| Co-Investigator(Kenkyū-buntansha) |
NAKAI Yutaka Tohoku university, Graduate School of Agricultural Science, Associate professor, 大学院・農学研究科, 助教授 (80155655)
NAKAMURA Yasue Tohoku university, Graduate School of Agricultural Science, Research assistant, 大学院・農学研究科, 助手 (10005613)
WATANABE Takeshi Niigata university, Faculty of Agriculture, Professor, 農学部, 教授 (10201203)
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| Research Abstract |
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
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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
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