插入烟草叶绿体启动基因片段的重组质粒转化大肠杆菌和枯草杆菌感受态与原生质体的比较

大肠杆菌(E.coli)N100携带的pK01-26质粒插入烟草(Nicotiana tobacHm var.)叶绿体启动基因片段的重组质粒。该质粒以大肠杆菌HB101为受体可以再转化,转化频率为4.93×10^-6,以枯草杆菌168为受体不能实现转化。上述两种受体菌株的原生质体,经处理,再生细胞壁后,分别获得转化子。经原生质体转化,以大肠杆菌HB101为受体的转化频率为2.7×10^-4频率为2.6×10^-5。以H     

球形芽孢杆菌Ts-1原生质体电诱导质粒转化研究

本文探讨了球形芽孢杆菌Bacillus sphaericus Ts-1原生质体电诱导质粒pHV 33转化的最佳条件：3个连续的间隔为1秒,时程为10μs,强度为21Kv／cm的脉冲,使其转化频率为2．44×102转化子／μgDNA,转化效率为3．16×10-6,质粒转化吸附的饱和浓度为5μg／l03CFU。利用此条件,将重组杀蚊毒蛋白克隆pJB41 7转入Bacillus subtillis 168M和Bacillumphaericus Ts-1中,使得B．Sublilis有了杀蚊活性,但未能提高Ts-1的毒性。     

烟草tga1a基因的表达对外源基因在植物中表达的影响

烟草DNA结合蛋白TGA1a可特异地作用于CaMV35S增强子的激活序列as-1(-83~-63), 并表现为转录激活功能. 为了研究tga1a基因的表达对外源基因在植物中表达的影响, 将它置于维管束特异性启动子rolC下游, 并与CaMV35S启动子控制的报道基因串联成一种反式调控系统, 构建了植物表达载体, 同时, 以CaMV35S和rolC分别控制的报道基因构建植物表达载体为阳性对照. 通过农杆菌介导方法转化烟草和分子鉴定, 证明报道基因存在于转化烟草基因组中, 分别测定了不同转基因单株的GUS活性, 结果表明: rolC控制下的tga1a的表达显著增强了CaMV35S控制下的报道基因表达, 其GUS活性明显高于CaMV35S或rolC单独调控报道基因的转化植株, 单株的最高GUS活性达到两个阳性对照的10倍以上. 组织化学定位证实该串联系统使GUS蛋白主要集中在维管束组织. 这一研究结果为提高外源基因在转基因植株中的表达水平和外源基因的组织特异性表达创立了一个新模式.     

根癌农杆菌介导的白腐丝状真菌——黄孢原毛平革菌的转化

利用农杆菌介导的方法成功地对黄孢原毛平革菌（Phanerochaete chrysosporium）进行了遗传转化。将含有潮霉素磷酸转移酶融合基因的双元质粒pCH61300转入根癌农杆菌（Agrobacterium tumefaciens）208中,然后用该转化菌分别感染黄孢原毛平革菌的分生孢子和原生质体,获得16株可能的转化子,经复筛,共获得6株潮霉素抗性水平为100μg/mL的稳定转化子,分生孢子和原生质体的转化频率没有明显差别。PCR检测结果显示,抗性基因已导入黄孢原毛平革菌细胞中;Southern杂交表明,TDNA以单拷贝形式整合到黄孢原毛平革菌基因组中。其中的一个转化子菌落形态与原野生型菌株相比有所不同,菌丝稀薄,分生孢子较少。利用分生孢子转化更为简便易行,无需特殊的设备和制备原生质体,此方法为深入开展该菌的遗传转化研究奠定了基础。     

玉米大斑病菌（Exserohilum turcicum）原生质体的分离及转化

玉米大斑病是严重危害玉米生产的一个世界性真菌病害。由于玉米大斑病菌（Exserohilum turcicum）在无性生长过程中迅速产生黑色素,致使原生质体难以分离。测试了包括Fungase、Funcelase、Novozyme、Glucanex、Driselase、Uskizyme、Kitalase在内的7种细胞壁降解酶及其组合、病原菌菌株和培养基对原生质体分离效果的影响。结果表明菌株的培养形态和菌丝的生长状态显著影响原生质体的分离效率;酶组合Kitalase+Glucanex+Driselase, Kitalase+Glucanex和Kitalase+Uskizyme能够有效地分离玉米大斑病菌的原生质体。初步的转化试验表明,质粒pAN71可以用于该病原菌的转化。这些结果将为E.turcicum和Exserohium属其它真菌的基因克隆提供一些有用的信息。     

维生素C生产用菌系2980产酸菌基因转移的筛选模型及转座子Tn5诱变

维生素C的生产目前国内广泛采用由产酸菌(Gluconobacter oxydans)与伴生菌(Bacillus megaterium)组成的2980菌系,在2980中G.oxydans单独生长传代困难,其生长和产酸需要B.megaterium参与。以Bacillus subtilis Ki-2-132(pUB110)作为伴生菌与原2980的G.oxydans组合,获得稳定产酸的新菌系。在此基础上建立了适合我国混菌发酵产酸菌外源基因(Kanr)转移的筛选模型。同时报道了以携带有自杀性载体P1：：Tn5的大肠杆菌E.coli W3110为供体菌对G.oxydans进行Tn5诱变的条件和结果。     

