DEVELOPMENT OF SHUTTLE VECTOR FOR GENE TRANSFER IN MICROCYSTIS AERUGINOSA

这项建议的目的是开发一种利用遗传系统来研究铜绿微囊藻UV027毒素和藻胆体产生的调控的方法。UV027是一种潜在的健康危害淡水蓝藻。我们所构建的Lambda ZAP II铜绿假单胞菌UV027基因组DNA文库将用于相关基因的筛选。异源探针将被用来分离与毒素的生物合成有关的多肽合成酶基因，微囊藻毒素是一种蛋白磷酸酶抑制剂。微囊藻毒素是以非核糖体的方式合成的。一旦确定，在可诱导启动子的控制下，多肽合成酶基因将被克隆到穿梭载体中，并用于转化缺乏制造微囊藻毒素能力的微囊藻菌株。改变的基因将被用来试图抑制产生毒素的菌株中微囊藻毒素的产生。藻胆体研究将通过使用异源探针分离别藻蓝蛋白的阿尔法亚单位的基因组来启动。一旦确定特征，该基因将被用于分离和鉴定别藻蓝蛋白操纵子。将突变基因克隆到我们构建的穿梭载体pMaL或pMaL-7中，转化微囊藻进行藻胆体组装研究。最近报道的几种蓝藻的调控基因和转录DNA共同序列将被用于寻找微囊藻的相似序列。一旦发现，将获得下游和上游序列，以确定哪些基因受到调控，以及涉及的调控机制(S)。还将研究控制蓝藻毒素产生和生长的潜力。

The object of this proposal is to develop a use a genetic system to study the synthesis and regulation of toxin and phycobilisome production in Microcystis aeruginosa UV027, a fresh water cyanobacterium implicated as a potential health hazard. A lambda ZAP II M. aeruginosa UV027 genomic DNA library, which we made, will be screened for relevant genes of interest. Heterologous probes will be used to isolate a peptide synthetase gene that is implicated in the biosynthesis of the toxin, microcystin, a protein phosphatase inhibitor. Microcystins are synthesized non-ribosomally. Once characterized, the peptide synthetase gene, under the control of an inducible promoter, will be cloned into the shuttle vector and used to transform Microcystis strains lacking the ability to make microcystins. Altered genes will be used to attempt to inhibit microcystin production in toxin-producing strains. Phycobilisome studies will be initiated by using a heterologous probe to isolate the genome for the alpha-subunit of allophycocyanin. Once characterized, this gene will be used to isolate and characterize the allophycocyanin operon. Mutated genes will be cloned into the shuttle vectors pMaL or pMaL-7, which we constructed, to transform Microcystis to study phycobilisome assembly. Regulatory gene and transcriptional DNA consensus sequences recently reported for several cyanobacteria will be used to search for similar sequences in Microcystis. Once found, downstream and upstream sequences will be obtained to determine which genes are being regulated and the regulatory mechanism(s) involved. The potential for controlling toxin production and growth of the cyanobacterium will also be examined.