秀珍菇单孢子菌株原生质体制备条件的优化

以秀珍菇单核菌丝为材料,用溶壁酶进行原生质体的制备,考察各因素对原生质体产量的影响,用单因素试验和正交试验确定最佳条件。结果表明,菌龄为8 d菌丝体,0.6 mol/L甘露醇+10 mmol/L Tris-HCl为稳渗剂,酶解温度29℃、酶浓度1.6%的条件下酶解160 min。原生质体产量达到3.5×10~7 CFU/m L,原生质体再生率达到1.4%。     

大球盖菇原生质体制备与再生条件研究

研究了大球盖菇(Stropharia rugoso-annulata)菌丝原生质体制备和再生条件,结果表明:在30℃、pH5.5、0.6mol/L甘露醇、1.5%溶壁酶条件下对培养3d的菌丝体酶解2.5h,原生质体产量达到1.36×107个/mL。原生质体适宜再生培养基为马铃薯200g,蔗糖20g,蛋白胨2g,酵母粉2g,KH2PO41.5g,K2HPO41.5g,MgSO4.7H2O 1.5g,维生素B10.1g,维生素B60.1g,0.6mol/L甘露醇。采用上述培养基,双层平板法进行再生试验,再生率达到0.96%。为大球盖菇原生质体技术深入研究和应用奠定了基础。     

PEG介导的柱状田头菇遗传转化体系建立

柱状田头菇（茶树菇）Agrocybe aegerita是一种美味的食用菌,具有极高的经济价值。随着其全基因组测序的完成,功能基因组学研究也逐渐展开,其中,高效的遗传转化体系作为技术基础成为研究重点。本研究以柱状田头菇原生质体为受体、潮霉素抗性基因（hph）作为筛选标记,以增强型绿色荧光蛋白基因（egfp）为报告基因,应用PEG介导法进行柱状田头菇遗传转化体系研究。结果表明,150μg/mL潮霉素可以完全抑制柱状田头菇的生长。30℃下用2%裂解酶液酶解菌丝3h,能够获得最大得率的原生质体。通过PEG介导将构建好的DNA片段转化入柱状田头菇原生质体,通过潮霉素抗性筛选获得转化子,转化得率达到7个/μg DNA。PCR验证和荧光显微镜观察,外源片段成功转入柱状田头菇中并稳定表达。本研究建立的PEG介导转化体系,为柱状田头菇基因功能研究提供了技术基础。     

玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

去壁酶与酶解方式对曲霉原生质体释放的影响

研究了纤维素酶、蜗牛酶、溶菌酶以及菌丝体培养方式和酶解方式对黑曲霉和米曲霉菌丝释放原生质体的效应。发现黑曲霉菌丝原生质体制备最佳条件为固体透析培养菌丝体,2%纤维素酶,在平皿中,28℃和80r/min条件下酶解3h;米曲霉原生质体制备最佳条件为2%纤维素酶+1%蜗牛酶+5mmol/L二硫苏糖醇,酶解时间6h,其它条件与黑曲霉的相同。     

纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化

旨在优化深绿木霉(Trichoderma atroviride)菌株HP35-3原生质体制备和转化条件,便于对该菌株进行遗传操作以提高其纤维素酶产量。分别对制备深绿木霉原生质体的菌龄、酶解时间、酶组分及比例和转化条件进行优化。结果显示,利用3mg/m L蜗牛酶、3 mg/m L溶菌酶和3 mg/m L裂解酶酶组分酶解菌龄10 h的菌丝2 h,获得的原生质浓度达到3.5×107个/m L以上,原生质体再生率为61%。利用原生质体进行PEG介导转化,当原生质体浓度为1×108个/m L、外源DNA为5μg时,转化率达到35个转化子/μg DNA。建立的高效原生质体制备及转化体系可用于深绿木霉的遗传转化及菌株改造。     

狗头枣叶片原生质体分离研究

以培养25d的狗头枣试管苗叶片为材料,研究了不同酶液浓度,不同甘露醇浓度对狗头枣组培苗叶片原生质体分离的影响.结果表明适合狗头枣组培苗叶片原生质体分离的适宜酶液配比为1.0 g/L纤维素酶、0.4 g/L果胶酶,0.6 mol/L甘露醇,黑暗条件下,酶解8h,可获得大量有活力的原生质体.其叶片原生质体的产量为2.12×106个/g,活原生质体获得率为80.88％.     

