studies, we found CP showed significant immuno-regulative activities. In order to evaluate this activity systematically and lay foundations for revealling its immuno-regulative machanisms and the Structure -Function relationship, we carried out the following research works: 1． The in vitro immunoactivities of CP were evaluated by using normal mice immunocytes with respects to cytotoxicity, lymphocytes proliferation, NK activity and the ability of peritoneal macrophage phagocytizing neutral red. The polysaccharide showed no cytotoxicity below the concentration of 300 μg/mL, and could promote B lymphocytes proliferation (P<0.01), enhance NK activity (P<0.05) and the ability of peritoneal macrophage phagocytizing neutral red (P<0.01) at the concentration of 10-300 μg/mL. The above effects were positively correlated with the concentration of the polysaccharides. But it could not promote T lymphocytes proliferation (P>0.05). 2． The in vivo immunoactivities of CP were observed on normal mice through the following indices: splenic lymphocyte transformation efficiency, delayed-type allergy, antibody-forming cells activity (AFC), rate of carbon clearance, rate of peritoneal macrophage phagocytizing chicken red blood cell (CRBC) and NK activity, and its influence on the decline of the mouse leucocyte count induced by Cy. The polysaccharide at medium-dose enhanced delayed-type allergy （P<0.05）and NK activity（P<0.05） and increased the rate of carbon clearance（P<0.05）, AFC activity（P<0.01） and the rate of peritoneal macrophage phagocytizing CRBC（P<0.01）. The polysaccharides also effectively resisted the decline of the mouse leucocyte count induced by Cy（P<0.01）. However, it couldn’t increase the splenic lymphocyte transformation efficiency（P>0.05）. 3． Attempting to isolate and prepare the minimal fragments retaining activity with identical structure for further studying on immuno-regulative mechanism and Structure-Function relationship, we carried out the study on hydrolysis of CP, isolation of hydrolysed fragments, and the activity evaluation of the isolated fragments. CP with concentration of 6% was hydrolysed at 65℃ for 8 min with sulfuric acid of 0.025 mol/L，then the hydrolysate was separated using Bio-Gel P2 chromatography, 5 portions of fragments were obtained. The immunoactivities of these fragments were evaluated by using normal mice immunocytes with respect to lymphocytes proliferation, NK activity and ability of peritoneal macrophage phagocytizing neutral red. One fragment with relative molecular mass of 2057Da was found retaining immunoactivity.

