苏云金芽胞杆菌XL6~-候选生物被膜调控基因00940序列分析及基因敲除突变体构建

细菌生物被膜(bacterial biofilm,BBF)可以提高细菌的紫外线抗逆性,调控细菌对环境胁迫的适应能力。本研究以课题组前期比较基因组工作中筛选出的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)XL6-(Gen Bank No.CP013000.1)的调控生物被膜形成的候选基因00940为基础,分析其基因功能,并构建基因敲除突变株。通过生物信息学分析发现00940基因可能编码谷氨酰胺合成酶,并受到转录因子Sig L、CcpA、Deg U和Lex A的调控。在枯草芽胞杆菌(Bacillus subtilis)中,Sig L是一种增强子,位于枯草芽孢杆菌的谷氨酸脱氢酶基因的下游,负责转录编码谷氨酸脱氢酶的roc G基因;CcpA转录因子参与代谢产物的分解;Deg U控制鞭毛形成和生物被膜形成的基因表达;Lex A蛋白在DNA损伤的情况下被诱导,是细菌SOS DNA修复系统的转录抑制因子,推测这些转录因子在Bt中也发挥相似的作用。通过PCR获得00940基因上、下游同源臂和卡那霉素(kan)抗性基因,利用重叠PCR获得完整的基因敲除片段。根据酶切位点将基因敲除片段和p MAD温敏载体进行重组反应,得到重组质粒。将重组质粒电击转化Bt XL6-,筛选获得了00940基因敲除突变株。以Bt Xl6-作为对照,对Bt Xl6-00940基因突变株进行表型分析,包括生长曲线测定、群游能力测定以及生物被膜形成能力测定。结果表明,00940基因的敲除对菌株的生长趋势没有影响,但是群游能力和生物被膜形成能力提高,初步确定00940基因的敲除提高Bt Xl6-菌株的生物被膜形成能力。基因敲除突变株的获得为进一步分析相关调控基因的功能提供科学依据和基础资料。     

利用抗性基因替换的方法将两个拷贝外源基因表达单元整合到枯草芽孢杆菌染色体基因组同一位点

本研究提供了一种新的整合多拷贝外源基因表达单元到枯草芽孢杆菌染色体同一位点的方法。以β-淀粉酶表达单元作为应用实例,本方法包括以下步骤：首先,构建含有一个拷贝β-淀粉酶表达单元的整合质粒pMLK83-CTBA,通过同源双交换获得单拷贝β-淀粉酶表达单元整合到枯草芽孢杆菌1A751染色体α-淀粉酶基因位点的菌株1A751[CTBA]Neo＋;然后,利用抗性基因替换质粒pVK71,将新霉素抗性基因替换为状观霉素抗性基因,得到1A751[CTBA]Neo-Spe＋;最后,整合质粒pMLK83-CTBA再以同源单交换方式整合到1A751[CTBA]Neo-Spe＋染色体,通过新霉素抗性和状观霉素抗性筛选出两个拷贝β-淀粉酶基因整合的重组菌1A751[CTBA2]Neo＋Spe＋。结果显示,利用此方法增加β-淀粉酶基因的拷贝数,能够显著和稳定地提高β-淀粉酶的表达量。     

α-乙酰乳酸脱羧酶基因在枯草芽孢杆菌中的整合型表达

α-乙酰乳酸脱羧酶在啤酒生产中能加快啤酒成熟,有重要的应用价值。本研究将枯草芽孢杆菌启动子P43克隆到质粒pUC19-ALDC中的α-乙酰乳酸脱羧酶基因之前,得到重组质粒pUC19-P43-ALDC。重组质粒pUC19-P43-ALDC与质粒pMLK83-BN同源重组,筛选得到枯草芽孢杆菌整合质粒pMLK83-ALDC。用此整合质粒转化枯草芽孢杆菌1A751,挑选出新霉素抗性且无淀粉酶活性的重组菌株。此菌株用LB培养基在37℃、220r/min摇瓶培养过夜,测得α-乙酰乳酸脱羧酶活力为15.6U/mL,说明整合的α-乙酰乳酸脱羧酶基因能够在重组菌株中稳定传代和表达。本研究首次在枯草芽孢杆菌中用整合型的方式重组表达了α-乙酰乳酸脱羧酶,提出了一种有潜力的生产α-乙酰乳酸脱羧酶的新方法。     

