黄曲霉菌原生质体的制备和再生技术优化

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×10^6个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。     

液泡ATP酶在肿瘤中的作用及机制

液泡ATP酶（vacuolar ATPases,V-ATPases）是真核细胞中高度保守的一类大型多亚基复合物,广泛分布于质膜及溶酶体、内体、囊泡等细胞内膜系统,能够借助水解ATP产生的能量控制H+的跨膜转运。V-ATPases通过对细胞内外多种结构的酸化作用调控着一系列重要的细胞活动,如膜运输、蛋白质加工和降解等。近年来,V-ATPases在肿瘤形成中的功能正逐渐成为研究热点。本文重点综述了V-ATPases通过调控细胞内外环境pH,从而在肿瘤发生过程中所行使的多种功能,例如V-ATPases抑制肿瘤细胞凋亡,参与肿瘤细胞自噬,促进肿瘤的侵袭、迁移与增殖,以及参与肿瘤耐药性的产生等。阐明V-ATPases在肿瘤中的作用机制有望为肿瘤治疗策略的探索、新型药物的开发以及相关科学研究的开展提供参考。     

植物钙依赖型蛋白激酶激活液泡膜上的阴离子通道

运用膜片钳技术研究植物钙依赖型蛋白激酶(CDPK)对细胞液泡离子通道的作用,发现它明显地激活液泡膜上一种电流方向与慢液泡(SV)通道电流相反的阴离子通道.用CsCl和GluK作通道阻断试验,结果表明它是一种Cl^-通道,单通道电导约为30pS,不同于SV的50～100 pS.对它在细胞信息传导中的作用作了分析.     

灰绿藜液泡膜焦磷酸酶基因的克隆及表达分析

采用同源克隆的方法,获得盐生植物灰绿藜的液泡膜焦磷酸酶基因(VP1)全长cDNA,命名为CgVP1。生物信息学预测分析表明,CgVP1基因包含一个2 292bp的开放阅读框,编码763个氨基酸。CgVP1不仅具有与植物液泡膜焦磷酸酶共有的氨基酸序列DVGADLVGKVE,而且CgVP1与其它植物的VP1相似性达86%。跨膜结构域预测显示,CgVP1氨基酸序列含有12个跨膜螺旋区,可能定位于细胞膜系统上。RT-PCR检测表明,200mmol/L NaCl条件下萌发生长的灰绿藜,再进行800mmol/L NaCl胁迫处理24h后,CgVP1基因表达显著增强。不同浓度KCl、CaCl2、MgCl2分别处理24h,KCl和MgCl2浓度增高,CgVP1基因表达下降,CaCl2则不影响CgVP1基因表达。研究结果表明,灰绿藜CgVP1基因表达对不同种类盐胁迫响应不同,NaCl胁迫可以上调CgVP1基因表达。该研究结果有助于阐明盐胁迫对盐生植物灰绿藜CgVP1基因表达的调控作用。     

小麦全蚀病菌原生质体制备的条件及再生菌株的致病性

分别利用崩溃酶、溶壁酶、纤维素酶和蜗牛酶酶解小麦全蚀病菌,进行原生质体的制备试验。结果显示,4种酶均能消化该菌细胞壁,获得一定数量的原生质体;产生原生质体效率最高的是溶壁酶,该酶在浓度为16 mg/mL时产生的原生质体数量最多,最佳的酶解时间为2~3 h,最适作用温度为28℃。制备的原生质体可以再生并与原始出发菌株具有相同的致病能力。     

斜卧青霉原生质体制备和再生的研究

研究了斜卧青霉原生质体制备和再生的条件,确定了培养方式、菌龄、渗透压稳定剂、酶的配比、酶解时间等因素对原生质体制备和再生的影响。最佳条件：菌丝体培养12h,用1％溶壁酶＋1％纤维素酶＋1％蜗牛酶的混合酶液酶解,将NaCl作为渗透压稳定剂,酶解时间为1．5h。在此条件下原生质体的形成量达到5．3×10^5个／mL,再生率为19．4％。     