链霉菌S227抗耐药菌活性成分的研究

本论文以从四川峨嵋山森林土壤中分离筛选获得的一株产抗耐药性活性化合物的链霉菌S227为材料，对发酵液中活性物质的分离纯化及抗耐药性活性进行了研究。 建立了抗耐药性活性的定性、定量检测方法。建立的管蝶法活性定量检测的标准回归方程为：D=4.8229Ln(C)+3.6326 R=0.9972 ；纸片法活性定量检测的标准回归方程为：D=5.5Ln(C)-12.794 R=0.999。 根据建立的样品活性的检测方法，测定了发酵液的初始活性。实验证明活性物质的温度、pH稳定性好。 通过活性的定性、定量追踪方法，分别利用等体积的石油醚、乙酸乙酯、正丁醇在不同的pH梯度下萃取，确定了pH3条件下正丁醇能最大程度的萃取活性物质，说明活性物质极性很大。对正丁醇萃取相经过两次硅胶柱层析及薄层层析分离得到具有抗耐药菌活性的纯化样品S227-4。 经过核磁共振氢谱、碳谱数据分析初步确定S227-4为四聚糖，通过糖的水解实验初步确定S227-4由葡萄糖和半乳糖组成。 纸片法活性检测表明S227-4具有抗耐药菌活性。采用MIC测定法对该样品抗耐药活性进行研究。在证明该样品本身不具有抗菌活性的基础上，以临床分离的耐药性金黄色葡萄球菌为指示菌，考察了该样品与抗生素联合使用时对耐药菌抗生素MIC（最小抑菌浓度）值的影响，结果表明在不影响菌体生长的浓度条件下，该样品能明显降低多株耐药菌对多种抗生素的MIC值，不同程度地恢复所测试耐药菌对相应抗生素的敏感性。如S227-4与青霉素钠联用可以使S. aureus 12352的MIC降低8倍，而与红霉素联用可以使S. aureus 12334的MIC降低128倍。 The purification process and the activity of the anti bacterial drug resistance compounds produced by Streptomyces S227 isolated from the forest soil sample of the Mountain E’MEI in Sichuan Province were studied in this thesis. Quantitative and qualitative activity assay methods of the active compounds were established. The regression equation of the tube method was D=4.8229Ln(C)+3.6326, R=0.9972. The regression equation of the paper method was D=5.5Ln(C)-12.794, R=0.999. According to the established activity assay method, the incipient activity of the broth was evaluated. And it was proved that the stability of the active compounds was good. By quantitative and qualitative activity tracing method, petroleum ether, ethyl acetate and butanol were used to extract the active compounds at different pH. The result showed that butanol was the most effective agent for active component recovery at pH3. From the butanol extraction a purified sample, S227-4, was isolated by silica gel column chromatography and thin-layer chromatography . S227-4 was proved to be a tetra- saccharide by 1H-NMR and 13C-NMR. And its monosaccharides include glucose and galactose by hydrolysate analysis. The anti-drug resistant activity of S227-4 was tested in vitro by MICs assay using different drug resistant Staphylococcus aureus strains isolated clinically. The sample itself showed no anti-microbial activity in growth inhibitory experiment, but when it was used together with different antibiotics, it could remarkably decrease the MICs of different clinically isolated drug-resistant bacterial strains to these antibiotics. For example, when S227-4 was used with penicillin, the MIC of S. aureus 12352 decreased 8 times compared with that when penicillin was used alone. Meanwhile when it was used with erythromycin the MIC of S. aureus 12334 deceased 128 times compared with erythromycin alone.