番茄果实中NEVER—RIPE基因的克隆及其反义表达载体的构建

从粉红期番茄果实中提取总RNA，根据Genebank中NEVER－RIPE（即NR基因）cDNA序列，设计合成特异性引物，用反转录－聚合酶链式反应（RT－PCR）进行cDNA扩增，获得一个715bp左右的扩增产物，克隆于pGEM-T easy载体，测序确定其正确性。将该扩增产物反向克隆到植物表达载体pBI121中，通过PCR及限制性内切酶酶切鉴定重组质粒。将重组表达质粒导入根癌农杆菌LBA4404中，PCR及限制性内切酶酶切确定质粒已被导入。     

Bt cry I A（c） 杀虫基因向棉内生细菌Bacillus cereus染色体中整合的研究

将来自Bacillus thuringiensis subsp.kurstaki的Bt CRy I A(c) 杀虫基因通过综合质粒载体pBC601，整合到棉花（Gossypium hirsutum)优势内生细菌Bacillus cereus(Bc9002)的染色体上，得到的工程菌对棉铃虫（Helicoverpa armigera Hb.)有杀虫活性。此综合质粒含有能在营养期表达Bt cry I A(c) 基因的强启动子，cry I A(c)杀虫基因，四环素抗性标记基因tet^r 8.0kb的EcoR I-Nco I B.cereus染色体片段。将综合质粒通过电击导入B.cereus(Bc9002)中，综合质粒因含B.cereus染色体片段，可与B.cereus染色体发生同源重组，从而将Bt cry I A(c)基因整合到B.cereus的染色体上。通过对转化子的DNA酶切分析，PCR扩增，SDS－PAGE凝胶电泳检测，ELISA检测，电镜观察，毒力测定。结果表明Bt cry I A(c)基因已经整合到内生细胞Bc9002的染色体上，并可高效表达。     

STAT3基因转染牛乳腺上皮细胞及生物学特性观察

应用PCR技术从含有人STAT3基因cDNA的质粒pOTB7中扩增STAT3基因片段。将其克隆入真核表达载体pEGFP-C1中,构建重组表达质粒。利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(Bovine Mammary Epithelial cells)。G418筛选阳性克隆,RT-PCR检测STAT3基因在牛乳腺上皮细胞中的表达情况,并通过流式细胞术(Flow cytometry)检测转染细胞的增殖能力、周期以及DNA含量。结果表明,转染24h后大约16%的细胞被导入含STAT3基因的重组表达质粒。在mRNA水平证实细胞内有STAT3基因的高表达。导入STAT3基因后,细胞增殖能力增强,染色体倍数发生变化,细胞的增殖寿命较对照细胞延长了13代,表明导入STAT3基因后,能延长体外培养的牛乳腺上皮细胞的寿命。     

构建食品级植酸酶黑曲霉工程菌

植酸是植物种子中肌醇和磷的主要存在形式,由于单胃动物体内缺乏能分解植酸的酶,以植酸磷形式存在的磷难以被单胃动物吸收。植酸酶作为饲料添加剂已经广泛应用,但在食品中的应用研究刚刚开始。本研究以黑曲霉(Aspergillus niger)CICC2462基因组DNA为模板,PCR扩增植酸酶基因片段,并在其两端加上糖化酶基因(glaA)的5'同源臂和3'同源臂,构建农杆菌Ti质粒基因置换载体pSZHG-phy。通过农杆菌(Agrobacterium tumefaciens)介导法转化黑曲霉,筛选以植酸酶基因替换糖化酶基因的同源重组转化子,以实现植酸酶基因的高效表达。结果表明,筛选获得4株同源重组转化子,同源重组率为100%,通过分光光度计法测定重组植酸酶菌株的最高发酵酶活为316.2 U/mL,为出发菌株的20.8倍,证明植酸酶phy基因是可以在黑曲霉糖化酶生产菌中实现高效表达。本研究为进一步构建食品级植酸酶工程菌提供了基础资料。     

草莓乙烯受体FaEtr1和FaErs1基因的克隆及其反义表达载体的构建

在已报道的FaEtr1和FaErs1基因序列的基础上,设计特异引物,分别克隆草莓(Fragaria ananassa)乙烯受体FaEtr1基因580bp部分特异序列和FaErs1基因445bp部分特异序列,将上述2个片段分别反向插入植物表达载体pBI121的CaMV35S启动子和Nos终止子之间,构建了反义表达载体pBI121Etr1和pBI121Ers1。将带有完整启动子和终止子的FaEtr1和FaErs1基因引入pCAMBIA1301中,最终获得FaEtr1和FaErs1的双价植物表达载体pFRS。将这3个重组表达质粒pBI121Etr1、pBI121Ers1和pFRS导入根癌农杆菌(Agrobactrium tumefaciens)EHA105中,PCR及限制性内切酶酶切确定质粒已被导入。     

