产黄青霉d8RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillum chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同谪定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。 异核体1中,病毒通过胞质融台转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的l／3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。     

产黄青霉原生质体融合杂种的产青霉素能力及非强制选择杂种的探讨

4个产黄青霉选育系谱的14列营养缺陷型原生质体融合率为0.01一8．88％。原生质体经紫外光照射后融合,有可能在非选择性的cM上分离出杂台二倍体。系谱内杂台二倍体的青霉素产量与直接亲本是否比原始亲本显著减产有关。系谱问杂合二倍体中双亲与产量有关的基因之间没有明显的显隐关系或累加效应。系谱I与I的杂合二倍体F16②—c24的分离子HP7②一1显示出比原始亲本更好的选育潜力。     

产黄青霉菌丝联合及病毒传递

通过营养要求、孢子颜色不同的带病毒和无病毒产黄青霉Penicillium chrysogenum菌株间的菌丝联合,在基本培养基(MM)上获得了营养互补的异核体。异核体菌落上出现亲本类型分离并产生深绿色角变或斑点。经孢子体积,DNA含量及营养类型测定证明角变和斑点为杂合二倍体。经电子显微镜观察,奥氏免疫双扩散,2.4％聚丙烯酰胺凝胶电泳及放射免疫测定检验了胞质融合或胞质和核融合后代病毒的传递,结果表明黄孢子后代带病毒而杂合二倍体及绿孢子后代均不带病毒。看来,这些绿孢子菌株(821和822)抗病毒感染。     

烟草种间的体细胞杂交──Ⅰ．通过原生质体融合将波缘烟草部分核基因组转移给普通烟草

通过“供体──受体”原生质体融合获得５个“波缘烟草＋普通烟草”体细胞杂种细胞系（ＴＵ＿１至ＴＵ＿５）。对５个杂种细胞系的细胞学观察表明,其中２个系为多细胞融合产物,其平均染色体数目分别为１６７．２和１９８．４;３个系为二细胞融合产物,其平均染色体数目分别为５４．３７０．６和６２．８。杂种细胞系的一个明显特征是所有观察的中期细胞均存在数量不等的畸变染色体。对５个杂种细胞系再生植株叶片苹果酸脱氢酶和酯酶的同工酶分析结果表明,它们均为程度不同的不对称核杂种,即只含有供体亲本（波缘烟草）的部分核基因组。以小麦ｒＤＮＡ为探针对杂种细胞系基因组ＤＮＡ的Ｓｏｕｔｈｅｒｎ杂交结果表明,杂种中双亲的载有ｒＤＮＡ的染色体可能均有部分消除;此外,杂种中新ＤＮＡ片段的存在说明双亲ｒＤＮＡ可能发生了分子间重组。两个由多细胞融合产生的杂种细胞系（ＴＵ＿２和ＴＵ＿５）再生苗不易生根而且形态上异常。３个由二细胞融合产生的杂种细胞系（ＴＵ＿１、ＴＵ＿３和ＴＵ＿４）再生苗可生根长大,形态上类似受体亲本;对其根尖的细胞学观察表明,染色体数目在５１─６６之间,说明均为只含有少量供体亲本染色体的不对称核杂种。     

原生质体融合提高农抗武夷菌素的效价

从农抗武夷菌素产生菌不吸水链霉菌武夷变种菌株Co-N-31诱变获得两个突变株M35(Leu^-,孢子颜色灰色)和M46(ser^-.孢子颜色灰白色),并以此两突变株为直接亲本在25％ PEG1000诱导下进行种内原生质体融合。M35和M46原生质体再生率分别为3.72％和0.248％,重组频率为55.20％。采用间接法选择营养标记互补的稳定的原养型重组子,并从中获得一株高产菌株F31-24;其效价比原始亲本Co-N-31提高了82％。薄层层析结果表明,菌株F31-24和Co-N-31的发效产物在Rf值为0.50和0.26处均有斑点,但含量有异。测定斑点生物活性证明其均有抑菌活性。温室试验表明,菌株F31-24发酵产物对小麦白粉病的防治效果优于菌株Co-N-31。     

啤酒酵母原生质体的制备、融合及再生

本文以16号(his~-)啤酒酵母和STA(ade~-)啤酒酵母作出发菌株进行原生质体融合,并探讨了菌龄、酶解时间,菌体分散程度和培养基种类等对原生质体融合的影响。     

用原生质体融合技术选育2—酮基—L—古龙酸高产菌株的研究

对革兰氏阳性的地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）Ｈ１９和革兰氏阴性的２－酮基－Ｌ－古龙酸产生菌Ｓ１９的原生质体的制备条件进行了研究,并采用聚乙二醇作诱导剂进行了两菌株的原生质体融合,用链霉素作为抗性标记对融合子进行了选择。从１７株产生２－酮基－Ｌ－古龙酸的融合子中选出了一株连续传代八次产酸高且产量稳定的融合子１５号。融合子１５号具有两个亲本菌株所具有的一些特性。     

