GAP1基因缺失对上面发酵酵母高级醇代谢能力的影响

以亲本上面发酵酵母工业菌株S17为对照,研究了GAP1一个等位基因敲除重组菌株S17G和GAP1两个等位基因敲除重组菌株S17G2的发酵性能.实验结果表明:与亲本菌株相比,重组菌株S17G的总高级醇生成量降低17.47%,α–氨基氮利用能力降低13.67%,麦芽糖利用能力降低31.08%;重组菌株S17G2的总高级醇生成量得到进一步降低,达到210.58 mg/L,较亲本菌株降低了21.98%,α–氨基氮利用能力降低了18.47%,麦芽糖利用能力降低了35.14%.重组菌株S17G、S17G2的其他发酵性能与亲本菌株S17相比没有发生显著的变化.     

麦芽糖酶基因MAL62的不同截断变体对面包酵母发酵性能的影响

为了探究麦芽糖酶基因MAL62的不同截断变体对面包酵母发酵性能的影响,以面包酵母工业菌株BY-14基因组为模板,通过PCR获得编码麦芽糖酶的3种不同长度氨基酸的DNA序列,研究改造后菌株的发酵力、麦芽糖消耗速率及麦芽糖酶活力的变化,从而揭示编码麦芽糖酶的氨基酸与其功能之间的关系。结果表明:含有编码麦芽糖酶不同长度基因的面包酵母菌株的发酵力、麦芽糖消耗速率及麦芽糖酶活力均优于含有未改造的麦芽糖酶的重组菌株,说明敲除催化区域外的氨基酸不会影响面包酵母中麦芽糖酶的主要性能和面包酵母菌株的无糖面团发酵能力。     

敲除iclR基因对大肠杆菌发酵L–色氨酸的影响

为了研究敲除icl R基因对大肠杆菌(Escherichia coli)发酵L–色氨酸的影响,以L–色氨酸工程菌大肠杆菌TRTH为出发菌株,利用Red重组技术构建了icl R基因(编码乙醛酸操纵子阻遏蛋白)缺失菌株TRTHΔicl R.摇瓶发酵实验结果显示:TRTH icl R的L–色氨酸产量和糖酸转化率分别达到(6.52±0.46)g/L和13.17%,,比原菌的分别提高了21.86%,和22.85%,;乙酸累积量为6.82,g/L,比原菌的降低了37.63%.30,L发酵罐发酵实验结果显示:TRTH icl R的L–色氨酸产量及糖酸转化率分别达到(13.01±1.05)g/L和6.51%,,比原菌的下降了60.34%,和68.27%,;乙酸累积量为18.21,g/L,比原菌的增加了33.42%.结果表明:在摇瓶条件下,重组菌株生物量较出发菌株高,代谢流分配适合L–色氨酸积累;但在发酵罐条件下,乙醛酸循环的增强导致重组菌株供能不足和乙酸的过多积累,最终使得生物量不足以及L–色氨酸产量下降.     

脱氮假单胞菌发酵生产维生素B12高产菌株的选育

以维生素B12生产菌株脱氮假单胞菌05-04为出发菌株,采用紫外线和5-澳尿嘧啶复合诱变处理,选育到一株抗5-澳尿嘧啶突变株5-BU-3-5,该菌株经摇瓶发酵,发酵单位较出发菌株提高了18.0%.该菌株特性优良,经57m3发酵罐试验,平均发酵单位提高8.9%,是一株性状较稳定可深入开发研究的优良菌株.     

基于关键酶表达的发酵调控与分子改造策略对E.coli产丁二酸的影响

考察E.coli JM001及其重组菌株E.coli JM002生物发酵生产丁二酸的性能。E.coli JM001在两阶段发酵产丁二酸过程中通过在有氧培养阶段添加乙酸钠,即可提高丁二酸的生产能力,厌氧阶段的丁二酸收率可达84%,但会有较多的副产物乙酸和丙酮酸积累。以E.coli JM001为出发菌株,敲除其磷酸烯醇式丙酮酸羧化酶(PPC)并导入来源于枯草芽孢杆菌的磷酸烯醇式丙酮酸羧化激酶(PCK)基因,构建了重组菌株E.coli JM002,该重组菌株在两阶段发酵的有氧培养过程中不需添加乙酸钠,转厌氧后菌株也具有转化葡萄糖合成丁二酸的能力,丁二酸收率可达86%,副产物积累很少。     

