T-DNA transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

Agrobacterium tumefaciens-mediated DNA transformation method was applied to transformNodulisporium sylviforme fusant HDF-68,a taxol-producing fungus.We constructed a binary vectorpBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA.Optimal co-cultivation of N.sylviforme with A.turnefaciens containing pBI121-43 led to 110～130 hy-gromycin-resistant transformants per million conidia.Putative transformants were found to be mitoticallystable.The molecular analysis of transformants demonstrated the random integration of single copy of theT-DNA into the host genome.This transformation system serves as a basic tool for insertional mutagenesisin N.sylviforme fusant HDF-68,and the development of such system lays a solid foundation for con-structing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Reducing Yield of Fusel Alcohols by Saccharomyces cerevisiae of Compound Mutagenesis Through UV-MPMS Method

Excessive fusel alcohol contents will cause the beer to produce off-flavors and cause dizziness and headaches.Reducing the contents of fusel alcohols in beer is very important to people's health.The excessive fusel alcohol contents in beer is a common problem in the industry.How to control the contents of fusel alcohols in a reasonable range is of great significance for improving beer quality.After one round of ultraviolet(UV)and one round of multifunctional plasma mutagenesis system(MPMS)mutagenesis,the yeast strains with lower fusel oil yield and more stablility could be screened.According to the relationship between the fusel alcohol Harris metabolic pathway of brewer's yeast and lactic acid metabolism,excellent strains were obtained by triple screening with lactic acid medium,calcium carbonate medium and 2,3,5-triphenyl tetrazolium chloride upper medium.The content of fusel alcohol in the finished beer fermentation test of screened strain Z43 was 52.1±0.142 mg•L^(-1),which was 43%lower than that of the starting strain,and other fermentation properties remained unchanged.After eight passages,it was verified that the strain was stable and heritable.These results showed that strain Z43 presented promising characteristics for use in the production of beer with a potentially low contents of fusel alcohols.

Stem cell gene therapy:the risks of insertional mutagenesis and approaches to minimize genotoxicity

Virus-based vectors are widely used in hematopoietic stem cell(HSC)gene therapy,and have the ability to integrate permanently into genomic DNA,thus driving long-term expression of corrective genes in all hematopoietic lineages.To date,HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency(SCID-X1),adenosine deaminase deficiency(ADA-SCID),adrenoleukodystrophy(ALD),thalassemia,chronic granulomatous disease(CGD),and Wiskott-Aldrich syndrome(WAS).However,adverse events were observed during some of these HSC gene therapy clinical trials,linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia.Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity,with the aim of developing safer vectors and lower-risk gene therapy protocols.This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors,discuss the available assays for predicting genotoxicity and mapping vector integration sites,and introduce newlydeveloped approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Effect of ultraviolet mutagenesis on heterotrophic strain mutation and bioleaching of low grade copper ore

The effect of ultraviolet mutagenesis on a heterotrophic strain(Providencia JAT-1) mutation was studied and bioleaching of low grade copper ore with mutant bacteria was investigated. The results show that the activity of bacteria was improved after ultraviolet mutagenesis; the best irradiation time was 120 s. Compared to the original bacteria, the cells density of mutant bacteria at stationary phase increased by 26% and ammonia produced by mutant bacteria increased by 12%. Higher activity of bacteria leads to a higher copper extraction rate. The bioleaching performance of Providencia JAT-1 was improved after UV mutagenesis. The copper extraction rate with mutant bacteria increased by 10.6% compared to the original bacteria. The ore surface was corroded and the fine particles were absent after bioleaching. Free copper oxide and copper silicates could be leached out easily by using JAT-1; a small part of the copper sulfide can also be leached out. Bioleaching using JAT-1 is more effective than ammonia leaching and copper extraction rate with mutant bacteria was 21.1% higher than that by ammonia leaching under the same condition.

Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and pro B Gene Deletion in Corynebacterium crenatum

Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.

Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation

Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.

Ethyl Methane Sulphonate (EMS) Induced Mutagenesis in Malaysian Rice (cv. MR219) for Lethal Dose Determination

Chemical and physical mutagenesis has been used to increase genetic variability in crop plants. More than 430 new varieties have been derived as mutants of rice (Oryza sativa L.) via the application of different mutagenic agents. Chemical mutagens such as ethyl methane sulphonate (EMS), diepoxybutane-derived (DEB), sodium azide and irradiation (Gamma rays, X-rays and fast neutrons) have been widely used to induce a large number of functional variations in rice and others crops. Among chemical mutagens, the alkylating agent, ethyl methane sulfonate (EMS) is the most commonly used in plants as it causes a high frequency of nucleotide substitutions, as detected in different genomes. In this study, seeds of potential genotype of the popular variety, (Oryza sativa L. spp. Indica cv. MR219) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1%, 1.25%, 1.5% and 2%. Sensitivity to EMS was determined by various measurements on the M1 generation. As concentration of applied EMS increased, will decrease in germination, seedling height, root length and emergence under field conditions was observed in M1 generation as compared to the non-treatment control. Plant height and root length also decreased with increases in EMS mutagenesis in an approximately linear fashion. The LD25 and LD50 values were observed based on growth reduction of seedlings after EMS treatment with 0.25% and 0.50% on the rice variety (Oryza sativa L. spp. Indica cv. MR219).

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane(Saccharum officinarum L.)

