子囊菌交配型基因MAT1-1的系统发育

子囊菌具有无性态与有性态的复杂性,以及人们对其系统发育和亲缘关系了解的局限性,进而导致菌物学家对子囊菌分类尚持不同意见。子囊菌的交配型基因（MAT）进化保守,且编码的蛋白质调控子囊菌的有性生殖过程。核盘菌Sclerotinia sclerotiorum (Lib.) de Bary隶属于子囊菌门Ascomycota、盘菌纲Discomycete,是一种典型丝状同宗配合真菌,控制该菌有性生殖的交配型基因MAT1-1和MAT1-2紧密连锁,且该菌并无有性态与无性态的复杂性。故此,本文根据所克隆的核盘菌交配型基因MAT1-1,利用PAUP*软件将82种含有Alpha-box交配型基因的子囊菌进行了系统进化分析,通过核苷酸及氨基酸水平的系统发育分析,并结合Ainsworth（1973）分类系统及最新的Deep Hyphae（2006）分类系统的对比研究,发现所构建的系统进化树与传统分类所表现的进化关系基本一致,且核盘菌交配型基因MAT1-1在进化过程中功能相对保守,该分析结果有助于对其他子囊菌交配型基因的克隆、系统分类与进化研究,同时对核盘菌的亲缘关系、病害预测及防治等具有重要意义。     

茯砖茶“金花菌”vcx基因克隆及表达分析

茯砖茶是一种紧压黑茶,冠突曲霉是茯砖茶发花阶段的主要微生物,其有性发育产生闭囊壳又名"金花菌",其数量和质量常用来判断茯砖茶的品质好坏。冠突曲霉可以用Na Cl来控制其产有性孢子及无性孢子,为了研究这一特殊产孢机理,及钙信号和液泡在这产孢过程中的作用。通过克隆冠突曲霉钙信号途径中的液泡上钙氢交换子vcx基因,并对这一基因进行结构和表达分析。结果表示:冠突曲霉中的vcx基因编码区1 326 bp,不含内含子,预测编码441个氨基酸,不含信号肽,是一个疏水蛋白,含有11个跨膜结构,符合钙氢交换蛋白的基本结构。通过对其在冠突曲霉的有性、无性、菌丝各生长阶段的表达量进行测定,结果显示:vcx基因对其发育产生正调控,可能参与了冠突曲霉的无性产孢及盐压力响应过程。实验结果不仅为该蛋白的后续研究提供了条件,而且为钙信号调控丝状真菌在盐压力应答下的产孢机制奠定基础。     

斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

【目的】蜕皮激素在昆虫变态发育中起着关键的调控作用。蜕皮激素活化为20-羟基蜕皮酮(20-hydroxyecdysone,20E)后与蜕皮激素受体二聚体〔蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle protein,USP)〕结合,启动20E诱导的级联反应。本研究旨在从互作蛋白角度探究EcR/USP自身调控的分子机理。【方法】以重要农业害虫斜纹夜蛾Spodoptera litura为研究对象,通过构建酵母双杂交筛选系统分别筛选了与EcR和USP相互作用的蛋白。【结果】文库转化效率达到3.0×106cfu/μg,文库滴度达到1.3×108cfu/m L,文库插入片段大小在500~2 000 bp之间。4种诱饵质粒(EcRA/p GBKT7,EcRB1/p GBKT7,USP1/p GBKT7和USP2/p GBKT7)对酵母细胞无毒性,并且无自激活性,说明构建的酵母双杂交文库质量可靠。用以上4种诱饵质粒筛选酵母双杂交文库,共得到110个互作蛋白,其中与EcRB1,USP1和USP2互作的蛋白分别有26,52和32个,未筛选到与EcRA互作的蛋白。随后,从中挑选了Dna J-5(Hsp40 homolog 5,一种热激蛋白分子伴侣),MBF2(mediator of Bm FTZ-F1 type 2,一种转录共激活子),polyubiquitin(多聚泛素类蛋白),esr16(ecdysteroid regulated 16k Da protein,一种蜕皮激素调控蛋白)和NEDD8-like(neural precursor cell expressed,developmentally down-regulated protein 8,一种泛素调节相关蛋白)5种蛋白,利用酵母双杂交和Far-Western印迹法进一步验证了蛋白间的互作关系。【结论】分子伴侣和泛素化修饰等在蜕皮激素受体调控中可能起着重要作用。本研究对深入理解昆虫变态发育的分子机理具有重要意义。     