一株丝状真菌对半纤维素利用的初步研究

本文筛选出一株能利用木糖产乙醇的丝状真菌Z7，对其利用木糖和半纤维素水解产物产乙醇的发酵条件进行了研究，并对Z7 利用玉米芯产木聚糖酶的条件进行了优化。全文分为三部分： 第一部分：目标微生物筛选、纯化及系统发育分析。以木糖为唯一碳源，采用梯度稀释和平板化线法从高温、中温酒曲中分离到16 株能利用木糖良好生长的丝状真菌；通过发酵试验复筛，获得一株能产乙醇的丝状真菌Z7；综合形态学和ITS 序列分析，初步鉴定为Aspergillus flavus。 第二部分：Z7 的乙醇发酵条件研究。以木糖为碳源，通过单因素试验确定最佳氮源和发酵温度；通过正交试验及SPSS 软件分析得到了不同N、P、K 成分对乙醇、残糖和菌体干重的影响。获得最佳的发酵条件为：（g/L）木糖50，尿素1, NH4NO3 1, K2HPO4 2 , KCl 0.5 , MgSO4.7H2O 0.5 , NaNO3 1 , pH 自然，培养温度33 ℃。以玉米芯半纤维素稀酸水解液为底物进行乙醇发酵，根据稀酸水解的单糖释放量和乙醇产量，确定115 ℃，1 h 为最佳玉米芯预处理条件；结合最佳发酵条件，添加1 g/L 的吐温20 能获得最大的乙醇浓度8.31 g/L。因此，Aspergillus flavus Z7 能利用半纤维素水解产物产乙醇，其中木糖的利用率80%以上。 第三部分：Z7 利用玉米芯产木聚糖酶条件优化。Aspergillus flavus Z7 在具有产乙醇能力的同时还具有产木聚糖酶的能力。本文通过单因素和正交试验得到最佳产酶培养基组分为：(g/L)玉米芯20，尿素2, 酵母膏2.5, K2HPO4 5，NaNO31, MgSO4.7H2O 1。单因素试验表明，用纱布代替塑料布密封摇瓶封口能显著提高产酶量；Z7 在碱性条件下具有更强的产酶性能。在最优条件下发酵，能产生最大木聚糖酶活122.23IU/mL。通过薄层分析，验证了Z7 产生的木聚糖酶具有水解木聚糖生成木糖及木寡糖的能力。 A strain of filamentous fungus which can produce ethanol by using the xylose was isolated in this research. The ethanol fermention conditions from xylose and dilute-acid hydrolyzate of the corn core were studied. The conditions of xylanase production by Z7 were also optimized. The paper involved three parts. Part1: Isolation, purification and phylogenetic analysis of the microbe. By using xylose as the single carbon source and the pla te streaking method, several filamentous fungi were isolated from the wine starter; through the fermentation test, a filamentous fungus Z7 which can produce ethanol was further recognized; furthermore, according to the morphologic observation and ITS seque nces analysis, Z7 was identified as Aspergillus flavus at the first step. Part2: Research on the condition of ethanol fermentation by Z7. By single factor experiment, the optional nitrogen resource and temperature of the fermentation were fixed; meanwhile, through the orthogonal array tests and the analysis of statistic software SPSS, the optional component of the culture medium and the fermentation condition were organized as follows: (g/L) xylose 50, urea 1, NH4NO3 1, K2HPO4 2, KCl 0.5 , MgSO4.7H2O 0.5, NaNO31, pH nature, temperature 33℃. Based on these optimal parameters, the fermentation of dilute-acid hydrolyzate of the corn core was carried on by Z7. According to the quantities of released sugar monomers and content of the ethanol, 115℃ in 1h is the best pretreatment condition; the maximal ethanol content can be obtained when 1g/L Tween 20 was added to. Therefore, the filamentous fungus Aspergillus flavus can use the hydrolysate of hemicellulose to produce ethanol, and the rate of xylose utilization was over 80%. Part3: Optimization of Z7’s xylanase producing condition from corn core. Aspergillus flavus Z7, which can utilize xylose or the hydrolysate of hemicellulose to produce ethanol, also had the ability of xylanase production. The optional component of the culture medium were fixed by the single factor experiment and the orthogonal array tests, and they were organized as follows: (g/L) corn core 20, Urea 2, Yeast extract 2.5, K2HPO4 5, NaNO31, MgSO4.7H2O 1; it was testified by the single factor experiment that sealing the shaking flasks with pledget other than plastic paper can obviously increase the xylanase activity; moreover, Z7 showed better xylanase production ability when in the alkali environment. Under the optional fermentation condition, the maximal xylanase activity 122.23IU/mL was proved. Through the analysis of thin- layer chromatography (TLC), the ability of xylanase from Z7, which can hydrolyze xylan to xylose monomer and oligomer, was vividly displayed.

Separation and detection of narcotic drugs on a microchip using micellar electrokinetic chromatography and electrochemiluminescence

A new approach for fast and sensitive electrochemiluminescence (ECL) detection of narcotic drugs on a microchip after separation by micellar electrokinetic chromatography (MEKC) is presented, taking the cocaine and its hydrolysate ecgonine as the test analytes. The mixture of hydrophilic BMIMBF4 ionic liquid (IL) and sodium dodecyl sulfate (SDS) was used directly as the buffer of MEKC with less noisy baselines, lower electrophoretic current and satisfactory separation performance.

The preparation and bioactivity research of agaro-oligosaccharides

Agaro-oligosaccharides were hydrolytically obtained from agar using hydrochloric acid, citric acid, and cationic exchange resin (solid acid). The FT-IR and NMR data showed that the hydrolysate has the structure of agaro-oligomers. Orthogonal matrix method was applied to optimize the preparation conditions based on alpha-naphthylamine end-labeled HPLC analysis method. The optimal way for oligosaccharides with different degree of polymerization (DP) was achieved by using solid acid degradation, which could give high yield and avoid solution neutralization process. Agaro-oligosaccharides with high purity were consequently obtained by activated carbon column isolation. Furthermore, the antioxidant and alpha-glucosidase inhibitory activity of three fractions were also investigated. The result indicated that 8% ethanol-eluted fraction showed highest activity against alpha-glucosidase with IC50 of 8.84 mg/mL, while 25% ethanol-eluted fraction possessed excellent antioxidant ability.

