Analysis And Inactivation Of Genes Involved In Signal Transduction In Anabaena PCC7120

This dissertation searched for Ser/Thr and Tyr kinases and phosphatases genes by bioinformation tools in Anabaena PCC7120, inactivated the genes involved in protein phosphorylation/dephosphorylation, and analyze the phenotypes of mutants. 1. Search for Ser/Thr and Tyr kinases and phosphateses in Anabaena PCC7120We found 52 genes encoding Ser/Thr and Tyr kinases (STPK) and 14 genes encoding ser/thr and Tyr phosphateses (PP). We divided all STPKs into 5 subfamilies according to their own structure characterizes. STK-I only possess regions similar to the typical catalytic domain of STPKs, STK-II contain WD40 repeats, STK-III contain all kinds of regulator domain in their C-terminal region, STK-IV have their catalytic domains located in the N-terminal region, While HSTK-I also have a his kinase domain of two-component system. Among 14 PPs genes, 8 of them belong to PP2C, 3 belong to PTPs and only 3 genes encoding PP1/PP2A/PP2B could be found. Only 11 genes can be found similar genes in the Synechocystis PCC6803 genome by genomic analysis. And via the analysis of genetic array, 12 genes can be classified into 6 gene clusters in the chromosome.2. Inactivation of genes which involved in signal transduction and analyze the phenotypes of mutantsAll the mutants in this study were inactivated by inserting a resistance gene. PCR products were inserted into pBluescriptSK using PstI and XhoI. The element bearing resistance to streptomycin(Sm) and spectinomycin(Sp) was excised from pHP45 and inserted into gene coding region. The disrupted gene was isolated as a Pstl and Xhol fragment and cloned into pRL271. Triparental mating was then performed. As a result, we obtained 91 recombined plasmids, 23 possible single-cross mutants and 53 possible double-cross mutants. PCR analysis of the genomic DNA from the resulting possible double-cross mutants indicated that 6 of them were wild-type, 4 of them were single-cross mutants and the remainder ones were true double-cross mutants. Observe the heterocysts and growing states of these mutants after nitrogen depletion and found that A112898-, All 1731- can not different heterocyst, A114141-, A111171- and A114761- can different heterocysts but still can not maintain normal growth. The results indicated that all these genes play roles in hetetocyst differention.