大豆24kDa油体蛋白与人bFGF融合基因转化拟南芥的研究

为探索大豆油体蛋白与人bFGF（碱性成纤维细胞生长因子）蛋白在植物油体中融合表达的可行性,以大豆总DNA为模板,通过PCR扩增得到大豆油体蛋白基因（Ddoil）及启动子（Ddprm）,构建Ddprm启动的Ddoil基因与植物偏好密码子改造的bFGF基因融合表达载体p1390Ddprm-Ddoil-bFGF,花侵染法转化拟南芥,对T2拟南芥抗性植株基因组进行PCR检测,对种子总蛋白进行SDS-PAGE分析、Western blot检测、ELISA检测及细胞活性测定,为bFGF在其他油料作物的油体系统的表达提供参考。     

VEGF基因油体表达系统构建及对拟南芥的遗传转化

为研究油体系统在植物中表达外源蛋白的优势,以油菜总DNA 为模板,通过PCR 得到油菜油体(Ycoil)及启动子序列(Ycprm)。同时,将多对合成的寡核苷酸链,通过PCR 方法拼接得到血管内皮生长因子全长基因(VEGF)。以此为基础,构建Ycoil、Ycprm、VEGF 融合表达载体p1390Ycprm-Ycoil-VEGF。将重组质粒转入农杆 菌GV3101,并用花浸染法对拟南芥进行浸染。在含潮霉素(20mg/L)的1/2MS 培养基上筛选转基因拟南芥种子(T0),获得了T1 代抗性植株,通过PCR 检测、SDS-PAGE 检测和Western blotting 检测,结果表明油菜油体及血管内皮生长因子的融合基因已经转入拟南芥中,并成功得到表达,转化率约为0.1%。     

Efficient construction of cDNA microarrays utilizing normalized cDNA libraries of Arabidopsis thaliana

We have demonstrated the utility of normalization for efficient cDNA microarray preparation using Arabidopsis as a model. Nonredundant cDNAs including 5722 species were efficiently collected from random 7914 expressed sequence tags (ESTs) in four normalized cDNA libraries. The prepared microarrays were successfully used to monitor gene expression. These methodologies should be applicable to the study of other species in plant biotechnology.     

拟南芥籽油超声波辅助提取工艺的响应面法优化

利用响应面法对拟南芥籽油超声波辅助提取工艺进行优化。在单因素试验基础上采用中心组合（Box-Behnken）试验设计方法,研究提取时间、超声功率、提取温度及其交互作用对拟南芥籽油提取率的影响。试验确定的超声波辅助提取拟南芥籽油的最佳条件为：料液比1∶6,提取时间55 min,超声功率400 W,提取温度55℃。在最佳条件下,拟南芥籽油提取率为38.01%。     

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.     

Theoretical analysis on pulsed microwave heating of pork meat supported on ceramic plate

Theoretical analysis has been carried out to study the role of ceramic plates (alumina and SiC) and pulsed microwave heating of pork meat (Pork Luncheon Roll (PLR) and White Pudding (WP)) samples. Spatial hot spots occur either at the center of the sample or at the outer face or at the face attached with alumina plate and application of pulsing minimizes formation of hot spots within meat samples. Pulsing of microwave is characterized by set point for temperature difference (ΔT_S) and on–off constraints for temperature (T′). It is found that alumina plate with higher ΔT_S and lower T′ may be recommended for thick meat samples (both WP and PLR) whereas for thin meat samples, lower ΔT_S with alumina plate/without plate may be preferred. It is also observed that SiC plate may be selectively used with ΔT_S = 20 K for both the pork meats. The distributed microwave incidence is found to be effective due to lesser degree of thermal runaway in absence of pulsing for both meat samples.     