利用原生质体融合技术筛选红霉素优良菌株

以红霉素高产菌HE-08-6及低产优组分菌HA-08-20为出发菌株,其原生质体经紫外诱变后在适宜条件下进行融合,再生培养后挑取融合子89株,经筛选得到3株具有二亲本优良性状的高产菌株HEA-08-01,HEA-08-02,HEA-08-03.其化学效价较出发菌株HA-08-20提高约25%,产品的红霉素A组分含量较出发菌株HE-08-6提高约26%.传代培养证明该菌株具有一定的遗传稳定性,在50L-Fus智能发酵罐上连续3批发酵结果表明,该优良菌种在提高红霉素产品质量及效价上取得较大进展,具有推广应用价值.     

利用原生质体融合技术选育高产菌株

以龟裂链霉菌(Sterpomyces rimosus)98#和76#为出发菌株,分别用溶菌酶制得2个亲本的原生质体,其中76#采用15 W紫外灯下30 cm照射25 min,100 %灭活,用PEG诱导进行了原生质体的融合.通过比较菌落外观、颜色挑选融合子328株,结合前期代谢情况进一步筛选高单位的融合菌株8株,经反复传代考察融合子的稳定性后得到1株新菌株102#,其发酵单位比亲本提高了13.0%.     

人Cu，Zn-SOD在重组毕赤酵母中表达的发酵工艺研究

以表达人Cu,Zn-SOD的重组毕赤酵母为出发菌株,通过摇瓶实验研究发酵初始pH值、诱导剂浓度、诱导温度和培养基组成等因素对目的蛋白表达水平的影响.实验结果表明,培养基组分对目的蛋白表达水平的影响最大.与YPG培养基相比,菌株在FM22培养基(含0.15%组氨酸及PTM4)中目的蛋白表达量提高5.5倍.在优化摇瓶发酵条件下(诱导剂浓度1%,诱导温度27℃,FM22培养基(含0.15%组氨酸及PTM4),初始pH值为6.0),发酵液上清酶活水平为895 U/mL,较初始提高8.5倍;以摇瓶实验结果指导5 L罐发酵,得到13 539 U/mL酶活,为摇瓶试验的15倍.     

过表达hom基因对谷氨酸棒杆菌发酵L-异亮氨酸的影响

谷氨酸棒杆菌(Corynebacterium glutamicum)中hom基因编码的高丝氨酸脱水酶为L-异亮氨酸合成过程的关键酶,本研究通过在L-异亮氨酸生产菌C.glutamicum YILW中过表达高丝氨酸脱水酶,考察过表达高丝氨酸脱水酶对发酵L-异亮氨酸产量的影响.以L-异亮氨酸生产菌C.glutamicum YILW基因组为模板克隆hom基因,将hom基因与表达载体pXMJ19连接构建出重组质粒pXMJ19-hom,再转入C.glutamicum YILW构建C.glutamicumYILW(pXMJ19-hom).通过5,L罐发酵研究重组质粒对工程菌的生长、耗糖、L-异亮氨酸产量及副产物积累等方面的影响.结果显示,重组酶的表达使菌株对L-苏氨酸的抗反馈抑制作用得到加强.最终L-异亮氨酸和L-蛋氨酸积累量分别为36.5,g/L和2.8,g/L,分别较出发菌株提高7%和33%,同时L-赖氨酸合成量仅为2.1,g/L,较出发菌株降低了63.8%.     

成孢延迟蛋白基因对枯草芽孢杆菌生物量和发酵酶活力的影响

探讨同种相噬对枯草芽孢杆菌内源酶发酵活力的影响.应用同源重组敲除的方法,构建了枯草芽孢杆菌168菌株的编码成孢延迟蛋白毒素前体的基因sdp C的knock-in突变株,比较了突变株和出发菌株的生物量、芽孢数和α-淀粉酶发酵活力.与出发菌株相比,突变株的生物量、芽孢数和α-淀粉酶发酵活力均显著提高,产酶高峰期酶活力提高23%.本研究结果显示,sdp基因突变不仅能够提高枯草芽孢杆菌的生物量,还能够提高其内源酶的发酵活力.     