丹参悬浮培养细胞原生质体的制备和活力检测

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针Fluo-3/AM装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%和离析酶0.5%;适宜的甘露醇浓度为0.4 mol/L;酶解时间为12 h;在600 r/min转速下离心5 min收集,纯化得到原生质体,其产量为1.1×106/g FW,FDA检测显示其活力为95%以上,荧光探针Fluo-3/AM可成功装载到原生质体中。     

大丽轮枝菌原生质体的制备及再生

为建立大丽轮枝菌(Verticillium dahliae)原生质体的制备和再生体系,采用单因素分析法分析了大丽轮枝菌摇培时间、裂解酶浓度、酶解时间和温度、渗透压稳定剂的种类和浓度及pH对原生质体释放的影响;从再生培养基、培养基Agar浓度和酶解时间对原生质体的再生条件进行了优化;并对优化条件下获得的原生质体进行了GFP瞬时表达。结果表明,将分生孢子培养18 h收集菌丝体,以1.2 mol/L的KCl作为渗透压稳定剂,在pH 6.0的条件下,加入10 mg/m L的裂解酶,30℃酶解4 h时,获得的原生质体产量最高,达到3.3×10~7个/mL;在Agar浓度为0.5%的TB3再生培养基中进行原生质体再生,再生率最高,可达22.45%;GFP瞬时表达结果表明,优化条件下获得的原生质体可用于遗传转化材料。     

卷枝毛霉孢子原生质体的制备与再生

为了构建高产γ-亚麻酸的卷枝毛霉稳定遗传转化体系,利用酶解法对卷枝毛霉(Mucor circinelloides sp.)EIM-10的孢子进行原生质体制备。研究酶液组成、渗透压稳定剂、酶解温度、酶解时间等对卷枝毛霉孢子原生质体形成和再生的影响,建立了制备卷枝毛霉孢子原生质体的最适条件:1%纤维素酶和2%溶壁酶为酶解体系,0.5mol/L NaCl作为渗透压稳定剂,酶解温度32℃,酶解时间2.5 h,再生培养基为0.5 mol/L NaCl高渗培养基。用双层平板培养法进行原生质体再生,在此条件下原生质体的形成量为1.2×106个/mL,再生率为70.5%。     

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

Molecular genetics of carbon-phosphorus bond cleavage in bacteria

Phosphonates (Pn) are a large class of organophosphorus molecules that have direct carbon-phosphorus (C - P) bonds in place of the carbon-oxygen-phosphorus ester bond. In bacteria two pathways exist for Pn breakdown for use as a P source: the phosphonatase and C - P lyase pathways. These pathways differ both in regard to their substrate specificity and their cleavage mechanism. The phosphonatase pathway acts on the natural Pn -aminoethylphosphonate(AEPn). In a two-step process it leads to cleavage of the C - P bond by a hydrolysis reaction requiring an adjacent carbonyl group. In contrast the C - P lyase pathway has a broad substrate specificity. It leads to cleavage of substituted Pn (such as AEPn) as well as unsubstituted Pn by a mechanism involving redox or radical chemistry. Due to its broad substrate specificity, the C - P lyase pathway is generally thought to be responsible for the breakdown of Pn herbicides (such as glyphosate) by bacteria. As a way to gain a more in-depth understanding of these Pn degradative pathways, their respective genes have been isolated and characterized. In the absence of a biochemical assay for the C - P lyase pathway such molecular approaches have been especially valuable. The roles of individual genes have been inferred from DNA sequence analysis and mutational effects. Genes for the C - P lyase pathway exist in a fourteen-gene operon that appears to encode both a binding protein-dependent Pn transporter and a C - P lyase. Genes for the phosphonatase pathway also exist in a gene cluster containing Pn uptake and degradative genes. A combination of biochemistry, molecular biology, and molecular genetics approaches has provided more detailed understanding of the mechanisms of C - P bond cleavage. Such basic information may provide a new handle for improvement of Pn degradation capabilities in bacteria, or in other cells in which the respective genes may be introduced and expressed.Abbreviations AEPn -aminoethylphosphonate - C carbon - kbp kilobase pair - kDa kilodalton - MPn methylphosphonate - P phosphorus - P _i inorganic phosphate - Pn phosphonate - psi phosphate starvation inducible     

桃褐腐病菌(Monilia fructigena)原生质体制备及再生条件

以桃褐腐病菌(Monilia fructigena)为供试菌株,研究了酶系组成、液体培养基、菌龄、酶解温度、酶解时间对原生质体制备的影响,以及等渗液、固体再生培养基、酶解时间对原生质体再生的影响。结果表明:Fries(1/2)液体培养基培养24h,在10mg/mL崩溃酶+5mg/mL纤维素酶+20mg/mL蜗牛酶+10mg/mL溶菌酶的混合酶液中28°C酶解4h为桃褐腐病菌原生质体制备的最佳条件。采用液体再生涂布平板法,以含Ca2+的STC为等渗液的液体培养基和含蔗糖及Ca2+的Fries(1/2)固体培养基为桃褐腐病菌原生质体再生的最佳条件。经过观察与测定,再生菌株保持了原有的培养性状和致病性,接种桃果实后发病率为100%。     

Photosynthetic efficiency and nitrogen distribution under different nitrogen management and relationship with physiological N-use efficiency in three rice genotypes