含有His 5基因用于酵母人工染色体重组筛选的置换型载体的构建

人工酵母染色体(YAC)可以克隆和分析大片段的染色体DNA,并且可以分离那些在大肠杆菌(Escherichia coli)中不可能得到的序列。实验将His5基因插入到一个Ura3基因的中间,构建了一个新的质粒pLRH33,从而打断了Ura3基因使之不能表达。利用His5基因两端各留下的部分Ura3基因片段,同源重组到YAC臂上原有的Ura3基因中,使重组后的YAC的标记性状从Rra^ His^-变成了His^ Ura^ 。用质粒pLRH33的5．5kb线性片段以一定比例和需要重组到YAC上的目的基因pBAC300的50kb片段相混合,同时作酵母菌转化,在His^-Ura^ 培养基上得到大量带His5基因的阳性克隆。在检测的1200个克隆中,有250个目的基因阳性克隆,占受检克隆的22．5％。表明质粒pLRH33可以有效地用作YAC重组的筛选标记：     

信号转导及转录活化因子基因(stat3)转染牛乳腺上皮细胞及生物学特性观察

应用PCR技术从含有人信号转导及转录活化因子(star3)基因cDNA的质粒pOTa7中扩增star3基因片段.将其克隆入真核表达载体pEGFP-C1中.构建重组表达质粒.利用脂质体介导法将重组表达质粒转染原代牛乳腺上皮细胞(bovinemammary epithelial cells).G418筛选阳性克隆.RT-PCR检测star3基因在牛乳腺上皮细胞中的表达,并通过流式细胞术(flowcytometry)检测转染细胞的增殖能力、周期以及DNA含量.结果表明.转染24 h后大约16%的细胞被导入含star3基因的重组表达质粒.在mRNA水平证实细胞内有stat3基因的高表达.导人star3基因后,细胞增殖能力增强,染色体变为三倍体,细胞的增殖寿命较对照细胞延长了13代.表明导入star3基因能延长体外培养的牛乳腺上皮细胞的寿命.     

瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因的克隆及功能分析

过敏性反应和致病性(hrp)基因存在于革兰氏阴性植物病原细菌中,决定病原细菌对寄主植物致病性和诱导非寄主及抗病植物过敏性反应(hypersensitive response,HR)。本研究从果斑菌(Acidovorax avenaesub sp.citrulli)的hrp基因簇中克隆了hpaA、hrcT、hrcC和hrpG基因,通过同源重组的方法,分别构建了其突变体。电镜观察发现,hpaA和hrpG基因突变体的鞭毛缺失且细胞形态发生显著变化,而hrcT和hrcC基因突变体的鞭毛和细胞形态未发生明显变化。在烟草(Nicotiana tabacam)和哈密瓜(Hami cantaloupe)叶片上的测定结果显示,hpaA、hrcT和hrcC的突变体均失去在烟草上的HR激发能力和在哈密瓜叶片上的致病性;hrpG在烟草上的HR激发能力和在哈密瓜上的致病性则显著减弱;进一步的生长曲线测定结果表明,hpaA、hrcT、hrcC和hrpG的突变体的定殖能力均显著下降。相应地,功能互补后突变体基本恢复至野生表型。证明瓜类细菌性果斑病菌hrp基因作为Ⅲ型分泌系统关键组份影响病原细菌对寄主植物的致病性和对非寄主及抗病植物的过敏性...     