产苯丙氨酸解氨酶重组大肠杆菌的培养

L-苯丙氨酸是人体8种必需氨基酸之一。酶法转化是目前生产L-苯丙氨酸的主要方法。为了提高苯丙氨酸解氨酶的产量,对可高效表达苯丙氨酸解氨酶基因的重组大肠杆菌JM105进行培养,对发酵过程中pH值对工程菌生长的影响进行了研究。结果表明,摇瓶培养条件下,控制发酵过程中发酵液的pH值可显著提高菌体产量,当控制pH值为7.0左右时,菌体量比对照高87%。在初始pH值为7.5的情况下,培养10h后将培养液pH值分别调整为5.5、6.0、6.5、7.0、7.5和8.0,则以pH值为7.5时的菌体量最高。10L发酵罐中控制发酵液pH值为7.5,菌体密度可达A600=20mol/L（DCW=11g/L）,比不控制pH值高出33%。试验结果为进一步开展该工程菌的高密度培养提供了一定的参考。     

羊种布鲁氏菌M5—90 omp31蛋白原核表达

克隆、测序布鲁氏菌疫苗株M5—90外膜蛋白基因OMP31,原核表达OMP31并对其检测。从羊种布鲁氏菌M5-90中PCR获得OMP31基因,连接到pBS—T克隆质粒并测序;将测序正确的基因片段克隆人大肠杆菌表达载体pET-28a,进行SDS—PAGE,而后Western—blot检测。结果M5-90疫苗株的OMP3I基因序列与羊种布鲁氏菌参考株16M的同源性为99．03％;外膜蛋白基因OMP31在大肠杆菌表达后能够被Western—blot检测到。结论：表达的布鲁氏菌疫苗株M5-90外膜蛋白OMP31可以被布鲁氏菌特异性血清识别,表现出良好的抗原性,为以后实验室诊断和疫苗的研究做好坚实的上游工作。     

活体烟草BY-2悬浮细胞微丝骨架的标记

微丝骨架是细胞骨架的重要成员,在细胞的多项生理活动中发挥着重要作用.本论文利用荧光标记鬼笔环肽技术和GFP融合蛋白技术,对活体烟草BY-2悬浮细胞中微丝骨架的标记方法进行了探索,结果表明,10nmol/L的Alexa488-Phalloidin为细胞微丝骨架标记的最佳浓度,利用PCR等技术构建pPZP-NtFABD2-GFP植物表达载体后,转化烟草BY-2悬浮细胞,激光共聚焦扫描显微镜观察发现,NtFABD2-GFP融合蛋白能够清晰地显示活体烟草悬浮细胞内的微丝骨架.这些结果为进一步深入研究微丝骨架在活体植物细胞中的功能奠定了基础.     

人CDK4基因的原核表达及重组蛋白的纯化和复性

将人CDK4基因克隆入原核表达载体pET28a（+）中, 经 酶切和测序鉴定正确的重组质粒pET28a-CDK4, 转化E.coliBL21(DE3)后获得表达菌株. 该表达菌株经IPTG诱导后, 高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白, 表达量占菌体总蛋白的52.6%, 包涵体经过洗涤、 尿素变性溶解、 His Trap HP Kit柱纯化、 稀释复性, 获得纯度达98%以上的蛋白. SDS-PAGE及Western blot分析表明, 在分子量34 000处有一特异性蛋白条带. 结果表明, 已成功的表达和纯化纯度达98%的重组人CDK4蛋白.     

Isolation, characterization and functional analysis of a cdc48 homologue from tobacco BY-2 cells

The fission yeast Schizosaccharomyces pombe was used to identify genes from tobacco BY-2 cells that may play roles in cell cycle regulation. A cDNA encoding a protein homologous to the yeast CDC48 was isolated and the gene was designated as NtCDC48 . The cDNA contains an open reading frame coding for a predicted protein of 808 amino acids which comprises of two typical ATPase modules (aa 245-374 and aa 518-646) . Overexpression of NtCDC48 in tobacco BY-2 cells led to an increase in the mitotic index as well as to the formation of diffused mitotic spindles. NtCDC48-GFP fusion proteins are distributed ubiquitously through Gl to M phases, yet their subcellular localization varied regularly along with the cell cycle progression. These results indicate that NtCDC48 may play an important role in the regulation of cell cycle in BY-2 cells.     