冠突曲霉产孢相关基因的克隆及功能分析

为探究冠突曲霉有性产孢调控的相关机制,从已构建的T-DNA随机插入突变体库中选取有性产孢突变株,采用Southern杂交鉴定T-DNA为单拷贝插入,并用Tail-PCR技术扩增获得T-DNA插入位点侧翼序列,分离获得突变基因。序列分析表明,该基因位于冠突曲霉基因组scaffold 7上,开放阅读框2 511 bp,含有4个内含子,预测编码666个氨基酸,含有NDT80_Pho G蛋白质超级家族保守结构域。BLASTp结果表明,该预测蛋白与Eurotium rubrum p53-like转录因子(EYE95652.1)相似性最高,identity=89%。显微观察发现T-DNA的插入影响了有性产孢结构的发育。本实验为进一步研究该基因在有性产孢过程中的分子功能及可能参与的调控途径奠定基础。     

冠突曲霉钙调素cam基因的克隆及表达分析

冠突曲霉是茯砖茶发花阶段的主要微生物,其在Na Cl作为渗透压调节下分别进入有性产孢和无性产孢两个不同的世代,为了考查钙信号途径在其特殊产孢过程中的作用,通过克隆冠突曲霉钙信号调控的关键基因钙调素cam基因,并对这一基因进行结构和表达分析。结果表示:冠突曲霉中的cam基因全长924 bp,开放阅读框为450 bp,含6个内含子,预测编码149个氨基酸。cam参与了冠突曲霉的产孢及盐压力响应过程,cam基因对其无性发育产生正调控。实验结果为钙信号调控丝状真菌盐压力应答下的产孢机制研究奠定基础。     

利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

【目的】筛选鉴定西花蓟马Franiklinella ocicdentalis体内与番茄环纹斑点病毒(tomato zonate spot virus, TZSV)NSs蛋白互作的介体因子。【方法】利用酵母双杂交技术筛选与TZSV NSs互作的西花蓟马蛋白;进行序列分析鉴定后,将捕获的蛋白与NSs基因回转到酵母细胞,利用营养缺陷型培养基鉴定蛋白互作情况。再利用GST Pull-down技术验证TZSV NSs与鉴定出的西花蓟马蛋白的体外互作关系。【结果】构建了TZSV NSs的酵母双杂交诱饵质粒pGBKT7-NSs,确定了诱饵质粒对酵母AH109细胞无毒性,并且无自激活活性。序列分析发现与TZSV NSs互作的西花蓟马蛋白为类电压依赖性阴离子通道(voltage-dependent anion-selective channel-like, VDAC)。酵母回转实验显示TZSV NSs与西花蓟马VDAC在酵母细胞内存在特异性互作。GST Pull-down结果表明TZSV NSs与西花蓟马VDAC在体外存在相互作用。【结论】通过酵母双杂交和GST Pull-down技术,分别在酵母细胞内和体外证实了TZSV NSs和西花蓟马VDAC存在特异性互作。这些结果有助于揭示西花蓟马VDAC蛋白调控西花蓟马持久传毒的机制,为虫传病毒病的防治提供理论基础。     

冠突曲霉AN1类锌指蛋白zfpA基因的克隆及表达分析

本研究采用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆出冠突曲霉中zfpA基因的cDNA全长序列并进行生物信息分析。该序列全长1 443 bp,开放阅读框长度为924 bp,从312~1 235 bp,没有内含子序列。推测编码的蛋白质含有307个氨基酸,同源性分析显示该基因与米曲霉的AN1锌指蛋白(XP_001826567)相似性达71%。利用荧光定量PCR技术分析了zfpA基因在营养菌丝体阶段、无性产孢阶段及有性产孢阶段的表达量差异。结果表明,这个基因在在无性产孢时期的表达量最低,有性产孢时期的表达量最高,相比无性产孢时期上调了4倍。本实验为深入研究zfpA基因对冠突曲霉的产孢调控机制奠定了基础。     

