The Identification Of MAAs Biosynthetic Genes And The Establishment Of Genetic Transformation Method In Nostoc Flagelliforme

Nostoc flagelliforme [Nostoc flagelliforme (Berk&Curtis) Bornet&Flahault], is a terrestrial economic cyanobacterium with high food, medicinal value and vital ecological significance. It is classified the first-grade state protection wild plant in China. Many researchers dedicated to the study of artificial culture of Nostoc flagelliforme, but the progression moved slowly. Our laboratory has cultured filamentous thallus similar to nature Nostoc flagelliforme with low dose of UV-B treatment and periodic desiccation. During the process, we found the content of MAAs increased significantly induced by low dose of UV-B. But the variety, biosynthesis and regulation mechanism of MAAs are not clear so far. The effects of MAAs on the morphogenesis of Nostoc flagelliforme remains to further investigation. Meanwhile, the genetic manipulation of Nostoc flagelliforme has not been established and it was a major obstacle to research its critical functional genes deeply. This study identified the variety and biosynthetic genes of MAAs, and established the genetic transformation method in Nostoc flagelliforme. It layed an important foundation of revealing the effects of MAAs on the morphogenesis of Nostoc flagelliforme mediated with low dose of UV-B. Main results are summarized as follows:1. The purified MAAs of Nostoc flagelliforme was detected at8.3min through HPLC separation. The molecular weight of MAAs was identified to be378by LC-MS, which is a different MAAs type form all reported MAAs. At the same time, we identified the biosynthetic genes of MAAs with the help of filamentous cyanobacteria Anabaena sp. PCC7120. There is a MAAs-synthetic gene cluster which contains five genes, separately named ORF1, ORF2, ORF3, ORF4and ORF5in Nostoc flagelliforme. ORF1and ORF2participate in the synthesis of mycosporine core. ORF3, ORF4and ORF5are responsible for attaching amino acids to mycosporine core via imine linkages. The transgenic Anabaena sp. PCC7120cells containing ORF1-2, ORF1-4and ORF1-5show higher UV-B endurance capacity than their wild type.2. By modification with the conjugal transfer methods of Nostoc punctiforme ATCC29133and Anabaena sp. PCC7120, we established a conjugal transfer method of Nostoc flagelliforme. The procedure is described as follows:sonicate the Nostoc flagelliforme cells, resuspend the pellet in fresh BG-11, then put it in the incubator overnight to allow the cells to recover. Collect the E. coli strain cultivated to log phase, mix with the concentrated recovery Nostoc cells, spread on the filter on BG-11plate. Then place the plate at low light intensity (5-10μmol photons m-2s-1) two days, normal light intensity (25-30μmol photons m-2s-1) four days. Transfer the filter to new plate containing the appropriate antibiotics and continue to incubate. The colonies appeared after one month.