基于响应面法对香菇原生质体制备条件的优化

香菇Lentinula edodes是我国食用菌年产量最大的单品,优化香菇原生质体的制备条件,获得高质量、高活力的原生质体可以为香菇育种和遗传多样性分析提供技术保障。本研究利用单因素试验对稳渗剂浓度、酶解液类型、酶解时间、酶解温度4个因素的最优作用条件和相关性进行检验和筛选;依据单因素试验的结果,对酶解时间、酶解温度、酶解液种类进行响应面法3因素3水平试验优化分析。单因素试验结果表明,使用2%的溶壁酶+蜗牛酶+纤维素酶混合酶制剂为酶解液、0.6mol/L甘露醇为稳渗剂的效果显著高于其他处理(P<0.05)。经Box-Behnken设计得出香菇原生质体最佳制备条件为酶解时间3.25h,酶解液浓度1.7%,酶解温度30.49℃,获得原生质体产量为14.02×10⁶ CFU/mL,与理论产量(13.862×10⁶ CFU/mL)偏差小,可为后续原生质体融合育种和多样性分析提供重要基础。     

丝状真菌AL18的原生质体制备和再生条件的优化

目的:为了建立起产苝醌类光敏剂丝状真菌AL18原生质体制备和再生体系。方法:采用单因素实验法研究了预处理方式、渗透压稳定剂和酶解条件对原生质体制备率和再生率的影响。结果:原生质体制备及再生的最佳条件是用0.3%的β-巯基乙醇预处理15 min,酶解液以0.6 mol/L的MgSO4·7H2O作为渗透压稳定剂,0.02 mol/L的磷酸盐缓冲液pH值为5.8,纤维素酶:蜗牛酶=2:3,酶的总浓度为15mg/mL,36℃酶解2h;以0.6mol/L的蔗糖作为再生培养基的渗透压稳定剂。结论:原生质体的制备率和再生率可分别达到1.42×107/mL和3.2%。     

巨大侧耳原生质体制备条件的优化

为优化巨大侧耳原生质体的制备条件,该研究以两株不同温型的巨大侧耳菌株PG46和PG79为材料,采用单因素和正交试验方法对原生质体制备的菌丝体菌龄、稳渗剂种类、溶壁酶浓度、酶解温度和酶解时间进行研究。结果表明:(1)在单因素实验中,巨大侧耳原生质体制备的适宜条件为菌龄5 d,溶壁酶浓度2.5%,0.6 mol·L^-1甘露醇,32 ℃(PG46)或27～35 ℃(PG79)酶解4 h。(2)正交试验验证并优化了单因素实验结果,组合2(菌龄5 d,溶壁酶浓度2.5%,0.6 mol·L^-1的甘露醇,32 ℃酶解4 h)可同时作为PG46和PG79原生质体制备的最适条件,原生质体产量分别为11.22×10⁶ CFU·mL^-1和7.28×10⁶ CFU·mL^-1。(3)F-test检验中,各因素对原生质体制备的影响程度依次为菌龄>溶壁酶浓度>酶解温度>酶解时间(PG46),菌龄>酶解时间>酶解温度>溶壁酶浓度(PG79)。综上所述,两株不同温型巨大侧耳菌株的原生质体制备条件基本一致,菌龄对两菌株原生质体得率的影响程度最显著。该研究结果可为后续巨大侧耳的杂交育种、遗传转化、全基因组测序等工作奠定基础,进一步推动巨大侧耳分子遗传学的发展。     