Promoter of Arabidopsis thaliana phosphate transporter gene drives root-specific expression of transgene in rice

The PHT1 promoter::GUS fusion gene was constructed and introduced into Arabidopsis and rice by Agrobacterium-mediated transformation. Strong beta-glucuronidase (GUS) activity was detected in roots and showed phosphate starvation induction both in Arabidopsis and rice. In contrast, GUS activity in aerial tissues such as those of the leaf and stem was low. In situ GUS staining of root tissue indicated that PHT1 was expressed in root hairs and the outer layer of the main roots, but not in root tips. The PHT1 promoter has a desirable character for biotechnological transgene expression in monocot rice plants.     

Formate dehydrogenase gene of Arabidopsis thaliana is induced by formaldehyde and not by formic acid

Variability of expression of formate dehydrogenase (FDH) caused by uptake of C-1 compounds was examined by using Arabidopsis thaliana as a model plant. Effects of uptake of several C-1 compounds were evaluated by Northern blot analysis using cDNA of A. thaliana FDH prepared by cloning on the basis of known sequence. As a result, expression of the FDH gene in A. thaliana was not intensely influenced by formic acid, an inherent substrate for FDH, but strongly induced by its reduced form, formaldehyde.     

Development of a genetic transformation system using new selectable markers for fission yeast Schizosaccharomyces pombe

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.     

Preferential binding of SBD from Arabidopsis thaliana SSIII to polysaccharides: Study of amino acid residues involved

SS III (SSIII) has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N‐terminal transit peptide for chloroplast localization followed by three in tandem starch‐binding domains (SBDs D1, D2, and D3, residues 22‐591). Its C‐terminal catalytic domain (residues 592–1025) is similar to bacterial glycogen synthase. Binding studies to raw starch and its individual components, AM or AP show that the SBD region binds preferentially to AM, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites which include amino acid residues Y394 (binding site 1) and W366 (binding site 2) acting cooperatively with the D1 domain in the binding process while G335 and W340 have a minor role. In addition, mutations in these residues also affect the kinetic parameters for the polysaccharide substrate of SSIII.     

拟南芥蔗糖合酶基因合成、重组表达及其在酶催化合成莱鲍迪甙A中的初步应用

本研究采用两步PCR的方法,合成拟南芥蔗糖合酶At SUS1的编码基因,并将其亚克隆到携带糖基转移酶UGT76G1基因的表达质粒p ET28a-UGT上,构建了双酶共表达质粒p EUGT-SUS。将质粒p EUGT-SUS转化到大肠杆菌Rossetta（DE3）中,在自诱导培养基中培养12 h,收集细胞超声破碎取其上清液为粗酶液用于催化甜菊甙合成莱鲍迪甙A的反应,结果确定重组酶At SUS1获得了活性表达,在双酶反应体系中,蔗糖和UDP在At SUS1作用下生成UDP-葡萄糖,供给UGT76G1所催化的糖基转移反应。考察了UDP初始浓度、反应pH、温度及反应时间对酶催化RA甙合成的影响。当UDP初始浓度为0.01 mmol/L,pH7.2和35℃反应条件下,反应12 h,10 g/L甜菊甙转化为莱鲍迪甙A的收率最高可达56.2%。为建立高效、经济的合成莱鲍迪甙A的酶催化工艺奠定了基础。      

Enantioselective Damage of Diclofop Acid Mediated by Oxidative Stress and Acetyl-CoA Carboxylase in Nontarget Plant Arabidopsis thaliana

Diclofop-methyl (DM) is a widely used chiral herbicide, which rapidly hydrolyzes to its major metabolite diclofop acid (DC) after application. With a carbon chiral center, DC not only is an important ingredient of herbicidal activity, but also has a long half-life in soil. Studies so far have only considered the activity of racemic DM in target organisms, and the enantioselective toxicity in nontarget plants of DM and DC has yet to be explored. In this study, the enantioselective phytotoxicity of DC mediated by oxidative stress and the key enzyme ACCase in the fatty acid synthesis system on the model plant Arabidopsis thaliana was investigated. Significant differences between the two enantiomers were observed in phytotoxicity including growth inhibition, oxidative damage and alteration of key genes expression of ACCase, with R-DC showing greater toxicity to Arabidopsis thaliana than S-DC. The results of molecular docking showed that there was a stronger affinity between R-DC and the target enzyme carboxyltransferase domain of ACCase, likely leading to the enantioselective phytotoxicity of DC. This study suggested that chirality of both parent compounds and metabolites should be considered to improve our understanding of the environmental fate and risks of chiral pesticides.     

