庆大霉素生物合成基因genY的研究

以绛红色小单孢菌G1008基因组为模板,构建genY基因缺失的同源重组质粒pFY103,经接合转移导入绛红色小单孢菌G1008和GK1101(△genK),筛选获得genY基因缺失工程菌GY105(△genY)和工程菌GKY205(△genK+genY),发酵、提取并经质谱检测分析代谢产物.结果表明,工程菌GY105和GKY205主要积累西索米星、庆大霉素C1a和C2b.证明genY基因缺失阻断了庆大霉素X2到G418的转化,说明genY基因参与庆大霉素生物合成过程中绛红糖胺C-6’位甲基化.     

提高庆大霉素产生菌F-212孢子萌发率的研究

本文研究了三十一种孢子萌发因子对庆大霉素产生菌棘孢小单孢菌F—212的孢子萌发率和芽长的影响。分别进行了单因子、双因子及四因子试验,并通过正交试验找出了促进孢子萌发的最适剂量组合,为进一步提高庆大霉素发酵水平创造了条件。     

棘孢小单孢菌F-212种子移种期的控制及其对庆大霉素生物合成的影响

本文探讨了庆大霉素生产菌棘孢小单孢茼F-212的一级和二级种子的不同生长阶段的菌丝形态及其控制方法,以作为提高庆大霉素发酵单位的工艺手段。     

庆大霉素高产菌株的抗性方法选育

以生产菌株绛红小单孢菌为出发株,应用N 离子注入及紫外线诱变技术,利用高浓度的庆大霉素梯度平板筛选,或配合含有高浓度庆大霉素的液体培养基的生态驯化,筛选到摇瓶效价分别比出发株提高75.2%和70.1%的N08和Wt63两个高产菌株,10 L发酵罐发酵产量分别达2 118 u/mL和1 843 u/mL,C组分含量符合中国药典2000版的规定.     

依纽小单胞菌2-脱氧蟹肌醇合成酶基因的克隆与表达

从伊纽小单孢菌(Micromonospora inyoensis)扩增参与紫苏霉素生物合成的2-脱氧蟹肌醇合成酶基因sisB,并分别将其克隆到筛选载体pUC18和表达载体pET-30a上,其开放阅读框长1 185 bp,编码含394个氨基酸残基(41.94 ku)的多肽链,将pET-sisB转化大肠杆菌E.coliBL21(DE3),使sisB基因实现表达.基因sisB的碱基序列与棘孢小单孢菌(M.echinospora)的基因gntB的碱基序列的同源性高达93%.预测的SisB蛋白序列与GntB蛋白序列的同源性为95.2%.基因sisB是继紫苏霉素抗性基因srm1(AY661430)和参与紫苏霉素生物合成的糖基转移酶基因sisD(DQ250992)和sisZ(DQ250994)后,从依纽小单孢菌克隆到的又一新基因.该基因在GenBank的接受号为DQ250993.     

一株主产庆大霉素C2a工程菌的构建

为更好生产单组分庆大霉素C2a,拟构建一株主产庆大霉素C2a的工程菌.通过序列比对分析,锁定genB2为构建该工程菌的关键基因.以绛红小单孢菌G1008基因组为模板,PCR扩增genB2的上下游序列为同源交换臂,构建同源重组质粒pZB303.通过接合转移,将质粒pZB303导入绛红小单孢菌G1008,安普抗性及PCR扩增筛选得到一株genB2框内缺失工程菌GB102.发酵并提取代谢产物,再经TLC,HPLC,MS分析.结果表明,工程菌GB102主要积累庆大霉素C2a.有望用于生产单组分庆大霉素C2a.     

胡萝卜抗冻蛋白基因的克隆及其在E.coli中的表达

对中国的一个胡萝卜品种进行了冷诱导前后幼苗总蛋白组成的比较、分析 ,SDS聚丙烯酰胺凝胶电泳的结果表明两者在相对分子质量 36 0 0 0附近有一条蛋白带的差别。由此推测实验的胡萝卜品种在冷诱导过程中有抗冻蛋白 (AFP)表达。根据相关的研究结果设计引物 ,以胡萝卜幼苗基因组DNA为模板 ,通过PCR扩增得到了长 10 99bp的抗冻蛋白基因。测序结果表明其与GenBank公布的afp序列仅有 3个碱基的差别。将此抗冻蛋白基因克隆进大肠杆菌表达载体pGEX4T1,在IPTG的诱导下表达出了含有AFP的约 6 0 0 0 0的融合蛋白 ,证明该AFP基因在原核表达系统中可以正常表达     

紫苏霉素抗性基因srm1的克隆及其特性研究

从伊纽小单孢菌（Micromonosporain inyoensis）扩增到包含紫苏霉素抗性基因srm1的DNA序列LY102（847bp）,并将其克隆到pUC19／18载体上．srm1基因是依靠它自身的启动子激活（ATG上游19bp序列）,在E．coli DH5α中表达,并不受lac启动子的控制．其开放性阅读框长819bp,编码含273AA的多肽链预测的Srm1蛋白序列与Gmd3（来自玫瑰小单孢菌）、GrmA（来自绛红色小单孢菌）和Sgm（来自兹昂小单孢菌）的同源性依次为92％,89％和85％．srm1,grmA,grmB和sgm4个抗性基因具有相同的核糖体结合位点GGAGG,但是它们的核糖体结合位点与翻译起始密码子之间的序列互不相同．基因srm1以自阻遏方式在翻译水平上进行调节。     