Nitrogen fertilization strategies were widely adopted to enhance grain production and improve nitrogen utilization in rice all over the world. For fertilization timing strategy, ear fertilization was usually employed in recent years. For fertilization amount strategy, nitrogen fertilization would continually increase to meet the demands of increasing people for food. However, under heavy ear fertilization as well as great nitrogen amount (NA), physiological N-use efficiency (PE, defined as grain production per unit nitrogen uptake by plants) decreased. Under three NA and two ratios of fertilization given during ear development period to total NA (ear fertilization distribution ratio, EFDR), net photosynthetic rate (Pn), Pn to nitrogen content per unit area (photosynthetic N-use efficiency, Pn/N), nitrogen accumulation in plant tissues and PE of three rice (Oryza sativaL.) genotypes, Jinyou 253, Liangyoupeijiu and Baguixiang were screened in the first and second seasons in 2002 so as to understand the fluctuation patterns of Pn/N and nitrogen distribution in leaf blades under great NA & EFDR and relationship with PE in rice. Results showed that under greater NA & EFDR, Pn in flag leaves at heading and plant nitrogen accumulation at maturity always increased and PE & Pn/N always decreased in spite of increased grain production. Rice distributed more nitrogen in leaf blade under greater NA and EFDR. PE indicated significantly (P<0.05) positive relationship with Pn/N and negative relationship with nitrogen distribution ratio in leaf blades at heading and maturity, and no association with Pn in two growing seasons. Results suggested that low PE in rice under great NA and heavy ear fertilization is associated to more nitrogen distribution in leaf blades and decreases in photosynthetic efficiency.     

二倍体马铃薯叶肉细胞原生质体培养的研究

以马铃薯抗青枯病二倍体材料ED13和CE171、炸片颜色好的二倍体材料HS66以及优良性状双单体材料DH401和DH405为供体材料,对马铃薯叶肉原生质体培养进行研究。叶片悬浮黑暗预处理和试管苗黑暗预处理两种预处理方式对原生质体活力无显著影响。以0.5 mol/L甘露醇为渗透调节剂,25℃酶解12 h条件下,适宜CE171和DH401纤维酶浓度略高于ED13、HS66和DH405,分别为0.3%和0.4%。ED13和DH401原生质体在VKM液体培养基中培养3～4周,经愈伤组织生长培养基培养2周,转至芽诱导培养基培养,2～3个月后形成具根茎叶的完整植株。HS66和CE171原生质体培养6～8周也能形3～4 mm愈伤组织,但没有分化出芽;DH405的原生质体不分裂。     

A study of autologous anti-idiotypic antibody-forming cells in mice of different ages and genetic backgrounds.

Antibody response to phosphorylcholine, an immunodominant epitope of Streptococcus pneumoniae R36a (Pn), is characterized by a public idiotype, T15, that is expressed on a large proportion of antibody molecules produced by all mouse inbred strains. The ability of the immune system to produce an autologous antibody to T15 upon immunization with Pn vaccine was investigated using a modified ELISA plaque assay for detection of single antibody-forming cells (AFC). The limit of ELISA assay for detection of specific anti-T15 AFC is approximately 300 cells/spleen. However, our studies failed to detect any autologous anti-T15 AFC in the course of the primary antibody response to Pn vaccine in young/adult (2-4 months) BALB/c and C57BL/6 mice. Aged mice (20-22 months) also failed to develop any specific auto-anti-T15 AFC upon the primary Pn immunization, despite the fact that the anti-Pn response in these animals changes both quantitatively and qualitatively. In order to generate specific anti-T15 AFC, BALB/c mice had to be immunized repeatedly with Pn vaccine (four weekly injections) or immunized directly with T15 protein in CFA. Different results were obtained with D1.LP mice that are low responders to Pn and express lower levels of T15 Id as compared to BALB/c. Young D1.LP mice produced high numbers of auto-anti-T15 AFC of both IgM and IgG isotypes following a single immunization with Pn vaccine. The kinetics of auto-anti-T15 response in D1.LP mice was similar to that of the antigen-specific response. These results demonstrate that the ability of the immune network to produce autologous antibody to a shared Id depends on the genetic makeup of the host, and that this response may be regulated by the level of Id expression.     

酿酒酵母W5及休哈塔假丝酵母20335原生质体制备条件的确定

确定了酿酒酵母W5及休哈塔假丝酵母20335原生质体制备的最佳条件。选取不同脱壁预处理时间及不同酶解时间,对酿酒酵母W5、休哈塔假丝酵母20335进行原生质体制备和再生,比较制备率和再生率。确定脱壁预处理30 min后,以终浓度2%的蜗牛酶,30℃、100 r/min酶解处理15 min为双亲株原生质体制备的最佳条件。利用原生质体融合的方法,以酿酒酵母W5和休哈塔假丝酵母20335为亲本株,构建可以利用木糖生产生物乙醇的新型酿酒酵母融合株,该前期工作为W5、20335原生质体融合工作奠定了重要的基础,对于将木质纤维素原料转化为生物乙醇的研究具有极其重要的意义。     

紫外诱变原生质体选育低温碱性脂肪酶高产菌株

以产低温碱性脂肪酶约氏不动杆菌(Acinetobacter johnsonii)LP28为出发菌株,采用EDTA和溶菌酶处理制备原生质体.确定其最佳处理条件为37℃的水浴下,以终浓度为0.15 mg/mL的溶菌酶处理45 min,最终可获得90%的原生质体形成率及0.9%左右的再生率.采用紫外诱变原生质体的方法,筛选得...     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.