芸苔属花粉—下胚轴原生质体融合再生杂种小植株

从青菜（Ｂｒａｓｓｉｃａ ｃｈｉｎｅｎｓｉｓＬ．）单胞中后期至二胞早期花粉分离出原生质体,用聚乙二醇法诱导其与甘蓝型油菜（Ｂ．ｎａｐｕｓＬ．）下胚轴原生质体融合。通过控制双亲原生质体的数量与比率,提高了异源融合率。融合体在离体条件下发生细胞分裂,形成愈伤组织,再生了小植株。染色体计数与酯酶同工酶酶谱分析初步证明获得了１ 株异源三倍体,２ 株异源四倍体。这是以游离花粉时期的原生质体与体细胞原生质体融合,取得“配子－体细胞杂交”成功的首次报道     

麻疹病毒融合蛋白(F)和血凝素(HA)在痘苗病毒中的表达

将麻疹病毒F和HA基因插入到痘苗病毒中,分别处于痘苗启动子P7.5与P11控制下,获得重组病毒vLmF和vCmH。用抗F多肽抗体和HA单抗进行ELISA检测,结果表明,两株重组病毒均能表达相应的麻疹蛋白。蛋白印迹显示重组病毒表达产物在分子大小,蛋白切割和糖化方面与麻疹病毒糖蛋白一致。两株重组病毒分别免疫家兔都能产生较高滴度的麻疹抗体,这些抗体具有中和作用和血溶抑制作用。此外,vCmH产生的抗体还具有血凝抑制作用。     

INTRASPECIFIC TRANSFER OF HERBICIDE RESISTANCE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYCEAE) VIA PROTOPLAST FUSION

Stable progeny doubly resistant to the herbicides sulfometuron methyl (SMM) and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (DCMU) were obtained at a frequency of 2% on fusion of protoplasts derived from mutants of Porphyridium sp. (UTEX 637) that were resistant only to SMM (strain SMR) or DCMU (strain DC-2). In the presence of both herbicides, only the fusion progeny could grow; both parental mutants were inhibited. In the absence of SMM, the activity of acetohydroxy acid synthase (AHAS) in the wild-type strain was similar to that in DC-2, exceeding that of SMR by up to 4.5-fold. AHAS activities of all fusion progeny were lower than those of the wild-type strain and DC-2 but higher than that of SMR. In the presence of SMM, AHAS activities of all tested fusion progeny ranged between those of the two parental mutants. This result indicates that both types of AHAS, the type resistant to SMM and the sensitive type, originating from SMR and DC-2, respectively, were expressed in the fusion progeny. In the presence of DCMU, the photosynthetic activity of SMR was completely inhibited, whereas that of DC-2 was unaffected. The photosynthetic activity of the fusion progeny in the presence of DCMU was slightly lower than that of DC-2. Both the cell volume and the DNA content of the fusion progeny were similar to those of the parents. However, the genetic nature of the fusion products has not yet been elucidated. To the best of our knowledge, this is the first report on transfer of herbicide resistance via protoplast fusion in algae.     

PROTOPLAST FUSION AND GENETIC COMPLEMENTATION OF PIGMENT MUTATIONS IN THE RED MICROALGA PORPHYRZDZUM SP.1

Protoplasts from two green pigment mutants of Porphyridium sp. (UTEX 637) containing a low phycoerythrin level were fused by exposure to polyethylene glycol (MW 6000) combined with a short heat shock (45° C, 5 min). Following regeneration on agar plates, red colonies arose in which complementation of the phycoerythrin deficiency had occurred. The complementation frequency was estimated to be 0.2%. Eight progeny showing red pigmentation were isolated and purified by consecutive transfers on agar plates. Characterization of the fusion progeny revealed that their phycobiliprotein and chlorophyll contents per cell were higher than those of their parental mutant strains and, in most strains, similar to that of the wild type. The fusion products proved to be stable over many growth cycles. The DNA content of the wild type and of the parental mutant strains was about 0.05 pg-cell^?1. Fusion progeny strains showed a variable DNA content: a few fusants contained the same amount of DNA as the wild type and the parental strains, while others had about 50% more DNA per cell. The DNA content of one of the progeny strains (CF1c) was double that of the wild type (0.1 pg. cell^?1). Cells of this fusion progeny contained one nucleus per cell, which suggests that nuclear fusion and the formation of a stable diploid followed cell fusion. Analysis of phycobilisome components of CF1c revealed complementation of linker polypeptides associated with phycoerythrin (γ subunits). CF1c contained, like the wild-type strain, four linker polypeptides; all of these were absent in one parental strain and one was absent in the second. To the best of our knowledge, this is the first report of protoplast fusion, formation of somatic hybrids, and the apparent completion of a parasexual cycle in a red microalga.     

影响枯草杆菌原生质体转化的因素

原生质体转化是将外源基因导入细菌的主要方法之一,其转化频率受制于多种因素。本研究以激BacillussubtilisDB104为宿主菌,以枯草杆菌的高表达型质粒pNQ122为外源DNA,研究了原生质体再生率、原生质体浓度和用于制备原生质体的细胞生长期对转化频率的影响,获得了在该系统中实现高频率转化的条件。该转化条件使外源基因在多个B.subtilis菌株中的转化成为可能,并使从B.subtilis中筛选中性蛋白酶基因获得成功。     

粘虫颗粒体病毒增效因子的分离纯化及其生化性质

粘虫颗粒体病毒经０．０２ｍｏｌ／ＬＮａＯＨ碱溶,先用ＳｅｐｈａｄｅｘＧ－２００凝胶过滤层析柱从病毒蛋白粗提中分离增效因子,然后选用ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析柱进一步纯化增效因子,得到少量电泳纯的增效因子蛋白样品。