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp SM9913

A psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913, was isolated from deep-sea sediment collected at 1,855 m depth. Two proteases produced by Pseudoaltermonas sp. SM9913 were purified, MPC-01 and MCP-02. MCP-01 is a serine protease with a molecular weight of 60.7 kDa. It is cold-adapted with an optimum temperature of 30-35degreesC. Its K-m and E-a for the hydrolysis of casein were 0.18% and 39.1 kJ mol(-1), respectively. It had low thermostability, and its activity was reduced by 73% after incubation at 40degreesC for 10 min. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa. Its optimum temperature for the hydrolysis of casein was 50-55degreesC. The K-m and E-a of MCP-02 for the hydrolysis of casein were 0.36% and 59.3 kJ mol(-1), respectively. MCP-02 had high thermostability, and its activity was reduced by only 30.5% after incubation at 60degreesC for 10 min. At low temperatures, Pseudoaltermonas sp. SM9913 mainly produced the psychrophilic protease MCP-01.

中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究

卤虫(Artemia)是一种广温、耐高盐的小型甲壳动物，广泛分布于内陆盐湖和沿海盐田中。卤虫的无节幼体作为重要的蛋白优质饵料，被广泛的应用于水产养殖生产。卤虫具有特殊的生物学特性，是研究甲壳动物胚胎发育的良好的实验材料，同时也是一种研究动物抗逆机制的模式动物。卤虫有卵生和卵胎生两种繁殖后代的方式，当环境条件适宜时，卤虫倾向于采取卵胎生方式，即直接产生无节幼体；而在恶劣的环境条件下，卵生方式占主要地位，产生处于滞育状态的、具有复杂外壳的休眠卵。卤虫的滞育卵具有独特的生物学特性和特殊的生理生化特点。其发育停滞，细胞分裂停止，酶活力下降，代谢活动受到抑制并可耐受各种极端恶劣环境，如缺氧、低温、紫外线、干燥等。即使在最适的环境中滞育卵的孵化率也很低，只有受到某些特定的非生物信号的刺激才自能终止这种滞育状态，恢复生理代谢；当环境条件适宜时，能够继续发育孵化成无节幼体。因此，卤虫的滞育卵在卤虫的整个生活史中占有重要的地位。另一方面，卤虫是极端环境生物，能够抵抗各种恶劣环境胁迫刺激，因此是研究抗逆机理的良好的实验动物。 本论文利用蛋白质组学技术，研究了卤虫滞育卵及滞育卵发育过程中的蛋白质组表达情况，并研究了卤虫幼体在重金属刺激后蛋白表达的变化情况。得到如下结果： 建立了中华卤虫滞育卵可溶性总蛋白的双向凝胶电泳对照图谱。在pH 4–7、分子量10-100 kDa范围内，检测到约 233个蛋白点，并利用高效液相色谱-质谱联用（LC-ESI-MS/MS）技术鉴定了其中的48个丰度较大及感兴趣的蛋白点，根据这些蛋白的生物学功能进行分类，功能类别包括细胞防御蛋白、抗氧化蛋白、细胞骨架蛋白、代谢相关蛋白等。在卤虫滞育卵中共分离鉴定到6个分子量和等电点存在差异的小热休克蛋白p26的异构体，生物信息学分析表明该蛋白有三种不同的功能位点，分别是蛋白激酶C磷酸化位点，Casein 激酶II磷酸化位点及 N-myristoylation 位点。 采用低温脱水的方法对滞育卵进行激活刺激，并对活化卵和滞育卵蛋白表达图谱进行了对比分析。结果表明对卤虫滞育卵的激活刺激引起了其蛋白表达的明显变化。活化卵图谱中蛋白点总数比滞育卵中明显增多，特别是在pI<5.5范围内。约70个蛋白点在激活刺激后上调表达，包括部分只在激活卵中表达的蛋白；25个下调表达，包括部分只在滞育卵中表达的蛋白；其余约60％（占滞育卵蛋白点数目百分比）的蛋白点表达量基本恒定。热休克蛋白家族、抗氧化蛋白家族成员等蛋白变化明显，小热休克蛋白p26、小热休克蛋白ArHsp21蛋白以及过氧化物还原酶异构体在激活卵中特异表达。 活化卵孵化过程中不同发育时期的蛋白表达又呈现出不同的特点，分别在孵化后6h、12h、18h和24h的蛋白质组学图谱上检测到267、285、195和210个蛋白点。孵化后6h和12h休眠卵蛋白表达个数相对较多，与胚胎发育过程中的器官发生和剧烈的形态变化相适应；孵化后18h和24h休眠卵蛋白表达明显下降，部分蛋白的表达关闭，部分蛋白开始富集表达。 利用双向凝胶电泳技术分析了中华卤虫幼体受到急性硫酸铜刺激后的蛋白表达变化情况。通过图谱对比分析，检测到了5mM硫酸铜刺激24h后，卤虫幼体中14个差异表达的蛋白点。利用LC-ESI-MS/MS技术鉴定了其中的7个蛋白，其中3个蛋白上调表达，分别是热休克蛋白70（7.5倍）, 肌动蛋白（2.3倍）和伴侣分子亚基1（3.0倍）。3个蛋白下调表达，分别是：精氨酸激酶（2.8倍）, 延伸因子2 (2.0倍） 和富含甘氨酸蛋白(2.0倍）。硫酸铜刺激后特异表达的一个蛋白被鉴定为过氧化物还原酶(Peroxiredoxin，Prx)。根据质谱检测提供的蛋白肽段信息和其他生物过氧化物还原酶保守氨基酸序列设计简并引物，结合RACE技术，从中华卤虫幼体中克隆到了过氧化物还原酶基因，该基因的cDNA全长为756个碱基，其中开放阅读框为594个碱基，编码198个氨基酸，其蛋白理论分子量为22.0 kDa，理论等电点为6.98。多序列比对结果显示中华卤虫Prx基因的推导氨基酸序列与美国卤虫和中国对虾的同源性高达98%和94%。实时荧光定量PCR结果显示，硫酸铜刺激后，该基因在卤虫无节幼体中的转录水平明显升高，在24h达到正常水平的3.0倍。