苏云金芽孢杆菌以色列亚种130kDa杀蚊蛋白基因在枯草芽孢杆菌中的表达

本文将苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis)130kDa杀蚊蛋白基因亚克隆到pNQ1 22载体上,通过枯草芽孢杆菌(Bacillus subtilis)原生质体转化,得到Kmt Cm2的正反向克隆子(pFZl和pFZ2)。 Western—blotting免疫杂交证明130kDa杀蚊蛋白基因在枯草芽孢杆菌中表达了具有免疫活性的130kDa杀蚊蛋白。 所表达的杀蚊蛋白在实验中具有杀蚊活性。     

木耳属种间杂交技术研究*I．木耳菌丝原生质体的形成与再生

本文比较了不同酶液、渗透压稳定剂、酶解媪度及菌丝培养基成份等因素对木耳属(Auricularia)中木耳(Auricularia auricula)和毛木耳(Auricularia polytricha)菌丝释放原生质体的作用及影响。用0.5％纤维素酶加0.5％蜗牛酶的混鸯酶液,以0.6M的MgSO4为稳定剂,在34℃下可自两种菌丝体获得大量原生质体。对原生质体再生条件的研究表明,纤维二糖和菌丝体培养物浸提物对再生有明显促进作用,再生率达20％左右。本文还用VBL型荧光增白剂观察了菌丝脱壁以及原生质体细胞壁再生的过程。     

含有拟病毒RNA的绒毛烟草斑驳病毒在克里夫兰烟原生质体内的增殖

利用酶联免疫吸附分析(ELISA)的双抗体夹心法和未标记免疫过氧化物酶法(PAP),研究了含有拟病毒RNA (Virusoid RNA)的绒毛烟草斑驳病毒(Velvet tobacco mottle virus,VTMoV)侵染克里夫兰烟(Nicotiana elevelandii A. Gray)原生质体的最适条件以及病毒在原生质体内增殖的一步生长曲线,建立了一种适宜VTMoV增殖的原生质体细胞体系。     

电击法诱导的欧白英原生质体转化和转化植株再生

本研究使用电击法成功地将带有标记基因NPTⅡ的质粒pCaMVNEO转入欧白英的原生质体,并获得了再生转化植株。通过用pDW2质粒进行的CAT基因短暂表达研究,确定了欧白英原生质体转化的电击条件为：电容30nF、电场强度l 500V／cm、时间衰变常数59．4微秒;质粒DNA浓度为20μg／2×10⁶原生质体。在以上条件下,欧白英原生质体的相对转化率为12．4％,绝对转化率为2.4×10^-4在大多数抗性愈伤组织和从抗性愈伤组织再生的植株中检测到新霉素磷酸转移酶活性。分子杂交结果也证明了在转化植株中存在NPTⅡ基因序列,而未转化的对照植株则没有。     

感受态还是芽胞？细胞命运决定的遗传调控网络

枯草芽胞杆菌作为一般认为安全(GRAS,Generally recognized as safe)菌株,被广泛应用于饲料、食品、生物防治等领域,同时,枯草芽胞杆菌作为表达宿主在工业酶的应用中扮演重要角色。然而,低效的芽胞形成率与感受态效率极大限制了枯草芽胞杆菌的应用潜力。尽管已有大量关于芽胞形成与感受态形成的分子遗传机制的研究报道,但是通过遗传改造提高枯草芽胞杆菌芽胞形成率与感受态效率的研究报道并不多。可能的原因是芽胞形成与感受态形成作为枯草芽胞杆菌生长后期两个主要的发育事件,受胞内复杂的遗传调控机制操纵,且两个遗传通路之间存在相互调控关系,对遗传改造工作形成挑战。随着基因工程与代谢工程研究的不断发展,积累了大量关于细胞生长、代谢与发育等方面的遗传信息,通过综合这些遗传信息构建细胞遗传调控网络,用于指导生产实践,已经成为当前研究的热点之一。基于此,本文简要概述了枯草芽胞杆菌芽胞形成和感受态形成的遗传通路,初步探讨了芽胞形成与感受态形成之间的遗传调控网络,及细胞在生长后期的遗传决定机制,并讨论了该遗传调控网络对枯草芽孢杆菌及其近缘种应用研究的指导作用。     

Bacillus subtilis generates a major specific deletion in pAM beta 1.

pAM beta 1, a 26.5-kilobase plasmid originally isolated from Streptococcus faecalis, was conjugally transferred from Streptococcus lactis to Bacillus subtilis. No conjugal transfer of pAM beta 1 from B. subtilis to S. lactis was observed. In addition, pAM beta 1 which had been reintroduced in S. lactis after cycling through B. subtilis had lost its conjugal transferability to Streptococcus cremoris, although under the same conditions noncycled pAM beta 1 was transferred at high efficiency. Restriction and Southern blot analyses showed that pAM beta 1 had suffered one major, specific 10.6-kilobase deletion and several minor but also specific deletions in B. subtilis. Comparing the major deletion derivative, delta pAM beta 1, with B. subtilis strains which have been reported to contain pAM beta 1 showed that these strains also contained delta pAM beta 1. Hybridization experiments showed that the deleted fragment was not transposed to the B. subtilis chromosome. Based on the size of the minor deletion derivatives from pAM beta 1, it is suggested that these use a different origin of replication in B. subtilis.     

