Feed science of the cyanobacterium Spirulina platensis

钝顶蓝藻的饲料科学

基本信息

批准号：
09460150
负责人：
TOYOMIZU Masaaki
金额：
$ 7.23万
依托单位：
TOUHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

Spirulina (Arthrospira) is one of the most promising species among the cyanobacteria to serve as a source of nutritional protein, that, in turn, once the stable production promised, may greatly expedite the process involved in producing industrial enzymes for animal feeds. Unfortunately, genetic engineering tools for Spirulina have been almost non-existent, as Spirulina appears refractory to common genetic manipulation techniques. (1) As the initial stages of the development of recombinant DNA methodology, we examined the effects on transformation efficiency of electroporation conditions such as electric-field strength (2, 4, 6, 8, 10, 12 kV cm^<-1>) and time constant (2.5, 5 ms). As a results, we found that transformation of S.platensis was most effective at a pulse duration 5.0 ms with an electric field of 4 kV cm^<-1>, and that foreign genes, CAT in pHSG399 can be expressed in this organism. (2) ＆ (3) Further, plasmids related to the broad host range vector, pSTV29, and plasmid shut … More tle vector, pRL6, containing two origins of replication which allow it to replicate both in cyanobacteria and E.coli were also useful in the sustaining Cm resistance in S.platensis ; Gene replacement using of 16s rRNA fragments in pHSG399 can be performed by electrotransformation showing the cells PCR-positive for CAT.(4) ＆ (5) For cloning the Spirulina transformants, we subjected it to protoplasting with glycine, and developed a simple and rapid method for sensitive fluorometric determination of the protoplasts : the optimum condition for forming S.platensis protoplasts were treatments with lysozyme solution for 2 hr of cells cultured at 25℃ in medium supplemented with 0.1 % glycine, whereas for in vivo identification and quantification of growing S.platensis with fluorescent measurements the major excitation and emission maxima were found to be 615 and 647 nm from their spectra, respectively. (6) Finally to clarify effects of dietary spirulina on growth performance, broiler chicks were fed an experimental diet containing spirulina at 0, 40, or 80 g/kg for 16 d. As a result, no significant differences among treatments were observed in body weights, nor weights or yields (as a percentage of body weight) for any of the selected traits, including liver, abdominal fat, kidney, and pectoralis profundus.We are currently investigating other requirements for the 'stable' S.platensis gene transfer system, which include : a) cloning and assessment of Spirulina promoters using a plasmid carrying a promoterless CAT reporter gene, b) establishing a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes followed by introduction of the complexes into the Spirulina cell by electroporation. Establishments of stable gene transfer system for Spirulina would have many advantages as a biological factory for producing recombinant enzymes. Less

螺旋藻（节旋藻）是蓝藻中最有希望作为营养蛋白来源的物种之一，一旦稳定生产，可能会大大加快动物饲料工业酶的生产过程。螺旋藻的基因工程工具几乎不存在，因为螺旋藻似乎难以适应常见的基因操作技术 (1) 作为重组 DNA 方法发展的初始阶段，我们研究了其对螺旋藻的影响。电穿孔条件，如电场强度（2、4、6、8、10、12 kV cm^<-1>）和时间常数（2.5、5 ms）的转化效率结果，我们发现转化率。钝顶葡萄球菌在脉冲持续时间5.0ms、电场为4kV cm ^ -1 时最有效，并且外源基因pHSG399中的CAT可以在该生物体中表达。 (2) 和 (3) 此外，与广泛宿主范围载体 pSTV29 和质粒关闭载体 pRL6 相关的质粒也很有用，该载体包含两个复制起点，使其能够在蓝细菌和大肠杆菌中复制在 S.platensis 中维持 Cm 抗性；使用 pHSG399 中的 16s rRNA 片段进行基因替换可通过电转化进行，显示细胞 PCR 阳性CAT.(4) & (5) 为了克隆螺旋藻转化体，我们将其用甘氨酸进行原生质化，并开发了一种简单快速的原生质体荧光灵敏测定方法：形成 S.platensis 原生质体的最佳条件是用溶菌酶溶液用于在 25℃ 补充有 0.1% 甘氨酸的培养基中培养细胞 2 小时，而用于体内生长的鉴定和定量对钝顶螺旋藻进行荧光测量，从其光谱中发现主要激发和发射最大值分别为 615 和 647 nm (6) 最后，为了阐明日粮螺旋藻对生长性能的影响，在 30 ℃ 下饲喂含有螺旋藻的实验日粮。 0、40 或 80 g/kg，持续 16 天结果，在体重、重量或产量方面没有观察到显着差异。体重的百分比）用于任何选定的性状，包括肝脏、腹部脂肪、肾脏和胸深肌。我们目前正在研究“稳定”钝顶链球菌基因转移系统的其他要求，其中包括：a）克隆和使用携带无启动子 CAT 报告基因的质粒评估螺旋藻启动子，b) 建立转座技术，该技术涉及在体外形成释放的 Tn5 转座复合物，然后通过以下方式将复合物引入螺旋藻细胞中螺旋藻稳定的基因转移系统的建立作为生产重组酶的生物工厂具有许多优势。