DEVELOPMENT OF SHUTTLE VECTOR FOR GENE TRANSFER IN MICROCYSTIS AERUGINOSA

本研究的目的是建立一个遗传系统来研究铜绿微囊藻（Microcystis aeruginosa）UV 027的毒素和藻胆体的合成与调控。A lambda ZAP II M.利用构建的铜绿假单胞菌UV 027基因组DNA文库，筛选相关基因。异源寡核苷酸探针将用于分离肽合成酶基因，该基因与毒素微囊藻毒素的生物合成有关，微囊藻毒素是一种蛋白磷酸酶抑制剂。微囊藻毒素是非核糖体合成的。一旦表征，在诱导型启动子控制下的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属菌株。改变的基因将被用来试图抑制产毒素菌株中微囊藻毒素的产生。藻胆体研究将开始使用异源探针分离的基因组的α-亚基的别藻蓝蛋白。一旦表征，该基因将用于分离和表征别藻蓝蛋白操纵子。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，研究藻胆体的组装。最近报道的几种蓝藻的调控基因和转录DNA的一致序列将被用来寻找类似的序列在微囊藻。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。控制毒素的生产和蓝细菌的生长的潜力也将被检查。

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.