小麦硫转运蛋白基因半定量RT-PCR检测方法的建立

为进一步研究干旱胁迫下小麦硫转运蛋白基因(ST)的表达,选用小麦-αT ubu lin基因(T a)为内参照,提取小麦根系总RNA,反转录cDNA,同批异管对2个基因片段进行PCR扩增.通过对PCR体系中M g2 浓度、退火温度及循环次数的优化和对体系重复性、准确性的分析,最终建立了一个稳定、方便的半定量RT-PCR体系.该体系可用于比较不同小麦样品间硫转运蛋白基因的表达,且因为两对引物均垮内含子,其稳定RT-PCR体系,也为实验室建立小麦mRNA反转录体系提供了参考依据.     

水分胁迫下小麦类脱水素基因表达的半定量RT-PCR分析

以‘郑引1号’小麦为材料,经水分胁迫后进行RT-PCR扩增,结果在胁迫条件下获得500bp的脱水素基因(wzy1-1)。利用半定量RT-PCR方法,分析‘陕合6号’和‘郑引1号’小麦在正常供水条件下及PEG6000胁迫后18、24和42h,以及复水6和12h时wzy1-1在叶片中的表达。结果表明:2个小麦品种中wzy1-1在水分胁迫后18h均有表达,至胁迫24、48h时表达量较18h均有所增多;复水6h后该基因表达迅速降低,至复水后12h表达消失。且该基因在‘陕合6号’(抗旱性较强)叶片中表达量较‘郑引1号’(抗旱性较弱)高,表明该基因的表达与小麦的抗旱性密切相关。     

黄瓜RGA基因的半定量RT-PCR表达分析

以黄瓜(Cucumis sativusL.)抗霜霉病品种东农129为材料,利用RT-PCR半定量法研究了接种霜霉病菌(Pseudoperonospora cubensisRostow)、喷施水杨酸(SA)和氯化钙(CaCl2)等不同处理对黄瓜抗病基因类似序列(RGA)表达的影响.结果表明:CsRGA1和CsRGA5基因的表达受霜霉病菌的侵染而启动或加强,外施SA和CaCl2都能够增强其表达;CsRGA4和CsRGA8属于组成型表达基因,其表达可能与霜霉病菌的侵染无关;CsRGA2的表达与外施SA和CaCl2缺乏密切关联.     

产乙醇运动发酵单胞菌的研究进展

运动发酵单胞菌作为天然生产乙醇的主要微生物之一,具有特殊的Entner Doudoroff途径和其他一些特殊的糖代谢和能量代谢方式,因此具有乙醇产率高和乙醇耐受力强的显著特点。通过简述运动发酵单胞菌的糖代谢和能量代谢、乙醇和高渗透压等耐性及其遗传改造三方面的研究进展,阐明其应用于燃料乙醇生产的巨大潜力     

重组运动发酵单胞菌的构建及木糖利用特性研究

将大肠杆菌(Escherichia coli)木糖代谢的关键酶基因.引入到运动发酵单胞菌中,获得能利用木糖发酵生产乙醇的重组工程菌株PZM.混合糖发酵过程中,重组菌利用葡萄糖和木糖生成乙醇的效率分别达到理论值的81.2%和63.1%.     

TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建立

构建包含RAcl基因cDNA片段的质粒,作为水稻肌动蛋白基因RAcl之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAcl基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAcl基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102—1护拷贝,阈值循环数（Ct）与PCR体系中起始模板量的对数值之间有着良好的线性关系（r=1．000）,扩增效率高（E=98．2％）。建立了基因RAcl实时定量PCR的质粒标准品。     

定量RT-PCR中人干细胞因子的RNA-CRS的构建

一种适用于定量RT-PCR、用ExonucleaseⅢ部分酶切剔除技术,构建人干细胞因子(hSCF)基因的RNA竞争性参考标准(RNA-CRS)的新方法：用RT-PCR技术,从HepG2细胞中扩增出全长人hSCF cDNA,并克隆入质粒pGEM-T获重组的pGEMSCF载体,经ExonucleaseⅢ和S1核酸酶适当处理,以导致hSCF cDNA中一小片段缺失,由此获得重组pGEMSCF模拟体(mimic),经体外转录得到hSCF RNA-CRS.测序表明：该RNA-CRS与hSCF mRNA比较,缺失了从第499位至608位共110个核苷酸,但二者RT-PCR反应可用同一对扩增引物,反应动力学极为相似.这种hSCF RNA-CRS可作为一种较理想的竞争性参考标准,适用于定量RT-PCR中,以对重组hSCF在真核细胞中的表达水平进行准确的定量分析.此方法亦可推广应用于其他真核基因的表达水平及/或调控的检测和研究.     