适用于稻瘟病菌基因敲除、过表达和荧光融合蛋白表达载体的构建和使用

本研究意在提供一系列适于稻瘟病菌(Magnaporthe oryzae)的基因敲除、过表达和荧光蛋白融合表达,并且操作简单、耗时短、稳定可靠的通用载体,为系统研究稻瘟病菌的基因功能提供便利.通过PCR、克隆、酶切、连接等分子生物学方法,克隆了2个在菌丝和附着胞阶段都强力表达的启动子(SOD1启动子和H3启动子);构建了14种载体:3种稻瘟病菌基因敲除载体(pBS-SUR、pBS-BAR和pBS-NEO)、4种丝状真菌基因过表达载体(pKD5、pKD6、pKD61和pKD8)和7种丝状真菌荧光融合蛋白载体(pKD5-GFP、pKD5-RED、pKD6-GFP、pKD6-RED、pKD7-RED、pKD8-GFP和pKD8-RED);并提供了这些载体在丝状真菌的基因敲除、基因过表达和荧光融合蛋白表达中的应用方法.使用构建的基因敲除载体通过原生质体及ATMT转化最多同时在稻瘟病菌中敲除了4个基因;用pKD5-RED、pKD6-GFP和pK)6-RED转化稻瘟病菌后,发现SOD1启动子和H3启动子控制下的绿色和红色荧光蛋白在稻瘟病菌中强力表达,Real-time PCR结果证实SOD1启动子指导下的eGFPmRNA表达量是β-tubulin的2.5倍,SOD1启动子指导下的DsRED2mRNA表达量是β-tubulin的5.4倍,而H3启动子指导下的DsRED2 mRNA表达量是β-tubulin的20.8倍;MoATG8-DsRED2的融合蛋白(使用pKD6 -RED)可以正确定位MoATG8于小泡附近;SOD1启动子驱动的DsRED2(使用pKD6-RED)可以在稻瘟病菌的菌丝、孢子、附着胞等各个发育阶段表达.这些实验说明14种载体可以用于稻瘟病菌的基因敲除、基因过表达和荧光融合蛋白表达,也可用于镰刀菌等其他丝状真菌的基因功能研究.     

New sources of resistance to race Ro1 of the Golden nematode (Globodera rostochiensis Woll.) among reputed duplicate germplasm accessions of Solanum tuberosum L. subsp. andigena (Juz. et Buk.) Hawkes in the VIR (Russian) and US Potato Genebanks

Cultivated Solanum tuberosum L. subsp. andigena is well known as a rich source of valuable traits for potato breeding, especially for resistance to diseases and pests. The golden potato cyst nematode, Globodera rostochiensis Woll., is considered to be one of todays most serious hindrances to potato production in Europe and North America. Thus, the breeding of new cultivars that have resistance to PCN is of great importance. The USPG (USA) and VIR (Russian) potato genebanks, as well as others, maintain many samples of primitive cultivated and wild potato species originating from Latin America. Many of these samples are assumed to be genetically duplicated because the material in both genebanks came from the same original source. A joint investigation of new genotypes of subsp. andigena forms resistant to potato cyst nematode (PCN) was carried out on samples of subsp. andigena at VIR with reputed duplicate samples at USPG. After careful screening, 14 samples which possessed resistance to PCN were identified. A high level of this resistance was transmitted to sexual progeny at a high frequency for all of the selections. Eleven of the accessions found to be resistant have reputed duplicates in USPG that were not previously known to be resistant. Thus, this research not only broadens the choice of parents available for resistance breeding, but identifies model materials for future research to test the parity of PCN resistance among reputed duplicate samples in the two genebanks.     

Sources of resistance to ascochyta blight in wild species of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik.)

Cultivated lentil (Lens culinaris Medik. subsp. culinaris) has a relatively narrow genetic base and many commercial cultivars are susceptible to ascochyta blight caused by Ascochyta lentis Vassilievsky. A total of 375 accessions of six wild species of lentil received from ICARDA and 18 cultivated genotypes were screened for resistance to A. lentis under both field and greenhouse conditions in Saskatoon, Canada. A mixture of three monoconidial isolates of A. lentis was used as an inoculum and the level of infection rated using the Horsfall-Barratt scale (0–11). Accessions with resistance to A. lentis were observed in all wild species except for L. culinaris subsp. tomentosa (Ladiz.) Ferguson et al. showing no resistant accessions. Several consistently resistant accessions were found among entries of L. ervoides (Brign.) Grande and L. nigricans, (M. Bieb.) Godr., both of which belong to the secondary gene pool and a few in L. culinaris subsp. orientalis (Boiss.) Ponert and L. culinaris subsp. odemensis (Ladiz.) Ferguson et al. belonging to the primary gene pool. Some accessions of L. ervoides exhibited lower disease ratings and AUDPC values than the resistant control cv. ‘Indianhead.’ Thirteen accessions, previously reported as resistant to Syrian isolates of A. lentis were also resistant to the Canadian isolates; some also had resistance to anthracnose. The highest frequency of resistance was found in accessions of L. ervoides which originated from Syria and Turkey. These wild accessions represent a useful and untapped source for improving disease resistance in lentil.     

