基因枪法获得GAI转基因橡胶植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

基因枪法将GAI基因导入巴西橡胶的研究

为了探讨利用基因工程技术进行橡胶树品质改良的可行性,用基因枪轰击巴西橡胶(Hevea brasiliensis)愈伤组织,将GAI矮化基因导入橡胶受体中,通过50mg L-1卡那霉素筛选鉴定,优化了基因枪法转化橡胶的各种参数。结果表明,DNA金弹与靶细胞的距离为9cm,每皿轰击一次时,胚状体诱导率可以达到1.87%。经过GUS组织染色和PCR扩增鉴定,初步确定GAI基因已经整合到橡胶基因组中。     

利用Red重组系统敲除大肠杆菌 O157:H7的waaL 基因

目的:利用λ噬菌体Red重组系统敲除大肠杆菌O157:H7的waaL基因。方法:以pKD4为模板扩增出与waaL基因上下游同源的、含有卡那霉素抗性基因的PCR产物。然后电击转化到大肠杆菌 O157:H7 中,利用Red重组系统,通过卡那霉素抗性基因两侧的waaL基因序列在体内与waaL基因发生同源重组,置换了 O157:H7 基因组中的waaL基因。并进一步利用卡那霉素抗性基因两侧的FRT位点,通过FLP位点专一性重组将卡那霉素抗性基因敲除。结果:成功构建了敲除waaL基因且不带卡那霉素抗性基因的菌株。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法*

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌DH5α,用 5′端与组氨酸基因同源 ,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物 ,然后电击转化DH5α,在λRed重组系统的帮助下 ,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组 ,置换了DH5α组氨酸操纵元中的hisDCB基因 ,最后利用卡那霉素抗性基因两端的FRT位点 ,通过FTP位点专一性重组将卡那霉素抗性基因去除 ,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大     

体内遗传标记成团肠杆菌固氮质粒pEA9

用卡那霉素抗性（Kan^r)基因对成团肠杆菌固氮质粒pEA9进行活体遗传标记。将来自质粒pEA9的3.0kb片段(nif ENX)克隆到pBR322载体中,再将卡那霉素抗性(Kan^r)基因插入到3.0kb的片段中,构建成供体质粒pST5。将该质粒转化到含有待标记质粒pEA9的E.a.339菌株中,然后在AP培养基中消除供体质粒,筛选得到40个失去了pST5并保持卡那霉素抗性的克隆,分析表明它们不是质粒pEA9和pST5的共整合体,而是卡那霉素抗性基因通过两个质粒在nifENX区域内的DNA间的同源重组整合到了质粒pEA9上。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

几种白菜类蔬菜卡那霉素抗性的研究

标记基因的利用是筛选和鉴定基因转化的细胞、组织和转基因植株的有效方法。针对目前遗传转化中常用的卡那霉素(Km)抗性基因,对几种白菜类蔬菜的卡那霉素抗性作了较系统的探索,发现5.0 mg/L Km可完全抑制大白菜、小白菜及菜心子叶的再生;幼苗Km抗性测验表明各品种均表现出随着Km浓度的提高,子叶黄化率升高、根茎变短、鲜重减轻的趋势。白菜类蔬菜的卡那霉素抗性检测为遗传转化和阳性后代筛选奠定了基础。     

利用Red重组系统敲除鲍曼不动杆菌asa A基因

目的利用Red重组系统敲除鲍曼不动杆菌ATCC 17978的asaA。方法设计上下游引物中包含asaA的同源序列,并以pKD4质粒为模板,扩增含有卡那霉素抗性基因的DNA片段。将该片段转化于表达重组酶的17978感受态细胞中,在卡那霉素筛选压力下,得到经两次同源双交换的具有卡那霉素基因标记的突变菌株。随后,在重组酶的作用下将抗性基因去除,最终得到无抗性基因标记的突变菌株ΔasaA。结果通过该重组系统,首先将卡那霉素抗性基因的DNA片段替换了基因组中asaA的DNA片段,然后将卡那霉素抗性基因的DNA片段消除,最终获得了asaA缺失的突变体。结论通过Red重组系统为鲍曼不动杆菌中其他基因的缺失突变提供方法与思路。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率,将一个植物的内含子插入卡那霉素抗性基因编码区的N端。合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达载体pYP1202并转化烟草,受外植体在含卡那霉素50－200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比较仅有26.7％,在200mg/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Engineering of Polyploid Saccharomyces cerevisiae for Secretion of Large Amounts of Fungal Glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the δ-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. δ-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Inactivation of kanamycin A by phosphorylation in pathogenic Nocardia.