水稻苹果酸脱氢酶基因的克隆及其在E.coli中的表达

以玉米苹果酸脱氢酶的基因为探针，从水稻幼苗cDNA文库筛选得到一条水稻新基因，它与玉米ZHO2序列的一致性高达87％，被命名为RcMDH(rice cytoplasmic malic dehydrogenase gene)，分析发现RcMDH具有完整的读码框，编码332个氨基酸，预计分子质量为36．5ku,导入大肠杆菌中表达，经检测证实该产物具苹果酸脱氢酶酶活性。     

N-乙酰氨基葡萄糖对绛红小单孢菌合成庆大霉素的影响

研究了N-乙酰氨基葡萄糖对绛红小单孢菌产素的影响,实验探索了N-乙酰氨基葡萄糖添加工艺,在250mL摇瓶发酵过程中培养48h,60h分别添加0.05%,0.15%N-乙酰氨基葡萄糖可使庆大霉素生测效价达2 030U/mL,较对照1 228U/mL提高了65%以上(三次平均基本一致);5L发酵罐试产三批次发酵终结,生测效价平均为1 727U/mL(厡工艺1 097U/mL)提高了57%,效果十分明显.同时测定了发酵过程中菌体代谢各种参数的变化,证明了N-乙酰氨基葡萄糖在绛红小单孢菌生物合成中的促进作用.本方法操作简单,基本不改变原工艺,生产成本低廉,对提高庆大霉素产量效果非常明显.     

Paenibacillus sp.K1 β-半乳糖苷酶基因的克隆及在大肠杆菌中的表达

从鲜牛奶中分离到1株产β-galactosidase的细菌,经16S rDNA序列比对鉴定为类芽孢菌Paenibacillus sp. K1。提取该菌株的染色体DNA,以pUC18（lac-）为载体,构建其DNA文库;在含有X-gal的LB平板上筛选该文库,得到6个蓝色菌落;对阳性克隆中插入的DNA片段序列测定,鉴定出1个编码全长为2028bp并携带有组成型启动子的β-半乳糖苷酶基因。将该基因导入大肠杆菌BL21（DE3）中,实现了β-半乳糖苷酶高效表达,其酶活为25.06U/mL,高于原始菌株的4.55U/mL,并进一步用亲和层析将该酶进行了纯化。     

几种氨基酸对庆大霉素生物合成的影响

在绛红色小单孢菌(Micromonospora purpurea)产生庆大霉素的过程中,运用正交设计筛选庆大霉素(gentam icin)生物合成促进剂——氨基酸.经摇瓶实验证明,在原培养基中添加一定量的甘氨酸、赖氨酸、酪氨酸、蛋氨酸,在旋转摇床上220 r/min 36℃培养96 h,有利于生物合成庆大霉素,较对照产量可增加30%左右,产素率提高40%左右.     

Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.     

Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.     

薄壳山核桃原花青素合成关键酶基因的克隆与表达分析

【目的】原花青素是广泛存在于植物中一种重要的次级代谢产物,其强抗氧化性增强了植物自身的抗逆能力,同时赋予植物清除人体自由基的保健作用。研究薄壳山核桃种仁中原花青素生物合成途径,对改良薄壳山核桃的种质与品质均具有重要意义。【方法】以薄壳山核桃‘波尼’115和135 d种仁混合样品为材料,通过RT-PCR扩增、克隆和测序后,获得了薄壳山核桃原花青素合成关键酶相关基因CiDFR、CiLAR和CiANR的基因序列,并进行了生物信息学分析和表达水平分析。【结果】CiDFR基因长1 148 bp,包含1 020 bp的开放阅读框(ORF),编码339个氨基酸;CiLAR基因长1 390 bp,包含1 050 bp的ORF,编码349个氨基酸;CiANR基因长度为1 104 bp,包含1 014 bp ORF,编码337个氨基酸。荧光定量PCR检测结果显示,CiDFR和CiANR基因在种仁发育中期(95～105 d)表达量较高,之后快速下降至较低值;CiLAR基因在95 d表达量较高,之后快速降低,在155 d样品中表达量又升至最高点。【结论】CiDFR和CiANR基因表达量与酚类代谢物含量变化相关...     

小鼠酪氨酸酶的克隆及在大肠杆菌中的表达

采用逆转录-聚合酶链式反应(RT -PCR)方法,从小鼠黑色素瘤总RNA中扩增得到小鼠酪氨酸酶的cDNA .该基因重组到T7启动子控制下的表达载体pET - 2 2b(+)中,构建表达质粒pET -TYR并转化大肠杆菌Rosetta (DE3) .SDS -PAGE及蛋白质序列测定表明,经0 .8mmol/L异丙基硫代- β-D -半乳糖苷(IPTG)诱导后,可高效表达重组的小鼠酪氨酸酶,表达量约占菌体总蛋白质的2 5 % ,表达产物为包涵体     

Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

A new MAPK gene, ZmSIMK1 （Zea mays L. salt-induced mitogen-activated protein kinase 1）, is isolatod from a maize eDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of eneoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 eDNA into pET-42a（＋）, and transformed into E. coli strain BL21（DE3）. A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1 mmol/L IPTG.