Feed science of the cyanobacterium Spirulina platensis

螺旋藻(Arthrospira)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，可能会大大加快生产动物饲料用工业酶的过程。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难以实现。(1)研究了电场强度(2、4、6、8、10、12千伏·cm~(-1)~(-1))和时间常数(2.5、5ms)等电穿孔条件对重组DNA转化效率的影响。结果表明，脉冲时间为5.0ms、电场强度为4kV cm~(-1)~(-1)的条件下，钝顶螺旋藻的转化效果最好，并且在pHSG399中可以表达外源基因CAT。(2)和(3)与广泛寄主范围载体pSTV29和质粒Shut…相关的质粒更多的TIL载体pRL6包含两个复制起点，使其能够在蓝藻和大肠杆菌中复制，这也有助于维持钝顶葡萄球菌对CM的抗性；(4)和(5)为了克隆螺旋藻转化子，我们用甘氨酸对其进行原生质体培养，并建立了一种灵敏的荧光检测方法：在含有0.1%甘氨酸的25℃培养的细胞中，用溶菌酶溶液处理细胞2小时，形成原生质体的最佳条件，而用荧光测量在体内鉴定和定量生长的钝顶螺旋藻，其主要激发和发射峰分别为615 nm和647 nm。(6)最后，为了阐明日粮中螺旋藻对生长性能的影响，给肉鸡饲喂含有螺旋藻的实验日粮，剂量分别为0、40或80g/kg，为期16天。结果表明，不同处理之间在体重、体重或选定性状(包括肝脏、腹脂、肾脏和胸深肌)的重量或产量(占体重的百分比)方面都没有显著差异。目前，我们正在研究‘稳定的’钝顶螺旋藻基因转移系统的其他要求，其中包括：a)使用携带无启动子的猫报告基因的质粒克隆和评估螺旋藻启动子，B)建立转座技术，包括在体外形成释放的Tn5转座复合体，然后通过电穿孔将该复合体导入螺旋藻细胞。建立稳定的螺旋藻基因转移系统作为生产重组酶的生物工厂具有许多优势。较少

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