Gamma ray-induced in vitro mutagenesis and selection for salt(NaC l) tolerance were investigated in sugarcane(Saccharum officinarum L.). Embryogenic callus cultures were irradiated(10 to 80 Gy) and subjected to in vitro selection by exposure of irradiated callus to NaC l(0, 50, 100,150, 200, and 250 mmol L-1). Increasing NaC l concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+and K+concentration. Higher accumulation of proline and glycine betaine was observed in NaC l stressed callus irradiated at 20 Gy. Na+concentration increased and K+concentration decreased with increasing salt level. Irradiated callus showed50–60% regeneration under NaC l stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%,number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

Quantitative evaluation of DNA damage caused by atmospheric and room-temperature plasma (ARTP) and other mutagenesis methods using a rapid umu-microplate test protocol for microbial mutation breeding

Mutagenesis is an important technique for microbial mutation breeding.As the source of mutations,DNA damage extent is a key indicator for the effectiveness of mutagenesis.Therefore,a rapid and easy DNA damage quantification method is required for the comparison of mutagenesis effects and development of mutagenesis tools.Here,we used the umu-microplate test system to quantitatively compare the DNA damage strength caused by atmospheric and room-temperature plasma(ARTP)and other traditional mutagenesis methods including:ultraviolet radiation(UV),diethyl sulfate(DES)and 4-nitroquinoline-1-oxide(4-NQO).The test strain of Salmonella typhimurium TA1535/pSK1002 was used to monitor the time-course profile of b-galactosidase activity induced by DNA damage caused by different mutagenesis methods using a microplate reader.The umu-microplate test results showed that ARTP caused higher extent of DNA damage than UV and chemical mutagens,which agrees well with the result obtained by SOS-FACS-based quantification method as reported previously.This umu-microplate test is accessible for broad researchers who are lack of the expensive FACS instruments and allows the quick quantitative evaluation of DNA damage among living cells for different mutagenesis methods in the study of the microbial mutation breeding.

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Development of peanut varieties with high oil content by in vitro mutagenesis and screening

Peanut (Arachis hypogaea L.) is an important oil crop globally and high oil content is one of the major targets in peanut breeding programs. Previous studies indicated that the osmotic pressure (OP) of the leaves of peanut plants subjected to drought stress was negatively correlated with kernel oil content. Based on this knowledge, we established a practical and reliable method for creating new peanut varieties with high oil content using in vitro mutagenesis and directional OP-based selection. Using embryonic leaflets of peanut variety Huayu 20 as explants, pingyangmycin (PYM) as the mutagen, and hydroxyproline (HYP) as the OP regulator, we developed 15 HYP-tolerant regenerated plants. For each regenerated plant, we selected offspring with oil content>55% (relative to 49.5% for Huayu 20). We developed and released three new peanut varieties with high yield and high oil content from the offspring of the HYP-tolerant regenerated plants. The three new varieties were named as Yuhua 4, Yuhua 9 and Yuhua 14 and their oil contents were 57.7, 61.1 and 59.3%, respectively. The results indicate that in vitro mutagenesis with PYM followed by directed screening with HYP is a useful approach for breeding peanut varieties with high oil contents.

Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.

Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9(CRISPR-associated protein9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na^+ /K^+ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na^+ efflux but also of K^+ and Ca^(2+) influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.

A Dynamic Model of Heavy Ion ~7Li Irradiation Mutagenesis Based on Maize Inbred Line Nutrition Difference

To reveal the saddle-type dose effect relationship, we propose a radiation mutagenesis model based on maize nutrition difference resulting from heavy ion ~7Li radiation. Through irradiation mutagenesis, apparent trait selection, amino acids and fatty acids content determination, and modeling, dynamic evolution from microscopic damage and repair initiation to the final macroscopic biological effects are considered simultaneously. The results show that the steady state nature is independent of evolution time and only relates to different radiation doses.Heavy ion ~7Li radiation could effectively cause maize phenotypic variation and could improve nutritional quality.This model not only gives a good fit to the experimental results on most types of amino acids and fatty acids, but also offers an adequate explanation of the experimental phenomenon underlying the saddle-type bimodal dose effect. By combining experimental results with theoretical analyses, we suggest that the synergy of the stimulus effect and momentum transfer is the main cause of the saddle-type dose effect bimodal curve. This provides an effective strategy for conducting maize germplasm innovation.

Mutagenesis of T richoderma V iride by Ultraviolet and Plasma

Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet （UV） and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

Mutagenesis of the Bacillus edaphicus Strain NBT and Its Effect on Growth of Chili and Cotton

A strain NBT capable of dissolving silicate minerals and promoting plant growth was treated with UV+LiCl. Thirty-two mutants tolerable to 2% NaCl solution were obtained. However, through the survival experiments in high osmatic pressure, high temperature and different acidities, two mutants of NBT-6 and NBT-19 were finally obtained. They could survive from 10% NaCl solution and tolerate 55℃, acidic (pH 4) and alkalic (pH 10) conditions. The mutants had the same ability to release K from silicate minerals as the starting strain NBT. Pot experiments with chili and cotton showed that both the mutants developed in the rhi-zosphere soils. The available P and K contents in the rhizosphere soils and plant biomass increased through inoculating these bacteria.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean(Glycine max(L.) Merr.) to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four-and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCNinfection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wildtype soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Identification of Residue Involved in Nucleotidyltransferase Activity of LinA from Staphylococci by Site-directed Mutagenesis

The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.