里氏木霉bgl1基因的克隆及其在酿酒酵母中的表达

采用cross—over PCR方法将里氏木霉β-葡萄糖苷酶基因的外显子进行体外拼接,成功获得了剔除两个内含子的β-葡萄糖苷酶基因bgl1;进一步构建了重组质粒pYX-tbgl,并在酿酒酵母Saccharomyces cerevisiae W303—1A中获得表达,得到的转化子能以纤维二糖为唯一碳源生长。     

发酵法生产聚羟基脂肪酸脂（PHA）的研究进展

本文简要综述了国内外发酵法生产聚羟基脂肪酸酯（PHA）的菌株、发酵条件、提取和鉴定方法,以供参考。     

糖化酵母糖化酶基因在酿酒酵母中的克隆与表达

糖化酵母结构基因ＳＴＡ编码分泌到胞外的葡萄糖淀粉酶（α－１，４－葡聚糖－葡糖水解酶，ＥＣ３．２．１．３），俗称糖化酶。通过限制性内切酶Ｓａｕ３Ａ部分酶切糖化酵母染色体，以穿梭质粒ＹＥＰ１３作载体，建立糖化酵母的基因文库，转化ｓｔａ°ｉｎｈ°型酿酒酵母受体菌Ｃ＿２，筛选出三株ｓｔａ￣＋转化子，检测出５．３ｋｂ大小的外源基因片段，继而采用薄层层析、ＫＮＲ染料测酶活性等方法对外源基因产物进行了鉴定并测定了酶活最佳反应条件。     

发酵法生产聚羟基脂肪酸酯(PHA)的研究进展

本文简要综述了国内外发酵法生产聚羟基脂肪酸酯(PHA)的菌株、发酵条件、提取和鉴定方法,以供参考。     

人β-防御素3基因的克隆及其在酿酒酵母中的表达

以质粒pPIC9K．hBD3为模板,扩增得到一段上游带信号肽因子（a-factor）的人3-防御素-3（hBD-3）基因,克隆至酿酒酵母穿梭质粒pYES2中,构建了酿酒酵母表达载体pYES2．a-factor-hBD3（pYa-hBD3）。将重组质粒转化至SaccharomycescerevisiaeINVSCl中,鉴定阳性重组子,经2％半乳糖诱导表达。实验发现表达所得hBD-3对金黄色葡萄球菌（ATCC6538）和大肠杆菌（ATCC10231）具有明显的抑菌活性。hBD-3基因片段在酿酒酵母中的表达,为进一步探讨hBD-3的生物活性及防御素应用安全性打下基础。     

FAS2基因过表达对酿酒酵母风味酯生成能力的影响

研究了共表达脂肪酸合成酶复合体中α亚基的编码基因FAS2和β亚基的编码基因FAS1对酿酒酵母产风味酯的影响。前期实验室构建的过表达FAS1基因的菌株α5F-FAS1和α5O-FAS1可以提高酿酒酵母产中链脂肪酸乙酯的含量,本研究以这两株菌为出发菌株过表达FAS2基因,相比较α5F-FAS1菌株,己酸乙酯、辛酸乙酯、癸酸乙酯分别提升了154.31%,65.22%和23.53%;相比较α5O-FAS1菌株,己酸乙酯、辛酸乙酯、癸酸乙酯、十二酸乙酯分别提升了17.34%,30.71%,27.12%和28.13%。结果显示共表达FAS1和FAS2基因可以更进一步的提升酿酒酵母中链脂肪酸乙酯的产量。同时,本研究还发现,过表达FAS1可以提高乙酸乙酯的生成量。为了探究本现象产生的初步机理,通过RT-PCR测定了合成与利用乙酰辅酶A相关基因的表达量,实验结果表明,过表达FAS1导致的乙酸乙酯生成量上升的现象主要是由于过表达FAS1使得ATF1的表达量上升造成的。     

DNA聚合酶基因pfu的克隆及初步表达

通过分子克隆技术从嗜热极端微生物Pyrococcus furiosus中克隆出高热稳定性的pfuDNA聚合酶基因,琼脂糖凝胶电泳显示基因片段的长度为2.3kb,将其转化入大肠杆菌中并获得初步表达.重组酶成功地扩增了800bp的DNA片段.     

