植物微原生质体的融合

文章就植物微原生质体融合技术的基本原理和技术的应用作了介绍和展望。     

金针菇原生质体分离制备、融合、再生及融合子检出

探讨了利用金针菇菌丝体分离、制备原生质体;不同的酶浓度和酶解时间对原生质体得率的影响;以及八种再生培养基的选择性试验.建立了金针菇原生质体的分离、制备、融合及再生的试验体系.试验共获得209株再生菌株,通过核染色检测,共获得37株具有双核和锁状联合的再生株.     

植物原生质体融合与作物品种的改良

Takebe等(1971)关于烟草原生质体全能性的证明,Rao和Michayluk(1974)及Wallin等(1974)聚乙二醇PEG细胞融合方法的建立,为作物品种的改良提供了新途径。任何两种原生质体,不管其亲缘关系的远近,     

大型真菌原生质体融合技术研究进展

原生质体融合技术是近些年来迅速发展起来的细胞工程育种新技术,它具有许多常规杂交方法所无法比拟的独到之处。随着生物学研究手段的不断创新,使得原生质体融合技术正在逐步完善。本文综述了原生质体融合技术在大型真菌遗传方面的研究进展。     

微生物的原生质体融合及应用

微生物的原生质体融合及应用辛明秀,马玉娥（北京师范大学生物系,北京１００８７５）（河北涞源第一中学,涞源１０２９００）原生质体融合起源于６０年代。１９６０年法国的Ｂａｒｓｋｉ￣［１］研究小组在两种不同类型的动物细胞混合培养中发现了自发融合现象。同时日...     

米曲霉原生质体的营养互补融合及二倍体的诱发分离*

采用1％溶壁酶加1％蜗牛酶的混合液获得的原生质体,以30％聚乙二醇(MW=6,000)、0.01M CaCl_2、0.05M Gly做为融合剂,对米曲霉进行了原生质体的营养互补融合,融合频率为0.27—0.47％。自4个菌株的4对杂交组合中获得了异核体,并分离到97株绿色融合株。二倍体的孢子经PFA和UV诱发分离后,获得了二株生长速度快、蛋白酶活性高和产孢能力强的单倍体菌株。     

金鱼草花粉管亚原生质体的分离及在培养中的行为

应用酶法从金鱼草花粉管中分离出大量的亚原生质体。这种亚原生质体培养在 D_2液体培养基中,不论是有核的或是无核的都能再生厚的细胞壁和生长出花粉管状的管状结构。这些管状结构除了它们的顶端区外也沉积厚的细胞壁。随着管状结构的生长,内含物逐渐移向管状结构的顶端。当生长停止后,内含物可能完全被耗尽;有时管状结构的顶端破裂,内含物释放至培养液中。无核和有核亚原生质体同样显示有正常花粉管的基因表达的特性,即在培养中有类似花粉管生长的行为。这一事实表明在萌发的花粉管中有预先合成的 mRNA 的存在。     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

继代核移植能成倍提高来自奶山羊卵胞质体的异种间核移植重构胚的发育率

为了提高异种间核移植重构胚的发育率,本研究以体内排放的奶山羊成熟卵为供胞质的受体细胞,以人、兔、波尔山羊等的异种或亚种体细胞的原代核移植（Primary Somatic Cell Nuclear Transfer,PSCNT）重构早胚（8-16细胞期）的卵裂球作供核体,观察经亚种或异种卵胞质体短期“修饰”的核再移植产生的继代（Secondary SCNT,SSCNT）重构胚的着床前发育潜能。结果：人、兔、波尔山羊的继代桑椹／囊胚发育率均显著地高于其PSCNT胚胎（人,14．81％VS．7．79％;兔,23．53％VS．12．50％;波尔羊,55．35％VS．24．53％）;这些早胚的各阶段发育时程仍遵循供核体动物正常受精卵的发育时程。结果启示：奶山羊成熟卵胞质对异种体细胞核亦具一定的去分化能力,能支持重构胚发育到囊胚;异种重构胚的发育特征是由供体核所决定的;继代核移植几乎能够成倍提高异种间重构胚的着床前发育率,提示核的去分化完全是在母型信息主导的调控之下完成的,而进一步发育的时序似乎是由核决定的：成倍延长在含母型信息主导调控环境中的时间能成倍提高SCNT重构胚的着床前发育率。     

甘蓝型油菜胞质体大量制备技术的研究

用Ｐｅｒｃｏｌｌ密度梯度离心法和吖啶橙（ＡＯ）结合光照处理两种方法,均从甘蓝型油菜下胚轴原生质体制备到胞质体。在２个Ｐｅｒｃｏｌｌ梯度、３００００ｇ（１８０００ｒ／ｍｉｎ）与１２℃离心６０ｍｉｎ的条件下,获得了含胞质体８０％以上的群体;以含８０、１００、１２０ｍｇ／Ｌ ＡＯ的酶液酶解下胚轴原生质体,纯化后分别给以３ｈ、２ｈ、１ｈ光照（光强度：４０００ｌｘ）可使原生质体几乎不能分裂,但保持５ｄ以上的     

Isolation and characterization of microprotoplasts from APM-treated suspension cells ofNicotiana plumbaginifolia

Summary Subprotoplasts with a DNA content of less than the G1 level (microprotoplasts) were isolated from micronucleated cells of transformedNicotiana plumbaginifolia (Doba line resistant to kanamycin) and characterized cytologically as well as by flow cytometry and Feulgen microdensitometry. Micronuclei were induced upon treatment of the suspension cells with the anti-microtubule drug amiprophos-methyl (APM). Protoplasts were fractionated on a continuous iso-osmotic gradient of Percoll; this resulted in several visible bands. Flow cytometric analysis of fluorescein and nuclear DNA contents after staining with fluorescein and DAPI respectively showed that the main band contained mostly evacuolated, intact (sub)protoplasts. Microprotoplasts contained one or a few micronuclei surrounded by a thin rim of cytoplasm and an intact plasma membrane. A maximum of 40% of the microprotoplasts in the fraction just below the main band had a DNA content less than the G1 level, in other fractions this maximum was 20%. Some of these contained an amount equivalent to that of one or a few chromosomes. The application of microprotoplasts for chromosome-mediated gene transfer in plants is indicated.     

烟草花粉管亚原生质体的分离和培养行为

应用酶法从烟草花粉管分离出大量亚原生质体。具核的和无核的亚原生质体之比约为1:1。这种亚原生质体在D_2培养基中培养后,不论有核的或是无核的都能生长管状结构和再生厚的壁。管状结构的生长有节律性,常呈结节状。随着管状结构的生长,细胞内含物逐渐流入生长中的管状结构内,有时会从薄的管状结构的顶端排出到培养液中。已生长管状结构的亚原生质体,具核的和无核的比例约为1:1.7,表明管状结构的生长和壁的再生与是否有核的存在无关。对酶液处理后花粉管亚原生质体从花粉管的释放和从单独的花粉管亚原生质体生长管状结构的过程,进行了活体连续观察和照相记录。实验结果说明,结节状的管状结构确实是从单独的一个亚原生质体形成的。管状结构的生长和壁的再生似乎与细胞质进入新生的管状结构有关。讨论了花粉管亚原生质体在植物遗传操作中应用的可能性。     

Production of mitochondrial DNA transgenic mice using zygotes

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.