Microfiltration of mosambi juice using low cost ceramic membrane

This work reports on microfiltration (MF) studies of mosambi juice using low cost ceramic membrane prepared from locally available inorganic precursors. Characterization of the prepared membrane was done by SEM analysis, porosity determination and pure water permeation experiments. The average pore diameter, total porosity and hydraulic resistance of the membrane were evaluated as 0.285 μm, 23.6% and 9.26 × 10¹¹ m²/m³, respectively. Dead-end MF experiments were performed for both centrifuged mosambi juice (CJ) and enzyme treated centrifuged mosambi juice (ETCJ). It was observed that after MF, important properties like TSS, pH, acidity and density of both CJ and ETCJ were almost unaffected. However, significant improvement in juice colour, clarity and AIS was observed. It was also observed that the clarified juice can be stored in refrigerated condition for more than 30 days without significant change in juice quality. Different membrane pore blocking models were used to analyze the observed permeate flux decline.     

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.     

蔗渣浆筛选系统升级改造

通过对筛选系统的升级改造,提高了筛选能力;同时降低筛选设备使用的电耗;改善漂白蔗渣浆的质量;减少因筛选原因造成的纤尘副品;减少纤维流失。在不影响抄纸生产质量的前提下,浆产量由原来的120t/d提高到135t/d,每小时电耗下降56kWh,漂白浆合格率提高到97%以上。     

成绳机压线瓦控制系统改造

成绳机是钢丝绳生产的关键设备之一,其合绳装置压线瓦一般采用手动调节螺杆压紧方式,不便于在钢丝绳合绳时对压线瓦压力进行有效的控制和监测,且当合绳出现质量问题时,压线瓦不能自动松开,设备不能自动停机,易造成产品报废。针对存在的问题,对压线装置进行改进,给出改造后的液压系统和控制系统框图。运行表明,采用通信技术和液压系统控制压线瓦,可保证生产不同规格的钢丝绳所需要的压力恒定,有效检测生产过程中钢丝绳直径的变化并自动调节,确保产品质量,提升设备自动化程度。     

平转浸出器传动系统整体改造

通过对平转浸出器转子链条安装形式的改造,解决了原有转子链条拆卸困难且危险性高等缺点;对减速机润滑系统的改进,解决了平转浸出器减速机故障率高的问题;通过安装转子平衡、限位装置,提高了平转浸出器传动系统的安全性和稳定性.     

陶瓷膜复滤古龙酸超滤液中试研究

利用陶瓷膜进一步浓缩古龙酸超滤浓缩液,考察了复滤过程中的运行压力、温度、复滤时间、洗水量等因素对膜通量和古龙酸收率的影响,并对膜污染和清洗方法进行了研究。结果表明,在运行压力0.30MPa,温度40℃,古龙酸料液进一步浓缩3倍的条件下,加0.67倍水,反算收率可达96.9%,滤渣的酸量降为进料的1/10,滤渣中的酸残留总量约为3%,整个过程膜过滤平均通量维持在24.5~26.5LMH,符合工业化生产实际需要。而且实验期内,古龙酸料液未对陶瓷膜造成不可逆污染,利用市售碱性清洗剂清洗,通量可以基本完全恢复。      

冲击波在陶瓷增强纬编双轴向多层衬纱织物及机织物复合材料中传递的表征

为了分析冲击波在复合材料内的传递规律,通过将纬编双轴向(MBWK)多层衬纱织物、机织物与陶瓷层交替铺层与不饱和聚酯树脂进行复合制备了复合材料,利用落锤冲击试验机对复合材料进行冲击测试,研究了试样冲击破坏后陶瓷片裂纹扩展及碎裂规律,探讨了冲击应力在不同结构复合材料内部的传播规律,并分析了MBWK织物增强复合材料的冲击应力...