Bacillus subtilis generates a major specific deletion in pAM beta 1

pAM beta 1, a 26.5-kilobase plasmid originally isolated from Streptococcus faecalis, was conjugally transferred from Streptococcus lactis to Bacillus subtilis. No conjugal transfer of pAM beta 1 from B. subtilis to S. lactis was observed. In addition, pAM beta 1 which had been reintroduced in S. lactis after cycling through B. subtilis had lost its conjugal transferability to Streptococcus cremoris, although under the same conditions noncycled pAM beta 1 was transferred at high efficiency. Restriction and Southern blot analyses showed that pAM beta 1 had suffered one major, specific 10.6-kilobase deletion and several minor but also specific deletions in B. subtilis. Comparing the major deletion derivative, delta pAM beta 1, with B. subtilis strains which have been reported to contain pAM beta 1 showed that these strains also contained delta pAM beta 1. Hybridization experiments showed that the deleted fragment was not transposed to the B. subtilis chromosome. Based on the size of the minor deletion derivatives from pAM beta 1, it is suggested that these use a different origin of replication in B. subtilis.     

Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E. coli

The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis-E. coli shuttle plasmid was designed to allow the insertion of either the Terl (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP-Terl complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex. The specificity effect is of less significance for an RTP-Ter complex functioning in B. subtilis.     

Sequence analysis and in vitro maturation of five precursor 5S RNAs from Bacillus Q.

Bacillus Q, which is closely related to B. subtilis, contains at least six different precursors of 5S rRNA. The complete nucleotide sequences of four of these precursors, as well as the major part of the sequence of a fifth one, have been determined. They all contain the same 5'-terminal non-conserved segment which is to a large degree homologous with the corresponding segment of the B. subtilis p5S RNAs (Sogin, M.L., Pace, N.R., Rosenberg, M., Weissman, S.M. (1976) J. Biol. Chem. 251, 3480-3488). On the other hand the 3'-terminal non-conserved sequences of the various Bacillus Q precursors show considerable differences both in length and in nucleotide sequence, while there is also little or no homology with the 3'-terminal non-conserved sequence of the B. subtilis precursors. Bacillus Q p5S RNAs do not possess tetranucleotide repeats around the sites which are cleaved during maturation, as does B. subtilis p5S RNA. Like in B. subtilis, however, the cleavage sites are contained within a double-helical region of the precursor molecules. Crude RNAse M5 isolated from various Bacillus strains can maturate the Bacillus Q p5S RNAs with high efficiency. Despite considerable differences in primary structure between the precursors from the various strains, each RNAs M5 preparation can maturate all these precursors with about the same efficiency.     

Interspecies transformation in Bacillus: sequence heterology as the major barrier.

The relative contribution of DNA restriction and of sequence heterology as barriers to interspecies transfer of DNA was studied in the heterologous transformation of Bacillus subtilis recipients by DNA was studied in the heterologous transformation of Bacillus subtilis recipients by DNA isolated from B. globigii. Transformants were obtained at very low frequencies in the evolutionarily nonconserved aromatic region; high cotransfer of linked markers was observed. New mutations were introduced into the B. globigii intergenote sequence in the resulting hybrids; these markers could be transformed with high efficiency by both B. globigii and B. subtilis DNA, representing a 10(5)-fold increase in heterologous transforming efficiency. A restriction activity in B. globigii crude extracts inactivated the biological activity of B. subtilis and hybrid DNA but not B. globigii DNA in vitro, demonstrating different sites for restriction and modification between these species. In vivo, however, B. globigii and hybrid DNA transformed the B. globigii sequence in a hybrid recipient with the same efficiency. These results show that sequence heterology is the major barrier to interspecies transformation and that, in this system, enzymatic restriction does not prevent interspecies transformation.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.     

微囊藻毒素对典型微生物生长及生理生化特性的影响

研究了不同浓度微囊藻毒素对典型微生物大肠杆菌和枯草芽孢杆菌生长及生理生化特性的影响。微囊藻毒素对大肠杆菌和枯草芽孢杆菌的生长和细胞活性具有一定的剂量效应,较高浓度微囊藻毒素对其生长和活性有短时间的抑制作用,随着处理时间的延长,细胞的生长和活性逐渐恢复。细胞内可溶性糖和可溶性蛋白的含量,处理组和对照组相比均有先上升后下降的趋势。结果表明,微囊藻毒素的处理对大肠杆菌和枯草芽孢杆菌具有一定的胁迫作用,细胞通过调节细胞内可溶性蛋白和可溶性糖的含量来抵抗外界胁迫,但随着处理时间的延长,细菌逐渐适应了这种胁迫,恢复正常的生长。