刺五加皂苷合成关键酶基因表达的半定量RT-PCR分析

根据已克隆的刺五加鲨烯合酶(squalene synthase,SS)、鲨烯环氧酶(squalene epoxidase,SE)和β-香树酯醇合成酶(β-amyrin synthase,bAS)基因序列信息设计引物,通过半定量RT-PCR分析了SS、SE和bAS基因在刺五加不同生长发育时期和不同器官中表达量的变化.结果表明,SS、SE和bAS基因在各生长发育时期和各器官中均有表达,但表达量差异显著(P<0.05),三者均在盛花期表达量最高,之后降低,进入果实成熟期后SS和bAS的表达量迅速回升,SE无显著变化.SS和bAS在叶片和根中的表达量较高,SE表达量的最大值出现在叶片和幼茎中.刺五加SS、SE和bAS基因的表达间存在显著的正相关关系(P<0.05).研究结果为进一步分析关键酶基因对刺五加三萜皂苷生物合成的影响奠定了基础.     

Separation of membrane fractions of Zymomonas mobilis

Abstract Membranes of Zymomonas mobilis ZM4 were separated by centrifugation on discontinuous density sorbitol gradient using a 2-step purification procedure. Four bands of respective densities 1.17 (L1), 1.20 (L2), 1.22 (H1), and 1.32 (H2) were obtained. NADH oxidase activity was detected in L1 and L2 fractions, indicating that they were derived from cytoplasmic membrane. H1 and H2 fractions gave a positive Limulus polyphemus lysate test of outer membrane endotoxin. Proteins of 2 cytoplasmic membrane bands and of 2 outer membrane bands showed respectively similar patterns when separated by electrophoresis.     

一株葡萄糖和木糖共发酵高效产乙醇重组运动发酵单胞菌的性能评价

木糖是木质纤维素原料水解液中的第二大组分,木糖和葡萄糖的充分利用是有经济性地生产纤维素乙醇的关键。通过基因克隆手段构建了一株可以高效利用木糖产乙醇的重组运动发酵单胞菌Zymomonas mobilis TSH01,并进行了利用单糖溶液、混合糖溶液及玉米秸秆水解液发酵产乙醇效率的研究。结果表明,利用单一葡萄糖或单一木糖溶液发酵时,当糖浓度为8%、发酵72 h后,糖利用率分别为100%和98.9%,乙醇代谢收率分别为87.8%和78.3%;利用8%葡萄糖和8%木糖的混合溶液发酵时,72 h后,葡萄糖和木糖的利用率分别为98.5%和97.4%,乙醇代谢收率为94.9%。利用含3.2%葡萄糖和3.5%木糖的玉米秸秆水解液发酵72 h后,葡萄糖和木糖的利用率分别为100%和92.3%,乙醇代谢收率为91.5%。此外,磷酸二氢钾对发酵过程中木糖利用率以及乙醇收率的提高有明显促进作用。     

运动发酵单胞菌在生物炼制中的研究进展

以木质纤维素生物质为原料的生物炼制技术已成为全球研发的热点和难点。欧盟国家和美国的中长期生物质能源发展路线图中均将木质纤维素生物炼制技术作为重要目标,但是目前整体水平尚处于中试阶段。我国的纤维素类生物质原料非常丰富,将其转化成燃料乙醇及生物基础化学品等具有较大的潜力,但当前要想实现商业化生产,还面临着很多瓶颈问题亟待解决。缺乏能够同时高效利用纤维素类水解物的发酵菌株,已成为纤维素生物质高效与高值转化的关键制约因素。运动发酵单胞菌是目前唯一一种通过ED途径兼性厌氧发酵葡萄糖的微生物,其独特的代谢途径使其成为构建产乙醇工程菌的优选宿主之一;同时由于该菌具有较高的糖利用效率等优点,也是其他生物基化学品生产的重要候选平台微生物,如山梨醇、葡萄糖酸、丁二酸和异丁醇等。本文从该菌的研究历程、分子生物学基础、菌种改良及该菌在生物能源及生物基化学品等生物炼制体系中的应用研究角度进行了综述,并提出该菌可作为纤维素生物质生物炼制系统的新的重要平台微生物。     

Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, mu, and biomass yield, Y(X/S)) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, q(p), and ethanol yield, Y(p/s)) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.     

High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

Response of Zymomonas mobilis levansucrase activity to sodium chloride

An activation of levansucrase-catalysed levan formation by NaCl, KCl and Na2 SO4 (0.03–0.7 M) was observed using cell-free extract of Zymomonas mobilis. A sigmoidal response of the rate of levansucrase-catalysed reaction to the sucrose concentration was significantly reduced in the presence of salts the Hill coefficient 2.10 and 1.0–1.2 respectively), possibly, due to the heterotropic activation of levansucrase as an allosteric enzyme. © Rapid Science Ltd. 1998     

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD’s SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD’s SP were also higher than the other two strains containing PhoC or ZMO0331’s SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.