甘蓝下胚轴原生质体高效成株系统的建立

本文采用萌发六天的甘蓝(Brassica oleracea)下胚轴材料游离原生质体,经纯化后的原生质体产量为1.45×10~6g~(-1)FW,于含有1.5mg/L BA,0.5mg/L NAA和0.5mg/L 2,4-D的KM 8p的培养基中进行液体浅层培养。原生质体培养3—4天后出现一次分裂,七天时统计分裂频率为50.3%,培养15天左右可形成细胞团,3—4周后即可形成肉眼可见的小愈伤组织,统计形成愈伤组织的植板率为1.25%。将愈伤组织转至含有1.5mg/L BA和0.2mg/L 2,4-D的MS固体扩增培养基上进行扩增,其后可在含有2mg/L BA和0.5mg/L ZT的MS分化培养基上分化出芽,其分化率为54.17%,分化芽可于生根培养基中生根形成完整植株,移栽后,在人工气候室中生长良好。在试验过程中,对取材的不同时间,酶解液的不同配比对原生质体产量的影响,以及不同培养基、不同培养密度、不同的激素配比和不同培养方法等方面对原生质体的再生和持续分裂的影响进行了讨论。     

Immobilized Aspergillus sydowii produces xylanase

Summary Spores ofAspergillus sydowii, immobilized in 2.5% caleium alginate was used as inoculum in batch cultures for production of xylanase enzyme using xylan as the sole carbon source. Partially germinated mycelium from these entrapped spores produced significant amount of the enzyme in a short period of 24 hours and the same inoculum could be used repeatedly for at least 5 cycles with less than 10% loss of enzyme activity.     

米曲霉和黑曲霉营养缺陷型的分离及原生质体的制备

米曲霉(Aspergillus oryzae)3042是目前国内酱油生产中广泛使用的菌种,而黑曲霉(Aspergillus niger)3350则是制醋业中广泛使用的菌种。前者具有较高的蛋白酶活性而后者具有较高的淀粉酶活性。在酱油生产中,为了提高原料利用率,改善酱油风味,希望获得一株既有较高的蛋白酶活性同时又具有较高淀粉酶活性的杂交菌株作为     

Studies on Isolated Protoplasts and Vacuoles: I. GENERAL PROPERTIES

Protoplasts and vacuoles have been isolated in large quantitiesfrom the parenchymatous placental tissue of immature tomatofruit. Their mechanical stability and osmotic stability havebeen investigated. A small proportion of initially discretebut adjacent vacuoles were seen to coalesce; the possible significanceof this phenomenon in the growth of plant cells is discussed.The overall chemical composition of isolated protoplasts hasbeen determined, and their lipids have been analysed by thin-layerchromatography.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Permeability Properties of Aspergillus nidulans Protoplasts

The regeneration of mycelial forms from protoplasts of mutant and wild-type strains of Aspergillus nidulans was followed microscopically under conditions with which the rate of regeneration depends upon a specific transport function. This method has shown that at least four uptake systems-one for divalent anions, two for purines, and one for sugars-remain functional after removal of the cell wall.     

Purification and Some Properties of a Xylanase from Aspergillus sydowii MG49

Volume 62, no. 10, p. 3915, Author's Correction (Ghosh and Nanda). Errors were made in the original identification of the microorganism used in this study and in the reporting of the corrected species identification. The culture resubmitted to the American Type Culture Collection was identified by Dr. Frank Dugan as Aspergillus flavus, not Aspergillus citrinum, and is available as ATCC 96916. [This corrects the article on p. 3915b in vol. 62.].     

Purification and Some Properties of a Xylanase from Aspergillus sydowii MG49

Volume 60, no. 12, p 4620-4623: errors appear to have been made in the original identification of the microorganism used in this study and in the cultures submitted in 1993 to both the American Type Culture Collection (ATCC) and the Indian Institute of Microbial Technology. A resubmitted culture has now been assigned to the species Aspergillus citrinum by Dr. Frank Dugan of the ATCC. The authors have confirmed that the resubmitted culture produces xylanase, so that the major findings of the original publication remain valid. [This corrects the article on p. 4620 in vol. 60.].