Among the five species of pathogenic Nocardia, i.e., N. asteroides, N. brasiliensis, N. farcinica, N. nova and N. otitidiscaviarum, all strains of N. brasiliensis and N. farcinica showed resistance to an aminoglycoside antibiotic, kanamycin A, showing the MIC (minimum inhibitory concentration) values of more than 100 micrograms/ml. This species-specific difference in sensitivity was found to be explained by the production of an inactivation enzyme, aminoglycoside 3'-phosphotransferase APH(3'). Structural studies by mass and NMR spectroscopy on the inactivated substance produced by a cell-free extract of the Nocardia confirmed the conversion of kanamycin A to an inactive substance, kanamycin A 3'-phosphate. The MIC values of N. otitidiscaviarum and N. nova for kanamycin A, on the other hand, ranged from 0.78 micrograms/ml to 100 micrograms/ml, and both species were non-producers of APH(3'). Sensitivity to the antibiotic and APH(3') productivity of N. asteroides varied depending on the strain.     

Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast.

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O-glycosylation. Secretion of GAI expressed in the pmt1-disruptant and in the kre2-disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O-linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O-linked oligosaccharides of GAI from the pmt1-disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2-Man4 were reduced in proportion to the reduction of Man1. The O-linked oligosaccharides of GAI from the kre2-disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O-linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.     

Efficient plastid transformation in tobacco using the<Emphasis Type="Italic"> aphA-6</Emphasis> gene and kanamycin selection

Here we report on the development of a new dominant selection marker for plastid transformation in higher plants using the aminoglycoside phosphotransferase gene aphA-6 from Acinetobacter baumannii. Vectors containing chimeric aphA-6 gene constructs were introduced into the tobacco chloroplast using particle bombardment of alginate-embedded protoplast-derived micro colonies or polyethylene glycol (PEG)-mediated DNA uptake. Targeted insertion into the plastome was achieved via homologous recombination, and plastid transformants were recovered on the basis of their resistance to kanamycin. Variations in kanamycin resistance in transplastomic lines were observed depending on the 5' and 3' regulatory elements associated with the aphA-6 coding region. Transplastomic plants were fertile and showed maternal inheritance of the transplastome in the progeny.     

Transformation of the tropane alkaloid-producing medicinal plant Hyoscyamus muticus by particle bombardment

We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants.     

Expression of GAI gene and disruption of PEP4 gene in an industrial brewer's yeast strain

Aims:  Construction of an industrial brewer's yeast strain, which could improve foam stability and reduce calorific values of beer.
Methods and Results:  An industrial brewer's yeast strain (Ts-10) was constructed by integrating glucoamylase encoding gene GAI amplified from Saccharomycopsis fibuligera by PCR into the locus of proteinase A (PrA) gene ( PEP4 ). The resulting recombinant strain identified by PCR could grow on YNB minimal medium plate with starch as sole carbon source. Its highest GAI activity was 91·69 U ml⁻¹, but it had no PrA activity. The real extract was reduced by 21·07% and the main residual maltotriose content was reduced by 14% in wort fermented with the recombinants strain. Its foam retention in beer was higher 39 s and the contents of potential off-flavour compounds, such as diacetyl, pentanedione and acetaldehyde were lowered by 16%, 13% and 14%, respectively, as compared with the industrial brewer's yeast YSF-5.
Conclusions:  An industrial brewer's yeast strain was constructed by introducing GAI gene and disrupting PEP4 gene.
Significance and Impact of the Study:  The recombinant strain (Ts-10) had better foam performance and mouthfeel in addition to low-calories values. It was free of heterologous DNA sequences and drug-resistance genes and could be safely used in beer production.