Improvement of antagonism and carbendazim tolerance in native Trichoderma isolates through ethyl methane sulfonate (EMS) mutagenesis

Genetic enhancement of TCT₄ and TCT₁₀ was aimed in the present paper. Trichoderma reesei (TCT₁₀/M₁₈) mutant isolate evolved by ethyl methane sulfonate mutations was found to exhibit altered properties compared to its parent isolates. This mutant grew well in the potato dextrose agar (PDA) medium containing carbendazim (50 ppm). RAPD-PCR results suggested the uniqueness of mutants, which was useful in differentiating mutant and wild Trichoderma isolates. These mutants established well in the rhizosphere of rough lemon seedlings. The seedlings treated with carbendazim followed by an application of carbendazim-resistant mutant (TCT₁₀/M₁₈) resulted in a better seedling emergence and a less dry root rot disease caused by Fusarium solani in nursery conditions.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

Physical and chemical mutagens improved Sporotrichum thermophile,strain ST20 for enhanced Phytase activity

ObjectivePhosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi.MethodologyIn current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile.ResultsWe report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production.ConclusionThe current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.     

Characterization of a high‐growth‐rate mutant strain of Pyropia yezoensis using physiology measurement and transcriptome analysis

A mutant strain of Pyropia yezoensis, strain E, was isolated from the free‐living conchocelis of a pure strain (NA) treated with ethyl methane sulfonate. The incremental quantities of young strain E blades were higher than those of NA after 14 d of cultivation, indicating that young blades of mutant strain E released more archeospores. The mean length and weight of large E blades were both over three times greater than those of NA after 4 weeks of cultivation. The photosynthetic parameters (Fv/Fm, Y[I], Y[II], and O₂ evolution rate) and pigment contents (including phycoerythrin and phycocyanin) of strain E blades were higher than those of NA (P < 0.05). The cellular respiratory rate of strain E blades was lower than that of NA (P < 0.05). In order to investigate the causes of changes in strain E blades, total RNA in strain E and NA blades were sequenced using the Illumina Hiseq platform. Compared with NA, 1,549 unigenes were selected in strain E including 657 up‐regulated and 892 down‐regulated genes. According to the physiology measurement and differentially expressed genes analysis, cell respiration in strain E might decrease, whereas anabolic‐like photosynthesis and protein biosynthesis might increase compared with NA. This means substance accumulation might be greater than decomposition in strain E. This might explain why strain E blades showed improved growth compared with NA. In addition, several genes related to stress resistance were up‐regulated in strain E indicating that strain E might have a higher stress resistance. The sequencing dataset may be conducive to Pyropia yezoensis molecular breeding research.     

化学诱变剂EMS对知母种子萌发的影响

以知母（Anemarrhena asphodeloides）种子为材料,分别采用1%、2%、4%、6%浓度的EMS处理6、8、12和24 h,在恒温培养箱内进行种子萌发实验,研究不同浓度的化学诱变剂甲基磺酸乙酯（ethyl methane sulfonate,EMS）处理对知母种子萌发的影响,筛选适宜的诱变浓度和诱变时间。结果表明：随EMS浓度的增加和处理时间的延长,知母种子发芽率呈现逐渐降低的趋势,以相对发芽率达到半致死浓度为标准,6%EMS浸种12和24 h可作为EMS诱导知母建立突变体库的适宜条件。     

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

花生EMS诱变后代的农艺性状与品质分析

为了研究花生EMS诱变后代的变化趋势,以期得到有益变异和创造新的花生种质,本研究以2个花生品系花U416、花U606为供试诱变亲本,利用0(CK)、0.5%、1.0%、1.5%、2.0%5个EMS浓度直接注入花生花器得到诱变后代,分析M1、M2的农艺性状与品质变化,结果表明,EMS对2个花生品系诱变效应明显,2个品系畸变率随EMS浓度增加而增大;EMS注入的当代植株结实率和M1的出苗率均低于对照,2个品系M2均产生了多种类型的突变植株,突变性状大部分可以稳定遗传。而这些遗传多样性的突变材料,为花生种质创新及品种遗传改良提供基础性材料。     

瑞氏木霉外源基因表达系统中受体菌株的改造

将解除了葡萄糖阻遏的纤维素酶高产菌株瑞氏木霉TrichodermareeseiRutC30经EMS诱变后,在酪蛋白平板上筛选出部分蛋白酶缺陷突变菌株T.reeseiRutC30M3。RutC30M3的胞外蛋白酶活力比亲本株降低约74%,但生长特性、产纤维素酶活力等性状与亲本株相同。用携带潮霉素磷酸转移酶基因(hph)的表达质粒pAN7-1分别转化瑞氏木霉RutC30和RutC30M3原生质体,转化结果显示RutC30M3(pAN7-1)对潮霉素的抗性比RutC30(pAN7-1)提高约20%,而Northernblot分析结果显示在两菌株中潮霉素基因转录水平相似,由此证明宿主菌蛋白酶缺陷对保护重组蛋白免于降解有明显作用。蛋白酶缺陷菌株RutC30M3适于作为瑞氏木霉外源基因表达系统中的受体菌株用于大量生产同源与异源蛋白。     

生命周期管理研究述评

生命周期管理起源于生命周期思想,它是生命周期思想在实践中的具体应用,是面向可持续生产和消费,对产品、工艺和服务的全生命周期环境影响进行的综合管理,是解决复合生态系统中结构无序、效率不高和代谢冗余的有效途径,是基于生命周期评价原则与框架的一种环境管理手段或环境管理体系。全面回顾了生命周期管理的起源与内涵,阐述了生命周期管理与生命周期评价的区别与联系,梳理了生命周期管理与环境管理体系的关系。对生命周期管理在产品、企业、行业及城市等层次上的具体应用进行了总结与述评,并对其今后需深入研究的方向进行了展望。