解脂亚罗酵母P12单倍体的制备

【背景】目前解脂亚罗酵母在实验研究和工业生产方面的应用越来越广泛,但相较于常规酵母而言,解脂亚罗酵母缺乏简便有效的遗传转化体系,致使其在基因表达调控方面存在较大困难。同时,酵母的染色体倍性也会对基因敲除效果产生影响,选择单倍体细胞作为功能基因改造的受体可以避免等位基因之间相互作用的影响,解决多倍体细胞基因敲除不完全的问题。【目的】以解脂亚罗酵母诱变菌株P12为研究对象,以不同方法分离得到单倍体菌株,建立解脂亚罗酵母单倍体的制备方法。【方法】分别采用固体和液体McClary产孢培养基诱导解脂亚罗酵母菌株产生子囊孢子,培养条件为30℃,固体7-14 d;液体200 r/min,2-4 d。以2%浓度的蜗牛酶33℃水浴裂解子囊孢子细胞壁3 h,通过染色镜检和PCR鉴定筛选单倍体细胞。【结果】镜检结果表明,解脂亚罗酵母在液体产孢培养基中产孢速度较快,相同视野下孢子数约为固体产孢培养基的3.7倍,在固体产孢培养基中产孢质量较好。初步探索并筛选得到6株解脂亚罗酵母P12 B型单倍体菌株。【结论】解脂亚罗酵母P12 B型单倍体菌株的获得可为后续继续开展基因工程操作奠定基础。     

稗草叶枯病病原尖角突脐孢菌的鉴定

采用形态学及分子生物学的方法对采集自湖南和北京的3株尖角突脐孢菌分离物进行了鉴定。结果表明,3株分离物(KF-1、HN-14和K-12)和保存于中国农业大学已定名的尖角突脐孢菌菌株G-9和X-27之间在菌落形态、产孢量及孢子大小和分隔方面存在较大差异。其中,K-12在PDA培养基上生长缓慢、产孢量小;菌株G-9、KF-1、X-27和HN-14生长迅速,产孢丰富。对菌株进行分子鉴定结果表明,菌株间ITS区序列相似度达98%以上,聚类分析也表明,种内各菌株之间的遗传距离明显小于种间的遗传距离;基于ITS1及ITS2序列,能将尖角突脐孢菌和突脐蠕孢属中其它种分开。由此可确定分离自湖南水稻田及中国农业大学科学园温室中自然发病的稗草病样上的3株病原真菌均为尖角突脐孢菌。     

蜡蚧菌Lecanicillium araneicola HK-1的鉴定及其对豆蚜的生物防治潜力

【目的】蚜虫是农业生产中最具破坏性的害虫之一,每年造成巨大的农业经济损失。本研究旨在对从死亡蚜虫虫体上分离获得的一株真菌HK-1进行鉴定,分析其生物学特性,并测定其分生孢子对豆蚜Aphis craccivora成蚜的毒力及其对常用化学农药的敏感性,以期为蜡蚧菌Lecanicillium的大规模生产和应用提供理论基础。【方法】结合形态特征和多基因联合分析对HK-1菌株进行鉴定;通过单独改变培养基、pH、碳源和氮源来测定菌株的菌丝生长速率、产孢量和孢子萌发率;采用浸渍法测定孢子悬浮液对豆蚜成蚜的致死率和致死中时(median lethal time, LT₅₀);采用生长速率法测定常用农药对该菌株的半效应浓度(median effective concentration, EC₅₀)。【结果】经鉴定本研究分离的菌株HK-1为蜡蚧菌Lecanicillium araneicola HK-1;生物学特性研究结果表明,L.araneicola HK-1在马铃薯葡萄糖琼脂(PDA)培养基上的生长、产孢量和孢子萌发的状态均较其他培养基上的表现好;pH 9....     