Isolation and identification of a novel coronavirus from wild bird

A novel coronavirus strain was isolated from laryngotracheal swab of wild partridge and designated as partridge/GD/S14/2003 (S14). Its whole genomic sequence was obtained (GenBank Accession number: AY646283) through RT-PCR amplification, cloning, nucleotide sequencing, and analysis by the DNASTAR program. To investigate the origin of the virus, we further analyzed the nucleotide sequences of the main structural proteins, and compared those with other available virus isolates. Our results showed that the highest nucleotide homologies between the S1 gene of S14 strain and those of nephrogenic-type strains JX1-99 and TJ2-96 were 94.6% and 93.4%, respectively. In addition, a relatively high genetic identity, 85% and 84.3%, respectively, was detected between S1 gene of S14 and those of strains QXIBV and LX4. The results suggested that the S14 strain may be originated from or related to nephrogenic-type and proventriculus-type infectious bronchitis virus (IBV). The highest nucleotide homology between the S2 gene of S14 strain and those of QXIBV and LX4 was 85% and 84.3%, respectively and all of them belonged to group II coronaviruses. The highest nucleotide homology between the M gene of S14 strain and those of strains SAIB20 and GD6-98 was 90.6% and 90.2%, respectively by which S14 belonged to group III. Although they displayed high level of genetic identity in S1 and S2 gene, there was lower homology of M coding sequences between S14 and BJ, and between S14 and QXIBV strains. Phylogenetic analysis of N gene indicated that group I strains might evolve from RNA recombination between strain H52 and Gray; while group II strains from strain H120 and D1466. S14 strain had the highest N gene homology with strain QXIBV which was 95.7%, thus classified as a group III member. Strains SAIB20 and GD6-98 which were closely related to the M gene of S14 strain belonged to group I and IV, respectively. A possible role of partridge S14 strain may play in the process of coronavirus evolution is discussed.     

Isolation, characterization and functional analysis of a cdc48 homologue from tobacco BY-2 cells

The fission yeast Schizosaccharomyces pombe was used to identify genes from tobacco BY-2 cells that may play roles in cell cycle regulation. A cDNA encoding a protein homologous to the yeast CDC48 was isolated and the gene was designated as NtCDC48. The cDNA contains an open reading frame coding for a predicted protein of 808 amino acids which comprises of two typical ATPase modules (aa 245?374 and aa 518?646). Overexpression of NtCDC48 in tobacco BY-2 cells led to an increase in the mitotic index as well as to the formation of diffused mitotic spindles. NtCDC48-GFP fusion proteins are distributed ubiquitously through G1 to M phases, yet their subcellular localization varied regularly along with the cell cycle progression. These results indicate that NtCDC48 may play an important role in the regulation of cell cycle in BY-2 cells.     

昆明地区1株HAM野毒株的基因分型

免疫捕获粪便悬液中的HAV，RT－PCR扩增目的的片段，将其连接到载体pUC18中，测序并对序列进行分析和比较，HAV野毒株W在核苷酸3024－3191与HM175/wt序列相同，表明W株为ⅠB亚型；该株5'NTR的核苷酸29－258与其它国际毒株有很大的不同，有碱基的、缺失和插入，其中插入的15个碱基是其它毒株所没有的。     

人细胞周期蛋白D1在大肠杆菌BL21中的表达及纯化

将人细胞周期蛋白D1全长cDNA克隆入原核表达载体pET-28c(+)中, 经酶切和测序鉴定正确的重组质粒转化E.coli BL21(DE3)后获得表达菌株. 该菌株经IPTG诱导高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白, 表达量占菌体总蛋白的23%. 包涵体经洗涤和溶解， 在变性条件下利用Ni²⁺螯合柱纯化、 尿素梯度复性后， 得到纯度达98%以上的纯化蛋白. SDS-PAGE显示纯化蛋白的分子量约为43 ０００， Western-b lot分析表明， 在相应分子量处有一特异性条带， 说明成功表达和纯化重组人细胞周期蛋白D1.     

利用5-氨基乙酰丙酸脱水酶缺失的重组大肠杆菌合成5-氨基乙酰丙酸

生物法合成5-氨基乙酰丙酸(5-ALA)大多通过添加5-ALA脱水酶(ALAD)的抑制剂乙酰丙酸(LA)减少5-ALA的降解,造成生产成本增高,发酵工艺复杂.本文利用ALAD缺失的大肠杆菌ZSEc2作为出发菌株,通过紫外诱变的方法,获得可利用外源血红素恢复正常生长的大肠杆菌突变株ZGEc1,并过表达来自沼泽红假单胞菌的5-ALA合成酶(ALAS)基因,最终建立一条不需要添加ALAD抑制剂的5-ALA的生物合成新路线.经过培养基初步优化,重组菌可在胞外积累约1,g/L的5-ALA.