粟长蠕孢菌的原生质体转化

本文报道了利用具有潮霉素抗性标记的质粒(pAN7-1)对粟长蠕孢菌原生质体进行转化的结果。经pAN7-1质粒DNA转化处理的粟长蠕孢菌原生质体在含潮霉素(200μg/ml)的选择性培养基上出现两类转化子。一类是正常转化子,其转化率为2个转化子/μg DNA;另一类是流产转化子,其产生频率为500—600个转化子/μg DNA。DNA杂交分析结果表明,在正常转化子中质粒DNA以首尾相接、重复排列的形式整合入受体菌染色体DNA。初筛获得的转化子多数以异核状态存在,经单孢分离纯化后可通过有丝分裂稳定传代。     

碱性蛋白酶基因在Bacillus licheniformis 2709中的整合和扩增

将含有去掉启动子的枯草杆菌碱性蛋白酶基因和Ｃｍ抗性基因的整合质粒ｐＭＰ以原生质体ＰＥＧ法转化Ｂ．ｌｉｃｈｅｎｉｆｏｒｍｉｓ２７０９在１０ｕ／ｍｌＣｍ平板上筛选整合子,并通过不断提高整合子的抗氯霉素能力来扩增染色体碱性蛋白酶基因,最终选到一株产酶比生产菌２７０９提高６０％的高表达整合菌ＢＬ－２０３,该菌在无选择压力下遗传性状相当稳定。     

运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法

【背景】萎锈灵抗性基因作为筛选标记在植物和真菌中得到广泛应用。【目的】构建可以使用萎锈灵作为筛选标记的香菇遗传转化技术。【方法】利用溶壁酶消化培养4 d的香菇菌株411-4的菌丝体获得原生质体,加入适量pL-cbx质粒和聚乙二醇溶液,混合物涂布于再生筛选培养基上,培养后挑选菌落进行验证试验。【结果】在原生质体数目108个和添加4μg质粒DNA的情况下,得到了40个抗性转化子。利用PCR实验和转代实验对转化子进行验证,结果显示38个转化子的抗性可稳定遗传,表明萎锈灵抗性基因整合进入了供试菌株的基因组中。【结论】利用香菇411-4菌株建立了一套运用萎锈灵抗性基因作为分子标记的遗传转化技术体系。     

信息库

1.用电穿孔法进行黑曲霉的转化作用 用电穿孔法可以将DNA转入完整的黑曲霉萌发分生孢子。此法不仅简单快捷,转化子菌落出现的时间也比原生质体转化法早些。分生孢子不经预处理,转化频率为1.2菌落/μg整合载体,或100菌落/μg质粒DNA。若将分生孢子用稀的真菌细胞壁分解酶溶液预处理,转化频率大约可提高两倍。用DNA贴印法分析染色体DNA,和用转化子DNA的限制性DNA图谱分析表明,整合载体已通过同源或非同源整合到染色体组中。这个结果和用原生质体转化法相同。在质粒DNA转中,无论电穿孔法或用常规方法,转化子中的质粒DNA大多数呈游离状态,只有很少数整合到染色体上。     

niaD基因与uidA基因共转化糖化酶高产菌株Aspergillus nigerT21

从Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ Ｔ２１分离到自发性的氯酸盐抗性株,再经氮源生长试验获得硝酸盐还原酶缺陷的ｎｉａＤ突变体Ｎ４４。用含有ｎｉａＤ的质粒ｐＳＴＡ１０转化Ｎ４４,转化频率为５个／μｇ（转化子／ＤＮＡ）。转化子的Ｓｏｕｔｈｅｒｎ印迹分析表明ｎｉａＤ基因同源整合到Ｎ４４的染色体ＤＮＡ中。ｐＳＴＡ１０与含葡萄苷酸酶基因（ｕｉｄＡ）的质粒ｐＮＯＭ１０２共转化Ｎ４４,共转化频率为４０％。共转化     

转座子Tn5096对刺孢吸水链霉菌北京变种PR220转座的诱变

用大肠杆菌／链霉菌穿梭质粒ｐＣＺＡ１６８多次转化农抗１２０产生菌刺孢吸水链霉菌北京变种ＲＦ２２０的原生质体,均未得到转化子。来自吸水链霉菌应城变种１０－２２突变株的链霉菌质粒ｐＩＪ７０２可以转化ＲＦ２２０,但转化频率只有数十个转化子／μｇＤＮＡ。用来自ＲＦ２２０本身的ｐＩＪ７０２对消除ｐＩＪ７０２后的ＲＦ２２０的原生质体进行了再转化,转化率没有明显的提高。用氨苄青霉素和甘氨酸协同处理ＲＦ２２０的菌     

超声波法转化酵母原生质体

本文报告了用超声波将外源ＤＮＡ导入酿酒酵母原生质体。我们以酿酒酵母Ｙ１６２作为质粒ＹＥＰ２４的受体菌,用２４ＫＨｚ、２０Ｗ的超声波处理酵母原生质体。上述超声处理对质粒ＤＮＡ没有明显破坏作用。随着作用时间的延长,酵母原生质体的存活率与转化率迅速下降。获得的最高转化率为１０３转化子／μｇＤＮＡ。用２０Ｗ或更高的功率及更长的时间处理完整酵母细胞没有获得转化子。     

枯草杆菌启动子—信号肽序列的克隆及序列分析

利用含红霉素抗生基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢａｎＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ     

PEG法介导转化诸葛菜下胚轴原生质体获得转基因植株

采用诸葛菜无菌苗的下胚轴组织为材料,分离原生质体,在原生质体培养基中作液体浅层暗培养,植板率为５％,植株再生频率为１００％。作者进而开展了遗传转化研究。为研究ＰＥＧ介导转化诸葛菜原生质体的影响因素,通过瞬间表达,实验了ＰＥＧ法转化子叶原生质体的过程,在此基础上将分离纯化后的原生质体与带ＨＰＴ基因的质粒ＤＮＡ（ｐＢＩ２２２）混合,ＨＰＴ基因作选择标记,ＰＥＧ介导转化;重新收集转化后的原生质体,以５×１０４／ｍｌ的密度在原生质体培养基中作浅层培养;培养１０—１５天后用２５ｍｇ／Ｌ的潮霉素（ｈｙｇｒｏｍｙｃｉｎ）进行筛选,一月后出现少量细胞团,转入含潮霉素５０ｍｇ／Ｌ的扩增培养基扩增愈伤组织,进而转入含５０—１００ｍｇ／Ｌ潮霉素的分化培养基诱导分化成苗,分化率为１００％,转入生根培养基中生根成完整植株。抗性植株再生率为４×１０（－５）。在获得再生转基因植株后,以再生植株叶片为材料,进行Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交,证实外源基因已稳定整合到植物基因组中并表达,再生转基因植株频率为１０（－５）。国内外首次转化诸葛菜属植物原生质体获得成功。     

酵母细胞的电击高频转化

用Ｂｉｏ－Ｒａｄ生产的基因脉冲仪进行酿酒酵母电击转化实验,得到的最适条件为：５ｋｖ／ｃｍ２５μＦ和２００Ω。电击后涂布前的培养时间为２小时。电击后细胞存活率为４６％时,每微克质粒ＤＮＡ得到１０６以上的转化子。用相同的质粒和受体菌进行原生质体法和醋酸锂法比较实验,转化率分别为２×１０４和３．５×１０２个转化子／μｇＤＮＡ。电击转化是最方便易行和高效率的方法。     

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.     

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.     

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.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

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.