RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

微生物法生成辅酶Q10的研究

本文根据我们实验室建立的发酵产物中辅酶Q10定性定量检测方法，筛选得到一株可以代谢产生较多辅酶Q10的野生菌株放射形土壤杆菌(Agrobacterium radiobacter No.50)。 为了提高放射形土壤杆菌的辅酶Q10的产量，本实验利用液体培养研究了单因素对菌株辅酶Q10产量的影响，并用正交法确定了最佳液态发酵条件。最佳发酵培养基是：葡萄糖20g,蔗糖40g, 硫酸铵10g,玉米浆30g, 酵母膏3g，K2HPO4 3g,MgSO4.7H2O 1g，蒸馏水1000mL,pH 7.0-7.2。最佳发酵条件是：转接斜面菌种到种子培养基, 转速220r/min、温度28。C培养24h后，转入发酵培养基(250mL三角拼装液量为50mL，pH 7.0), 接种量为10％，转速220r/min、温度28。C，培养120h。在此条件下，菌体湿重约为50g/L，辅酶Q10含量约为20mg/L。 本文以放射形土壤杆菌为出发菌株进行诱变育种，以期获得辅酶Q10高产菌。根据微生物育种原理、参照辅酶Q10的代谢调控机制，以野生型放射形土壤杆菌(Agrobacterium radiobacter No.50)为出发菌株，采用紫外线和亚硝基胍复合诱变技术，依次筛选得到菌体提取物M抗性菌ARM－7、烟草提取物T抗性菌株ARMT-26、Vk3抗性菌株ARMTV-25、链霉素抗性菌株ARMTVS-32，菌株ARMTVS-32产量达到了36.8mg/L，与原始出发菌株相比，产量提高了77％。 研究了茄尼醇、对羟基苯甲酸、橘子皮提取物D、胡萝卜提取物E、烟草提取物对ARMTVS-32合成辅酶Q10的影响，结果表明这些物质对菌体合成辅酶Q10有一定促进作用，添加0.2g/L茄尼醇时，辅酶Q10含量提高了17%，达到了40.7mg/L；添加1.2g/L橘子皮提取物D时，辅酶Q10含量提高了13.8%，达到了39.6mg/L；添加0.5g/L胡萝卜提取物E时，辅酶Q10含量提高了25.3% ，达到了43.6mg/L；添加8g/L烟草提取物时，辅酶Q10含量提高了12.6%，达到了39.2mg/L。 Production of Coenzyme- Q10 (CoQ10) by fermentation is considered as a process with broad prospects.Quantitative Analysis of CoQ10 in the culture of microbe by TLC—UV spectrophotometry was developed, by using this method we got the strain Agrobacterium radiobacter,which was isolated from forest soil of southwest of China. The effect of the single factor on CoQ10-production ability of the strain was examined by liquid cultured, and its best optimum cultivation conditions were established by orthogonal method. The results showed that the optimum fermentation conditions were as following: carbon sources glucose 20g/L,sucrose 40g/L; nitrongen sources (NH4)2SO4 10g/L,maize liquid 30g/L;yeast extract 3g; K2HPO4 3g/L,MgSO4.7H2O 1g/L; initial pH was 7 and volume of medium(medium volume vs flask volume) was 50mL/500mL, incubating for 120h on a rotary shaker at 220 rpm and 28℃.Under these conditions, the biomass and CoQ10 concentration reached 50g/L and 20mg/L respectively. According to the biosynthesis mechanism of CoQ10 and breeding theory, CoQ10 over-production strains were screened by UV--NTG. mutation using Agrobacterium radiobacter No.50 as parent strain. A microbe-juice resistant mutant ARMTVS-32, which also could resist tobacco-juice, VK3 and streptomycin, was screened out from an agar plate. The CoQ10 content of ARMTVS-32 reached 36.8mg/L, which was 77% higher than the initial strain. In addition, We discussed the effects of some organic substrates on the synthesis of CoQ10 in ARMTVS-32. The results showed that solanesol, orange juice D, carrot juice E and tobacco juice could promote the CoQ10 accumulation in the cells. The CoQ10 content of ARMTVS-32 reached 40.7mg/L when added 0.2g/L solanesol,it reached 39.6mg/L when added 1.2g/L orange juice D，it reached 43.6mg/L when added 0.5g/L carrot juice E. it reached 39.2mg/L when added 8g/L tobacco juice.

施钾水平对烟叶含钾量的影响

通过盆栽试验 ,采用定位叶片的方法 ,进行不同钾肥用量试验 ,研究了增施钾肥对石灰性土壤上烤烟吸钾和土壤供钾的影响及提高石灰性土壤上烤烟含钾量的可能途径。结果表明 ,增施钾肥可以显著提高烟叶含钾量 ;而当施钾量达一定水平时 ,只有大幅度增加钾肥施用量 ,烟叶含钾量才显著增加 ,但对烟叶产量影响不大。施钾对烟叶含钾量的提高作用在生育后期最为显著。保证生育后期充足的钾素供应对提高烟叶含钾量可能具有重要意义