皮状丝孢酵母发酵大米草水解液生产微生物油脂

大米草是一种外来引种植物，本来引进的目的是为了保滩护岸、促淤造陆。然而大米草过度繁殖，侵袭海洋沿岸滩涂，所以综合开发大米草有着重要的经济意义和生态意义。本实验研究着力于将大米草水解液通过皮状丝孢酵母发酵而制取生物油脂。并且随着近年来石油价格的攀升和对于汽车尾气对环境造成的日趋严重的污染，生物柴油的开发受到高度重视。微生物油脂则是生物柴油生产的很好的原料，因为它无需占用耕地，而能够把农业加工剩余物或是其它的含纤维素的生物质转化为油脂。 本实验采用产油酵母菌皮状丝孢酵母（Trichosporon cutaneum）直接发酵经过简单脱毒处理的大米草水解液生产微生物油脂。在122℃，固—液比为10%（w/v），分别用1%、2%、4%和6%稀硫酸水解大米草，结果表明在6%的酸浓度下水解40 min后还原糖含量最高，达到22.8 g/L。而同时以1%稀硫酸在140℃下水解大米草，2.8h后，水解液中葡萄糖产量达到最高14.2g/L。确定了摇瓶发酵最适工艺条件：摇床转速200 rpm，接种量10%（v/v），装液量为200 mL (1000-mL三角瓶)。在此基础上，确定了水解液最适浓缩倍数和pH值，水解液浓缩倍数为4.0时产油量高达6.0 g/L，产油率为46.3%。本文为高效利用大米草提供了新的途径。 关键词:皮状丝孢酵母,大米草,水解,发酵,微生物油脂