链霉菌IMS002的分类鉴定及其抗真菌活性物质解析

【目的】对一株分离自植物根际土壤的具有抗真菌活性的链霉菌IMS002进行菌株分类鉴定,通过活性追踪分离纯化并鉴定有机相中的活性物质。【方法】通过16S rDNA和5个不同基因(atpD,gyrB,recA,rpoB,trpB)串联聚类分析以及生理生化实验分析,对链霉菌IMS002进行菌株分类鉴定,用扫描电子显微镜观察该株链霉菌的菌丝及孢子形态,以尖孢镰刀菌(Fusarium oxysporum)为指示菌进行生物活性追踪,通过硅胶柱层析、凝胶柱层析及高压液相色谱(HPLC)对活性物质进行分离和纯化,使用液质联用高分辨质谱仪、500 MHz核磁共振波谱仪以及圆二色光谱仪确定该物质的化学结构。【结果】IMS002经初步鉴定与产二素链霉菌(Streptomycesambofaciens)具有较近的亲缘关系,其发酵液对尖孢镰刀菌具有良好的抑菌效果,经分离和纯化以及现代波谱技术分析,确定有机相中的抑菌活性组分为Borrelidin。【结论】链霉菌IMS002能够产生化合物Borrelidin,该化合物对尖孢镰刀菌具有抑制活性。     

分离菌株DT-08C的鉴定及其除草活性和对作物的安全性

【背景】猪殃殃为作物田的主要恶性杂草之一,目前的防除仍以化学方法为主,对人体和环境造成严重危害。微生物除草剂具有靶标性强、对环境安全等优点,对发展新型农业现代化具有重要的现实意义。【目的】筛选无污染、无危害、强除草的生防菌株,为微生物除草剂提供新的菌种资源。【方法】通过组织分离法分离纯化菌株,采用孢子悬浮液测定菌株除草活性和安全性,运用核糖体内转录间隔区(internaltranscribedspacer,ITS)和转录延伸因子(EF-1α,EF-1/EF-2)基因鉴定,并通过MEGA7.0软件构建系统发育树,最后通过菌落直径和菌丝干重结合血球记数法分析菌株适宜生长和产孢的环境条件。【结果】经分离纯化筛选获得一株菌,命名为DT-08C。经形态学特征和rDNAITS序列分析,分离菌株DT-08C鉴定为芍药镰刀菌(Fusariumpaeoniae),命名为Fusarium paeoniae DT-08C。该分离菌株对猪殃殃的致病率达到47.22%-93.93%,对蚕豆、豌豆、玉米、白菜、番茄、黄瓜和辣椒安全。最适菌落生长和产孢的培养基分别为PSA和燕麦片培养基。【结论】菌株DT-08C对猪...     

鸡舍环境中tri5基因阳性镰孢菌的分离及其产毒特性

【目的】探讨利用tri5-PCR鉴定产毒镰孢菌的可行性以及tri5阳性菌株的产毒特性和产毒条件。评估鸡舍空气和固体基质中镰孢菌分离株中产单端孢霉烯族毒素潜力的发生率。【方法】利用编码单端孢霉烯族毒素合成酶的起始基因(tri5基因)对139株镰孢菌分离株进行PCR检测,并对tri5阳性菌株进行产毒培养,通过免疫亲和柱-高效液相色谱方法来检测培养产物中的T-2毒素和HT-2毒素含量。【结果】共筛选到42株tri5阳性菌株,其中分离自鸡舍空气中的10株tri5阳性菌株经产毒培养后7株菌株产生T-2毒素(1.36-5 ng/mL)或HT-2毒素(6.1-17.1 ng/mL)。在5℃-20℃间隔24 h变温、光照与黑暗间隔24 h交替、前期振荡后期静止的培养条件下培养9 d镰孢菌的产毒量最高;镰孢菌的产毒量与温度和时间显著相关,而与菌丝体干重无显著相关性。【结论】与传统鉴定产毒镰孢菌的方法比较,tri5-PCR更适用于快速、准确地检测大量鸡舍环境样本中的产毒镰孢菌。本研究为养殖环境的产毒镰孢菌的危害预警和控制提供技术支撑和理论依据。     