供钾方式对烟叶含钾量的影响

通过大田试验 ,研究了不同供钾方式对烟叶含钾量的影响及提高石灰性土壤上烤烟含钾量的可能途径。结果表明 ,施钾能显著提高烟叶产量 ,其中以粉肥结合喷优丰处理效果最好。在干旱条件下 ,增施钾肥可以显著提高烟叶含钾量 ,尤其能较大幅度提高中上部烟叶含钾量。粉肥对提高烟叶含钾量的效果稍优于粒肥。采用喷施“优丰 98- 2”进行根外补钾 ,可显著提高烟叶含钾量。在石灰性土壤上 ,干旱是提高烟叶含钾量的障碍因子。仅凭土壤施钾来提高烟叶含钾量 ,效果不稳定 ,受气候影响较大。土施结合喷施叶肥 ,是提高该区烤烟烟叶含钾量的有效途径。

Biofilm-Engineered Nanostructures

The use of biofilms as nanostructure-engineering materials is discussed and exemplified using ZnO nanorods. Three examples are presented for illustration, the immobilization of ZnO-nanorod arrays on the inner wall of a polystyrene centrifuge tube using S. thermophilus, the morphological organization of ZnO "filters" using S. thermophilus. And the design and implementation of a ZnO-decorated Ag framework using E. coli.

卷烟厂制丝线生产过程建模与计划仿真

对当前卷烟厂制丝线计划与调度管理中存在的不足进行了介绍,并在建立制丝生产线生产过程模型的基础上,提出了用于解决问题的计划仿真系统,对所采用的仿真策略进行了详细介绍。采用这一策略实现的仿真系统能够很方便的适应于其他行业类型的仿真。

PASSIM卷接机组新型电控系统的设计与实现

PASSIM卷接机组原控制系统采用电路板进行逻辑控制,抗干扰性能差,故障率高,造成生产效率降低、原材料消耗增大、维修工作繁重等。为此,设计了一套新型卷接机组电气控制系统。该系统采用PLC进行过程控制,以工控机为上位机完成人机通信,采用交流伺服驱动,并通过高速信号处理专用系统完成重量检测控制及烟支质量检测功能;利用PROFIBUS、CAN及MPI多种总线方式完成各单元间的通讯,实现信号和数据间的传递和共享。改进后的PASSIM机组运行稳定可靠,采样速度快,实时性强,且维修方便。机组的有效作业率由85%左右提高到90%以上,降低了卷烟纸和烟丝等原材料的消耗。

面向烟草行业的MES系统模型研究与设计

MES系统模型构建是系统实施和正常运行的前提和条件,也是MES方案设计和系统配置的基础和难点。本文结合烟草生产过程特点,以某卷烟企业为研究和应用背景,建立了MES系统模型。实际系统运行效果良好,验证了模型的合理性。

卷烟厂制丝线计划仿真系统开发

文章首先分析了卷烟厂制造执行系统(MES)的特点以及发展现状,在此基础上对制丝线进行了介绍,指出了当前制丝线计划与调度管理中存在的不足,并提出了用于解决问题的计划仿真系统,最后在一个模拟的卷烟厂制丝线环境下实现了该系统。

卷烟厂制丝线生产过程建模与计划仿真技术研究

本课题结合国家863高技术计划“面向流程与混合行业的可配置MES产品及行业解决方案”项目2007AA040702和“吉林省延吉卷烟厂制丝生产线生产管理系统”项目选题进行研究与软件开发。是项目研究的主要内容之一，本人主要负责仿真模型建立模块中各项功能的设计与开发。 烟草行业作为一个特殊的行业，它面临着来自市场和国家计划的双重压力，面对这些压力，为了更好的应对市场，烟草企业采用制造执行系统(Manufacturing Execution System，MES)来对卷烟的整个生产过程进行管理。用于完成制丝的制丝生产线是烟草企业最重要的环节，它的工作性能直接决定了卷烟的质量及生产效益。其中，计划的合理性程度以及具体的执行情况是影响其工作性能的关键因素。当前制丝线MES系统的计划调度模块是通过基于规则的调度策略来获得调度方案以对制丝线的生产进行管理，这些规则是根据实际的经验从生产现场抽取得到，在一定程度上保证了计划调度的合理性，但不能保证获得符合实际情况的最优的调度方案，并且在此过程中没有考虑异常事件发生时的应对措施，由于这种调度策略的局限性以及生产线中异常事件发生的随机性，当前制丝线MES系统的计划调度模块已无法很好的管理制丝线的生产。 为解决上述问题，本文在对卷烟厂制丝线生产过程进行建模的基础上，提出并开发了制丝线的计划仿真系统。通过仿真模拟整个制丝生产过程，不仅能确定计划制定的合理性程度，还能预测异常事件发生时对整个计划执行的影响程度，进而采取相应的措施来保证生产的效率。本文的主要研究工作如下： 1、概述了当前主要的生产过程建模方法和离散事件仿真方法，对每种方法的特点进行了概括和总结，在此基础上提出了本文所采用的建模和仿真方法。其中的仿真方法较已有方法在程序实现上做了改进。 2、在对现有的烟草企业制丝线生产过程进行分析的基础上，采用面向对象的建模方法对其进行建模，为生产计划仿真提供基础。 3、设计了生产计划仿真系统的整体框架，对各功能模块设计、系统设计及其实现技术作了细致的阐述。 4、开发实现了该计划仿真系统并针对延吉卷烟厂新厂区制丝线进行计划仿真，分析表明，仿真效果良好。

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected phiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.