酵母双杂交筛选与GsCBRLK相互作用的蛋白质

GsCBRLK(calcium/calmodulin-binding receptor-like kinase from Glycine soja)在ABA及盐胁迫诱导的钙离子信号通路中起到关键的调节作用。为深入研究GsCBRLK蛋白的作用机制, 文章采用膜酵母双杂交系统, 以GsCBRLK为诱饵蛋白, 筛选与其相互作用的蛋白质。通过构建野生大豆盐胁迫条件下的cDNA文库、膜酵母双杂交系统筛选、复筛、回转验证、生物信息学分析以及酵母体内互作验证等手段, 最终获得2个(SNARE 和 14-3-3 蛋白)与GsCBRLK诱饵蛋白相互作用的蛋白质。     

玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选

为阐明玉米中心蛋白(ZmCEN)的生物学功能,采用酵母双杂交技术对其互作蛋白进行研究。提取玉米(Zea mays L.)自交系‘郑58’幼苗的总RNA,利用SMART技术反转录合成ds cDNA,构建以pGBKT7为载体的酵母双杂交cDNA文库;依据ZmCEN基因的CDS序列设计引物,构建重组诱饵载体(pGBKT7-ZmCEN)转化酵母菌株Y2HGold,检测诱饵载体的毒性与自激活能力后,筛选与玉米中心蛋白(ZmCEN)互作的猎物蛋白。将筛选的互作蛋白NAC67和TONNEAU1b(TON1b)再次验证相互作用,并选取互作蛋白TON1b,采用BiFC实验分别构建ZmCEN-pSPYNE和TON1b-pSPYCE BiFC半分子重组载体,转化拟南芥原生质体,进一步验证它们在细胞内的互作;并利用Uniprot和KEGG在线网站对互作蛋白进行gene ontology(GO)注释分析。结果表明:玉米全株幼苗的cDNA文库库容量达到2.56×107 CFU,文库滴度5.36×108 CFU/mL,符合建库要求。经检测诱饵载体无毒性也无自激活功能,所筛选的cDNA文库经测序和Blast比对分析以及共转验证,最终得到28个与诱饵蛋白ZmCEN互作的蛋白质。GO注释显示互作蛋白参与的生物过程有21种。BiFC结果显示,蛋白TON1b与ZmCEN在拟南芥原生质体细胞内互作而形成互补,从而产生黄色荧光,进一步证实了两者存在互作关系。酵母双杂交系统cDNA文库的成功构建与筛选,为进一步研究玉米ZmCEN及其与互作蛋白的作用机制奠定了基础。     

大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定

用大黄鱼Wap65-2基因成熟肽区构建酵母双杂交诱饵质粒,并对诱饵质粒进行自激活活性及毒性检测。从大黄鱼(Larimichthys crocea)肝组织c DNA中扩增Wap65-2基因成熟肽片段(Lc Wap65-2m),将其克隆至p GBKT7载体中,获得诱饵载体p GBKT7-Lc Wap65-2m,经PCR、酶切和测序验证无误后,转化至酵母菌株Y187中,在营养缺陷培养基中观察重组蛋白的毒性作用和自激活活性,同时利用Western blot分析重组蛋白的表达。成功扩增Lc Wap65-2m,并克隆至p GBKT7载体中;Western blot检测结果表明,p GBKT7-Lc Wap65-2m在酵母细胞中表达诱饵蛋白Lc Wap65-2m;表型筛选结果显示,Lc Wap65-2m无毒性作用和自激活活性。成功构建了Lc Wap65-2m酵母双杂交诱饵载体,为进一步利用酵母双杂交技术筛选与Wap65-2相互作用的蛋白奠定基础。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.