石楠锈孢锈菌重寄生菌的鉴定及产毒培养基筛选

将3株重寄生拟盘多毛孢PG52、PG53和PG90鉴定到种,筛选出三者最佳产毒培养基,旨为毒素的分离鉴定奠定基础。通过PAUP软件构建系统发育树,并结合形态特征将3株拟盘多毛孢鉴定到种;以不同培养基制备的粗提物处理石楠锈孢锈菌和茶藨生柱锈菌的锈孢子,利用台盼蓝染色法和生测法筛选最佳产毒培养基。分子鉴定结果显示PG52与PG90属于同一个分支,与Pestalotiopsis kenyana和Pestalotiopsis Oryza在同一个大分支,PG53与Pestalotiopsis telopeae在同一个分支;从形态特征来看,PG53的形态与Pestalotiopsis telopea的形态描述基本一致;PG52和PG90的形态特征与Pestalotiopsis kenyana的形态描述一致;PG52于改良Fries培养基制备的粗提物处理孢子效果最好,PG53于改良M-1-D培养基制备的粗提物处理孢子效果最好,PG90于改良M-1-D培养基制备的粗提物处理孢子效果最好。PG52和PG90同属一种拟盘多毛孢菌为Pestalotiopsis kenyana;PG53为Pestalotiopsis telopeae的菌株。PG52菌株的最佳产毒培养基为改良Fries培养基;PG53菌株的最佳产毒培养基为改良M-1-D培养基;PG90菌株的最佳产毒培养基为改良M-1-D培养基。     

蛇足石杉中产石杉碱甲内生真菌的分离和鉴定

【目的】从野生蛇足石杉(Huperzia serrata)中分离筛选产石杉碱甲的内生真菌。【方法】采用薄层层析及高效液相色谱法对内生真菌代谢产物进行测定和分析以期分离获得产石杉碱甲菌株,运用形态及ITS序列分析方法对产石杉碱甲菌株进行鉴定,并利用连续传代方法考察菌株遗传稳定性。【结果】经筛选获得一株产石杉碱甲内生真菌NSH-5,经形态学鉴定及ITS序列分析鉴定为轮枝镰孢菌(Fusarium verticillioides),其石杉碱甲产量为11.76 mg/100 m L,菌株经20次连续传代后遗传稳定。【结论】NSH-5菌株为一株具有产石杉碱甲能力的轮枝镰孢菌,该菌株的发现为生物合成石杉碱甲提供了新的菌种资源。     

古银杏内生真菌的分离及其抑菌活性

采用组织分离法从古银杏健康组织中分离得到55株内生真菌, 其中28个分离菌株在PDA培养基上不产孢子, 占总分离菌株的50.9%, 其它菌株根据其在PDA培养基上的培养特征, 10株被鉴定为青霉、6株为曲霉、4株为交链孢霉、3株为简梗孢霉, 另外酵母、毛霉、小单头孢霉、镰孢霉各1株。考察内生真菌培养上清对7种受试指示菌的抑制作用, 共筛选得到23株至少对一种指示菌的生长有抑制作用的菌株, 其中11株为不产孢真菌, 占活性菌株的47.83%。对活性最强的一株菌进行形态学和分子生物学鉴定, 将其确定为Xyla     

酿酒酵母单倍体菌株分离筛选及其产腺苷甲硫氨酸的初步研究

获得产腺苷甲硫氨酸的二倍体酿酒酵母CGMCC 2842遗传育种单倍体亲本。采用不同产孢培养基考察了酿酒酵母的产孢率,并对酿酒酵母CGMCC 2842进行了生孢培养分离子囊孢子得到单倍体菌株,确定单倍体配型,测定不同单倍体菌株腺苷甲硫氨酸含量。从分离的七株单倍体菌株(6株a型和1株α型)中筛选出一株产腺苷甲硫氨酸较高的a配型的单倍体菌株,经250 m L摇瓶发酵48 h后产腺苷甲硫氨酸1.10 g/L。筛选得到了一株产腺苷甲硫氨酸较高a型的单倍体菌株,为菌株的进一步遗传育种改良和腺苷甲硫氨酸微生物发酵法规模化生产奠定了基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.