抗冷冻蛋白基因遗传转化草菇的研究

采用RT PCR技术从瑞典的北极云杉卷叶蛾幼虫中扩增出抗冷冻蛋白基因 ,利用基因枪法遗传转化草菇。PCR检测和Southern杂交结果证明 ,抗冷冻蛋白基因已整合进草菇基因组。低温胁迫试验结果表明 ,转基因草菇具有较强的耐低温能力。转基因草菇生物学特性观测结果显示 ,大多数草菇转化子的生长速率明显地慢于对照的宿主菌株 ,多数转化子的菌丝也明显地比宿主菌丝细弱。转化子筛选结果表明 ,采用三轮的转化子筛选程序 ,即第一轮在固体培养基上筛选、第二轮和第三轮在液体培养基中筛选 ,有利于获得真实转化子和淘汰假转化子。转基因草菇一代低温胁迫结果证明 ,转基因草菇后代仍然具有较强的低温耐受能力 ,这说明转基因草菇的耐低温性能在世代之间是稳定的     

草菇蛋白酶的分离、纯化及等电点的测定

草菇是我国食用菌主要栽培品种之一,属典型高温真菌。低温将诱导草菇细胞内的蛋白质降解,导致草菇菌丝自溶、死亡。蛋白酶在草菇低温自溶过程中起了重要作用。在分离、纯化草菇蛋白酶的基础上,采用等电点聚焦电泳测定了蛋白酶的等电点,为草菇低温自溶与蛋白酶之间的关系的研究奠定了基础。     

草菇蛋白酶的分离、纯化及等电点的测定

草菇是我国食用菌主要栽培品种之一,属典型高温真菌。低温将诱导草菇细胞内的蛋白质降解,导致草菇菌丝自溶、死亡。蛋白酶在草菇低温自溶过程中起了重要作用。在分离、纯化草菇蛋白酶的基础上,采用等电点聚焦电泳测定了蛋白酶的等电点,为草菇低温自溶与蛋白酶之间的关系的研究奠定了基础。     

草菇蛋白酶的分离、纯化及等电点的测定*

草菇是我国食用菌主要栽培品种之一,属典型高温真菌。低温将诱导草菇细胞内的蛋白质降解,导致草菇菌丝自溶、死亡。蛋白酶在草菇低温自溶过程中起了重要作用。在分离、纯化草菇蛋白酶的基础上,采用等电点聚焦电泳测定了蛋白酶的等电点,为草菇低温自溶与蛋白酶之间的关系的研究奠定了基础。     

草菇低温诱导蛋白研究初探

对低温处理的草菇Volvariella volvacea菌丝体的可溶性蛋白进行分析,发现草菇菌丝在低温协迫中有新的可溶性蛋白产生,应用电泳技术分离纯化了草菇菌丝体中的一个低温诱导蛋白,经等电聚焦分析,该蛋白质的等电点6.79,SDS-聚丙烯酰胺凝胶电泳分析该蛋白质是由分子最为70kD和48kD的两条多肽所组成。     

草菇低温诱导蛋白研究初探*

对低温处理的草菇Volvariella volvacea菌丝体的可溶性蛋白进行分析,发现草菇菌丝在低温协迫中有新的可溶性蛋白产生,应用电泳技术分离纯化了草菇菌丝体中的一个低温诱导蛋白,经等电聚焦分析,该蛋白质的等电点6.79,SDS-聚丙烯酰胺凝胶电泳分析该蛋白质是由分子最为70kD和48kD的两条多肽所组成。     

草菇MADS-box转录因子Vvrin1基因过表达转化子菌株的构建及其生长速度的初步差异分析

新鲜草菇味道鲜美,香味浓郁,而且营养丰富,是具有中国特色的食药用菌。草菇采后极易开伞,低温贮藏（10℃以下）则容易自溶、渗水腐、发出异味,是最不易贮藏的食用菌之一。本课题组先前的研究表明草菇MADS-box转录因子Vvrin1基因可能在草菇菌柄的伸长、菌盖的开伞过程中起到一定的作用。因此,本研究以Vvrin1基因为研究对象,构建该基因的过表达载体,通过农杆菌介导的方法进行转化草菇异核菌株H1521菌丝块。通过潮霉素抗性平板筛选,PCR扩增潮霉素抗性基因及Vvrin1基因过表达特异片段,qRT-PCR分析拟转化子菌株内的Vvrin1基因表达量,最终获得了8个比较可靠的转化子。进一步对这些转化子的表型进行初探,发现其中7个转化子的生长速度比出发菌株H1521快,具有显著性差异（P<0.05）,且转化子菌丝更浓密,菌落表面颜色更深,推测草菇MADS-box转录因子Vvrin1基因可能参与了菌丝阶段生长速度和色素合成或积累的调控。研究结果为进一步研究草菇MADS-box转录因子Vvrin1基因的功能提供了菌株材料及数据支持。     