假单胞菌中聚羟基脂肪酸酯合成酶基因的克隆与分析

大多数二型聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHA)合成酶(PhaC)发现于假单胞菌中,其基因组成形式为两个PhaC基因中间有一个PHA降解酶PhaZ基因.根据已知的假单胞菌PHA合成酶基因区域的特殊结构,设计了采用PCR方法从假单胞菌中克隆PHA合成酶基因的克隆方案,并成功地对一株硝基还原假单胞菌(Pseudomonasnitroreducens)和一株石竹伯克霍尔德氏菌(Burkholderiacaryophylli)中的PHA合成酶基因进行了克隆.将克隆得到的phaC1,phaZ和phaC23个基因所编码的氨基酸序列与其他6株已知PHA合成酶基因的假单胞菌的相应序列进行比较,发现这3个蛋白质在假单胞菌中的相似性很高,由phaC1,phaZ和phaC23个基因所组成的基因区域在假单胞菌中非常保守.从对PhaC1,PhaZ和PhaC2的系统进化树分析可以发现,这3个蛋白质与整个PHA合成酶基因区域的系统进化树有着一致的结构.这表明假单胞菌中的PHA合成酶基因区域可能起源于共同的祖先,而其形成可能经历了一次PHA合成酶基因加倍的过程.     

纳豆激酶基因在酿酒酵母中的表达

目的:从纳豆芽孢杆菌基因组DNA中扩增纳豆激酶基因,以实现该基因在酿酒酵母中的表达.方法:通过PCR方法扩增纳豆激酶基因,利用DNA重组技术构建重组质粒pYES2-NK,转化酿酒酵母H158感受态细胞;经β-半乳糖诱导表达后,用纤维蛋白平板法检测纤溶酶活性.结果:克隆到大小为1192bp的纳豆激酶基因,编码397个氨基酸;活性检测表明酿酒酵母发酵液的上清液具有纤溶酶活性.结论:纳豆激酶基因在重组酿酒酵母中实现了分泌表达.     

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae

We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14–1 from chromosome XIV of S. cerevisiae.     

pMPY-ZAP: A reusable polymerase chain reaction-directed gene disruption cassette for Saccharomyces cerevisiae

Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae. We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S. cerevisiae without previously cloning them. In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker. An indefinite number of disruptions can therefore be made in the same strain.     

Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.     

Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.     

Recovery of gene function by gene duplication in Saccharomyces cerevisiae

A prototroph revertant (Rev9) selected from an ATCase^? mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild-type gene. Sequence analysis of the 5′ end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.     

α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的策略

综述了宿主细胞的表达特点，外源基因的来源，表达载体和转化方法对α-乙酰乳酸脱羧酶基因在酿酒酵母中稳定表达的影响，并在此基础上提出了相应的策略。     

瑞氏木霉内切葡聚糖酶基因在酿酒酵母中的表达研究

将PCR合成的瑞氏木霉 (Trichodermareesei) β 内切葡聚糖酶I(EGI)cDNA基因片断分别插入酵母met1 0和 pgk1启动子和终止子序列之间 ,构建了在不同启动子控制下 ,具有不同拷贝数的eg1表达分泌质粒pRS41 5ME、pRS41 5PE和 pRS42 5PE。通过电转化使重组质粒转移至实验室酿酒酵母H1 5 8菌株中 ,分别得到了 3株酵母转化子H1 p、H2p和H1m。在 3株酵母转化子中 ,重组 β 内切葡聚糖酶I都能在酶自身信号肽序列引导下进行分泌型表达。在YPD培养基中 3株重组酵母生长速率大致相同 ,H1m ,H1 p与H2 p的内切葡聚糖酶活力分别为 70 .4,1 2 6.7和 1 2 5 .0U/mL。     

Mapping of gene controlling thiamine transport in Saccharomyces cerevisiae

A recessive mutation leading to complete loss of thiamine uptake in Saccharomyces cerevisiae was mapped on the left arm of chromosome VII, approximately 56cM centromere-distal to trp5. As the analysed locus is relatively distant from its centromere and from the markers used, its attachment to chromosome VII was confirmed by chromosome loss methods.