Conjugated linoleic acid content and organoleptic attributes of fermented milk products produced with probiotic bacteria

The effect of probiotic bacteria on the formation of conjugated linoleic acid (CLA), microbial growth, and organoleptic attributes (acidity, texture, and flavor) of fermented milk products was determined. Four probiotic bacteria, Lactobacillus rhamnosus, Propionibacterium freudenreichii subsp. shermanii 56, P. freudenreichii subsp. shermanii 51, and P. freudenreichii subsp. freudenreichii 23, were evaluated individually or in coculture with traditional yogurt cultures (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus). The lipid source was hydrolyzed soy oil. L. rhamnosus, in coculture with yogurt culture, resulted in the highest content of CLA. Growth and CLA formation of propionibacteria were enhanced in the presence of yogurt cultures. Texture and flavor attributes of fermented milks produced with propionibacteria were significantly different than the fermented milks processed with yogurt cultures. The fermented milks processed with probiotic bacteria in coculture with yogurt cultures demonstrated similar acidity, texture, and flavor as the fermented milk produced with yogurt cultures.     

水稻种子相关细菌的系统发育分析与促生能力评价

对15株分离自水稻冀粳14号(90-3)的种子表面及其内生细菌进行了多功能筛选评价和16S rRNA系统发育分析.结果表明,供试菌株都能够溶解无机磷,不能分解有机磷;筛选获得6株具有明显分泌植物生长素吲哚-3-乙酸(IAA)能力的菌株;12株菌株明显产生铁载体,占所测菌株总数的80％,其中7株铁载体产量达到中等水平,通...     

Variation Among and Within Dogrose Taxa ( Rosa sect. caninae ) in Fruit Weight,Percentages of Fruit Flesh and Dry Matter,and Vitamin C Content

Important fruit traits were screened in a plant breeding program concerning a newly domesticated crop in Sweden of rose hips. The taxa Rosa dumalis subsp. coriifolia , R . dumalis subsp. dumalis , R . rubiginosa and R . villosa subsp. mollis, collected from 23 localities in Scandinavia, were investigated for fruit weight, percentage of fruit flesh, percentage of dry matter and vitamin C content. R . dumalis subsp. coriifolia, R . dumalis subsp. dumalis and R . villosa subsp. mollis had significantly larger fruits and a higher percentage of fruit flesh than R . rubiginosa. Analysis of intraspecific variation showed that R. dumalis subsp . coriifolia was the most variable taxon, followed by R . dumalis subsp. dumalis and R . villosa subsp. mollis , whereas R. rubiginosa showed the most restricted variability. A highly significant positive correlation was found between fruit weight and percentage fruit flesh which in turn showed a moderate negative correlation with percentage dry matter.     

用两个抗虫基因分别转化水稻及抗虫株系的获得

用水稻（Ｏｒｙｚａ ｓａｔｉｖａＬ．）的幼胚、愈伤组织作受体,采用ＰＩＧ基因枪法,成功地将苏云金杆菌毒蛋白基因〖Ｂｔ毒蛋白基因,ｃｒｙＩＡ（ｂ）〗和马铃薯蛋白酶抑制剂基因（ｐｉｎⅡ基因）连同抗除草剂Ｂａｒ基因分别转入不同的水稻中,获得大量的转基因植株。Ｓｏｕｔｈｅｎ杂交分析途中外源抗虫基因均分别整合到水稻的基因组中。Ｎｏｒｔｈｅｒｎ杂交分析显示它们在水稻叶片中表达的水平较高。外源基因在一代中遵循３     

促性腺激素释放激素受体(GnRHR)基因多态性及其与济宁青山羊高繁殖力关系

设计8对引物, 应用聚合酶链式反应-单链构象多态性（PCR-SSCP）技术检测促性腺激素释放激素受体（gonadotropin releasing hormone receptor, GnRHR）基因外显子1、外显子2和外显子3在高繁殖力山羊(Caprar hircus)品种（济宁青山羊）和低繁殖力山羊品种（安哥拉、波尔和内蒙古绒山羊）中的单核苷酸多态性,同时研究该基因对济宁青山羊高繁殖力的影响。结果发现引物P4扩增片段具有多态性,其余7对引物的扩增片段都不存在多态性。对于引物P4扩增片段,在济宁青山羊、波尔山羊和内蒙古绒山羊中均检测到AA、AB和BB基因型,而在安哥拉山羊中只检测到AA和AB基因型,未检测到BB基因型;测序分析发现BB型与AA型相比在外显子1有1个突变（757G→A）,但未引起氨基酸改变;济宁青山羊AA、AB和BB基因型频率分别为0.623、0.300和0.077,BB型济宁青山羊平均产羔数比AB型和AA型分别多0.69只（P　<　0.05）和0.82只（P　<　0.05）,AA型和AB型济宁青山羊产羔数的最小二乘均值差异不显著（P　>　0.05）。     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.