草菇(Volvariella volvacea)、银丝草菇(V.bombycina)原生质体分离研究

本文对草菇、银丝草菇菌丝原生质体制备的最佳条件作了探讨。结果表明,培养两天的草菇菌丝,以0.5MKCI或0.5MMgSO_(4.7)H_2O作渗透压稳定剂,1.5％Lywallzyme(v/m),35℃下酶解1.5—2小时,原生质体产量可达1.5×10~6个/ml以上,培养3天的银丝草菇菌丝,以0.5MKCl作渗透压稳定剂,1.5％Lywallzyme,28℃酶解2—3小时,原生质体产量可达2.8×10~6个/ml以上。此研究对以后的原生质体融合育种打下了基础。     

草菇低温诱导蛋白研究初探

对低温处理的草菇Ｖｏｌｖａｒｉｅｌｌａ ｖｏｌｖａｃｅａ菌丝体的可溶性蛋白进行分析,发现草菇菌丝在低温协迫中有新的可溶性蛋白产生,应用电泳技术分离纯化了草菇菌丝体中的一个低温诱导蛋白,经等电聚焦分析,该蛋白质的等电点６．７９,ＳＤＳ－聚丙烯酰胺凝胶电泳分析该蛋白质是由分子量为７０ｋＤ和４８ｋＤ的两条多肽所组成。     

五种矿质元素对草菇菌丝生长和活性氧清除能力的影响

【背景】菌种退化是草菇生产中面临的难题,探究一种简单高效的复壮方法是草菇产业亟须解决的问题。【目的】探讨外源添加5种矿质元素对草菇退化菌株的菌丝性状和活性氧(reactive oxygen species, ROS)清除能力的影响。【方法】筛选出5种矿质元素的最佳浓度并添加于PDA培养基中,测定草菇菌落形态、菌丝生长速度、菌丝生物量、ROS含量、抗氧化酶活性及抗氧化物质含量。【结果】MnSO4、Na2SeO3、CaSO4、FeSO4可有效提高草菇退化菌株D1和D2的菌丝生长速度和生物量;降低O2·-、H2O2等ROS的含量,并增加还原型谷胱甘肽(glutathione, GSH)、氧化型谷胱甘肽(glutathionedisulfide,GSSG)的含量及超氧化物歧化酶(superoxidedismutase,SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽...     

苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

基于抗性恢复的高效克隆超短片段重组质粒的构建

分子克隆作为一种常规技术被广泛应用于DNA及蛋白质的研究。在传统的分子克隆中,主要通过限制性内切酶先分别消化目的 DNA片段及载体,再纯化回收,然后用DNA连接酶将二者连接。而对一些超短基因片段(300 bp),通过酶切及切胶纯化后,回收率极低,导致插入表达载体比较困难。文中介绍了一种新的利用质粒抗性恢复进行克隆的方法,大大提高了克隆效率,为短基因片段的分子克隆提供了一种高效的方法。     

大鼠D-双功能蛋白基因原核表达载体的构建及表达

利用原核表达系统构建大鼠D-双功能蛋白表达载体。设计基因拼接引物,通过RT-PCR合成DBP基因cDNA序列,将酶切、纯化的DBP基因与经相同处理的表达载体pET-28 a相连接,转化感受态大肠杆菌DH5α,筛选阳性重组子。将通过酶切及序列分析鉴定阳性的重组子质粒转入感受态大肠杆菌BL21-gold表达菌中,经IPTG诱导表达,通过SDS-PAGE分析目的蛋白表达情况。结果显示,成功获得了包含DBP基因的双链cDNA序列,酶切、序列分析及Western blotting证实成功构建了DBP基因的原核表达载体。通过原核表达系统,DBP蛋白以包涵体形式产生,复性后可获得高表达的目的蛋白。     

Molecular genetic key for the identification of 17 Ixodes species of the United States (Acari:Ixodidae): a methods model.

