基于CRISPR/Cas9的蛹虫草无抗性标记转化技术的构建

传统的真菌遗传改造方法需要抗性标记,但目前可使用的抗性标记基因非常有限,导致蛹虫草遗传改造面临着抗性基因数量不足的问题,且尚未能实现多个目的基因的连续敲入或敲除,因此在蛹虫草中建立高效的无抗性标记转化技术显得尤为重要。本研究利用CRISPR/Cas9技术对蛹虫草的Cmura5基因进行编辑,通过内源5S-1、5S-2和U6启动子对gRNA进行转录,结果表明使用U6启动子对Cmura5基因的编辑效率达到了100%。在尿嘧啶缺陷型菌株Cmura5^-中,回补野生型Cmura5基因可实现正向选择,即野生型菌株可以在基础培养基上生长。利用设计的同源臂对Cmura5基因进行回收,可以实现反向选择,即野生型在含有5-氟乳清酸培养基中生长受到抑制。以尿嘧啶缺陷型Cmura5^-为出发菌株,利用无抗性标记转化技术,导入一个重组质粒效率为75%;连续导入2个重组质粒效率为80%;连续导入3个重组质粒效率为100%;连续导入4个重组质粒效率为50%,平均转化效率为75.7%,每一轮的标记回收率均在100%,实现了4个外源基因在蛹虫草中同时表达。     

The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434

A new plasmid vector, pNSI, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the Tc^R gene of pNS1 is under the control of the o_Rp_R operator-promoter of phage 434, which is regulated by the represser gene c1. The cloning sites of pNSI (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes derepressed expression of the Tc^R gene from p_R thus conferring the Tc^R phenotype on the harboring Escherichia coli strain. The use ofpNS1 is facilitated by the presence of another selectable marker, Ap^R its small size, and its known nucleotide sequence; no special host strain is required.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system

We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens

Ubiquinone (UQ), a lipid-soluble component, acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system in many organisms, and has been widely used in pharmaceuticals due to its antioxidant property. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study, dps gene, which encodes decaprenyl diphosphate synthase, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and coq2 gene of Saccharomyces cerevisiae, ppt1 gene of Schizosaccahromyces pombe and ubiA gene of Escherichia coli, all of them encoding 4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an operon under the control of a single promoter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production than that of the other recombinants and the nonrecombinant bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced 25.2 mg of ubiquinone-10 per liter which was 1.68 times higher than that of nonrecombinant type. While in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced 30.8 mg L⁻¹ of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10 yield and productivities of the recombinant bacterium pBIV-dpsca increased 88.9% and 77.7%, respectively, under microaerobic fed-batch conditions.     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

Molecular characterisation of the Stc mutation of Escherichia coli K-12

The previously described Stc ^- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF⁻ to OmpF⁺. Restriction mapping of chromosomal DNA from Stc ⁺ and Stc⁻ strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc⁻ phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc⁻ phenotype involves increase in ompF expression.     

纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响*

目的:纤维素酶水解天然纤维素产生易被微生物利用的葡萄糖是进行生物炼制的关键。丝状真菌分泌的纤维素酶大多数是经过糖基化修饰的,研究丝状真菌纤维二糖水解酶(Cel7A)的催化功能域N-糖基化修饰对其分泌及酶活的影响,有助于优化纤维素酶的表达。方法:利用定点突变将草酸青霉和深绿木霉Cel7A催化功能域的N-糖基化位点去除,构建突变体PoCel7A^*和TaCel7A^*。以草酸青霉为宿主构建分泌表达PoCel7A^*、TaCel7A和TaCel7A^*的重组菌,检测N-糖基化去除对Cel7A分泌和酶活力的影响。结果:PoCel7A催化功能域的N-糖基化去除对其蛋白分泌和酶活力无影响。TaCel7A催化功能域的N-糖基化去除不影响其蛋白分泌;但突变体的pNPCase、FPase和Avicelase酶活力分别下降了21.2%,15.2%和17.6%。去除Cel7A催化功能域N-糖基化,加强了细胞内UPR响应。外源蛋白TaCel7A和TaCel7A^*的表达也加强了胞内UPR响应。结论:不仅可以为丝状真菌Cel7A的酶工程改造提供理性设计思路,而且为进一步了解糖基化在纤维素酶降解纤维素过程中的作用及机理奠定一定基础。     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium.

Addition of cytochrome b₅ to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro. To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b₅ in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt⁻; and YG7108, ogt⁻, ada⁻). These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction). The four-fold increase in mutagenicity in the presence of cytochrome b₅ was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1. The greatest enhancement was obtained with a tricistronic construct in which the b₅ cDNA preceded the P450 and reductase cDNAs; placing the b₅ cDNA after the reductase cDNA was substantially less effective. These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for -complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

Abstract The role of helper elements in the mobilisation of pBR recombinant plasmids ( tra ⁻, mob ⁻, ori T⁺ and tra ⁻, mob ⁻, ori T⁻) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of ori T⁺ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an ori T⁻ pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR ori T⁻ plasmids in vitro.     

铁棍山药微型块茎遗传转化体系的建立

怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础, 但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv. ‘Tiegun’)的微型块茎为受体材料, 对影响遗传转化的因素进行优化, 建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体pCAMBIA1301-DoSERK2含GUS标记基因和潮霉素(Hyg)抗性筛选基因, 沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500 mg·L ^-1; 再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L ^-1, Kan的最佳浓度分别为120和160 mg·L ^-1。对转化植株进行PCR和GUS组织化学检测, 结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法, 对其它品种山药的转化也具有参考价值。     

Deinococcus misasensis and Deinococcus roseus, novel members of the genus Deinococcus, isolated from a radioactive site in Japan

Two gamma- and UV-radiation resistant, Gram-positive, red- or pink-pigmented, rod-shaped, strictly aerobic, oxidase- and catalase-positive bacterial strains, TDMA-25^T and TDMA-uv51^T, were isolated from fresh water collected at Misasa, a radioactive site in Japan. Phylogenetic analysis based on 16S rRNA gene sequences placed both in a distinct lineage in the family Deinococcaceae, and the highest degrees of sequence similarity determined belonged to Deinococcus maricopensis LB-34^T (88.8–89.3%), Deinococcus pimensis KR-235^T (86.4–86.7%) and Deinococcus yavapaiensis KR-236^T (86.1%). The DNA G+C content of the strains was 53–58 mol%. The major respiratory quinone was MK-8. The predominant fatty acids were C_15:0 iso, C_16:0 iso, C_13:0 iso, C_17:0 iso, C_16:0, C_13:0 anteiso, C_15:0 and C_12:0 iso. The strains degraded gelatin, casein, starch and Tween 80. Unique physiological characteristics, differences in their fatty acid profiles, and genotypic and phylogenetic features, differentiated strains TDMA-25^T and TDMA-uv51^T from closely related Deinococcus species. Hence, the two strains are described as novel species of the genus Deinococcus. The names Deinococcus misasensis sp. nov. (type strain TDMA-25^T=JCM 14369=NBRC 102116=CCUG 53610) and Deinococcus roseus sp. nov. (type strain TDMA-uv51^T=JCM 14370=NBRC 102117=CCUG 53611) are proposed.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.