Feed science of the cyanobacterium Spirulina platensis

钝顶蓝藻的饲料科学

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09460150/
• 关键词: Spirulina; foreign gene; electroporation; CAT gene; general recombination; feed resources; protoplast; fluorometric determination; 増殖応答

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）是蓝藻中最有前途的物种之一，可作为营养蛋白质的来源，一旦稳定生产，可能会大大加快生产用于动物饲料的工业酶的过程。不幸的是，螺旋藻的基因工程工具几乎不存在，因为螺旋藻似乎对常见的基因操作技术很难。(1)作为重组DNA方法学发展的初始阶段，我们研究了电穿孔条件如电场强度（2、4、6、8、10、12 kV cm-1<-1>）和时间常数（2.5、5 ms）对转化效率的影响。结果，我们发现，在脉冲持续时间为5.0 ms，电场为4 kV cm 2时，钝顶螺旋藻的转化最有效，<-1>并且外源基因pHSG 399中的CAT可以在该生物体中表达。(2)（3）此外，与广宿主范围载体pSTV 29相关的质粒和质粒shut ...更多信息 含有两个复制起点的质粒pRL 6也可用于维持钝顶链霉菌的Cm抗性;在pHSG 399中使用16 s rRNA片段的基因置换可通过电转化进行，显示细胞对CAT呈PCR阳性。(4)（5）为了克隆螺旋藻转化子，我们用甘氨酸对转化子进行原生质体处理，并建立了一种简便、快速、灵敏的荧光测定法：原生质体形成的最佳条件为：在25℃、添加0.1%甘氨酸的培养基中，用溶菌酶溶液处理2 h，而对于生长中的钝顶链霉菌的体内鉴定和定量，用荧光测量发现主要的激发和发射最大值分别在615和647 nm处。(6)最后，为了阐明螺旋藻对肉仔鸡生长性能的影响，在试验日粮中分别添加0、40和80 g/kg螺旋藻，试验期为16 d。结果，在体重、体重或产量方面，未观察到处理之间的显著差异（作为体重的百分比）。我们目前正在研究“稳定的”钝顶螺旋藻基因转移系统的其他要求，包括：a）使用携带无启动子CAT报告基因的质粒克隆和评估螺旋藻启动子，B）建立一种转座技术，包括在体外形成释放的Tn 5转座复合物，然后将该复合物引入螺旋藻细胞通过电穿孔。建立稳定的螺旋藻基因转移系统，将有利于螺旋藻作为生产重组酶的生物工厂。少

项目成果

Toyomizu, M., Sato, K., Taroda, H., Kato, T.and Akiba, Y.: "Effects of dietary spirulina on meat color in muscle of broiler chickens."British Poultry Science. (in press). (2001)

Toyomizu, M.、Sato, K.、Taroda, H.、Kato, T. 和 Akiba, Y.：“膳食螺旋藻对肉鸡肌肉肉色的影响。”英国家禽科学。

Toyomizu,M.,Sato,K.,Taroda,H.,Kato,T.and Akiba,Y.: "Effects of dietary spirulina on meat colour in muscle of broiler chickens."British Poultry Science. (in press). (2001)

Toyomizu, M.、Sato, K.、Taroda, H.、Kato, T. 和 Akiba, Y.：“膳食螺旋藻对肉鸡肌肉肉色的影响。”英国家禽科学。

Toyomizu,M.,Suzuki,K.,Kawata,Y.,Kojima,H.and Akiba,Y.: "Effective transformation of the cyanobacterium Spirulina platensis using electroporation."Journal of Applied Phycology. (in press). (2001)

Toyomizu, M.、Suzuki, K.、Kawata, Y.、Kojima, H. 和 Akiba, Y.：“利用电穿孔有效转化蓝藻钝顶螺旋藻。”应用藻类学杂志。

Toyomizu,M.,Sato,K.,Taroda,H.,Kato,T.and Akiba,Y.: "Effect of dietary spirulina on meat colour in muscle of broiler chickens."British Poultry Science. (in press). (2001)

Toyomizu, M.、Sato, K.、Taroda, H.、Kato, T. 和 Akiba, Y.：“膳食螺旋藻对肉鸡肌肉肉色的影响。”英国家禽科学。

Toyomizu, M., Suzuki, K., Kawata, Y., Kojima, H.and Akiba, Y.: "Effective transformation of the cyanobacterium Spirulina platensis using electroporation."Journal of Applied Phycology. (in press). (2001)

Toyomizu, M.、Suzuki, K.、Kawata, Y.、Kojima, H. 和 Akiba, Y.：“利用电穿孔有效转化蓝藻钝顶螺旋藻。”应用藻类学杂志。