A taxonomic key, based on restriction enzyme analysis of the second internal-transcribed spacer (ITS-2) in the nuclear ribosomal DNA gene, was developed for identification of 17 Ixodes tick species in the United States. This key includes: Ixodes affinis Neumann, Ixodes angustus Neumann, Ixodes baergi Cooley and Kohls, Ixodes brunneus Koch, Ixodes cookei Packard, Ixodes dentatus Marx, Ixodes jellisoni Cooley and Kohls, Ixodes kingi Bishopp, Ixodes minor Neumann, Ixodes muris Bishopp and Smith, Ixodes pacificus Cooley and Kohls, Ixodes scapularis Say, Ixodes sculpularis Neumann, I. spinipalpis Hadwen and Nuttall, Ixodes texanus Banks, Ixodes uriae White, and Ixodes woodi Bishopp. A 900-bp fragment of the ITS-2 was amplified using the polymerase chain reaction. This fragment was then digested with the restriction enzymes MspI and CfoI, and the digested fragments were size fractionated on a 2.5% high-resolution agarose gel. A dichotomous key was developed based on digested fragment sizes relative to a standard set of size markers. Little intraspecific variation in restriction fragment banding patterns was detected.     

一种有效回收小分子DNA片段的方法

介绍了一种有效回收小于200bp DNA片段的方法。用改进的冻融-离心回收法对155bp的DNA片段进行回收,并且与常规冻融-离心回收、TaKaRa回收试剂盒的结果做对比,用琼脂糖凝胶电泳检测回收结果,紫外吸收法定量分析。结果证明:改进的方法是一种经济、方便、可靠的回收小分子DNA片段的方法。     

琼脂糖凝胶的合适浓度对于回收酶切后质粒载体的重要性

目的：探讨琼脂糖凝胶的不同浓度对回收不同大小的酶切后质粒载体纯度的影响。方法：将2种质粒载体酶切,并经不同浓度的琼脂糖凝胶电泳分析,通过比较酶切前和酶切后载体的相对位置,研究胶浓度对回收酶切后载体纯度的影响。结果：不同浓度的琼脂糖凝胶中,酶切前和酶切后载体的相对位置会发生变化,对能否成功回收到纯度高的酶切后DNA片段有重要影响;质粒大小不同,胶浓度的影响也不同。结论：合适的胶浓度对于回收酶切后质粒载体具有重要意义,应选择合适的胶浓度回收酶切后质粒载体。     

Construction and mapping of safflower chloroplast DNA recombinants and location of selected gene markers

Summary DNA was isolated and purified from chloroplasts of safflower (Carthamus tinctorius L.), digested with HindIII restriction endonuclease, and ligated into the HindIII site of the plasmid pUC9. Recombinant DNAs were isolated from ampicillin resistant white colonies which grew in the presence of the appropriate indicator, digested with HindIII, and then identified by comparison of agarose gel electrophoretic mobilities. HindIII digests of chloroplast DNA were used as a standard. Such recombinants were radiolabeled and hybridized with Southern blots of PstI, SalI, KpnI, and HindIII single and double digests of safflower chloroplast DNA. A physical map was subsequently generated showing the location of each recombinant on the circular plastid genome. Recombinants containing heterologous chloroplast gene markers from spinach or Euglena were also radiolabeled and mapped. The relative mapping positions of these genes are in good agreement with those which have previously been published for spinach and several other higher plants.     

Mitochondrial DNA polymorphism in male-sterile cytoplasm of rice

Summary Mitochondrial DNAs (mtDNAs) were isolated and purified from ten strains of rice plants with male-sterile cytoplasm. The mtDNAs were digested with the restriction endonuclease PstI and the fragment patterns produced were analysed by 0.7% agarose gel electrophoresis. Restriction fragment length polymorphism was observed among the mtDNAs analysed; there were seven different patterns for the ten examined. Our results indicate that there are a variety of mtDNAs in cytoplasmically male-sterile rice.     

艾滋病毒核酸探针的制备

 采用PXY104(含化学合成的HIV-env26肽基因4重串联体结构)为材料。温和裂解法提取质粒DNA,制备电泳纯化,Hind Ⅲ和EcoR Ⅰ酶解,低融点琼脂糖凝胶电泳回收HIV-DNA片段作为核酸探针;用[α-~(32)P]dATP通过缺口移位法标记,比放射性为4.05—6.69×10~7cpm/μgDNA通过分子杂交能测出lPg的靶DNA,初步试验结果表明,在本文试验条件下,可用该探针检测与之互补的核酸分子。