Molecular Structure Of MAAs In Nostoc Flagelliforme And The Transcription Factor Regulating Its Synthesis

The terrestrial cyanobacterium Nostoc flagelliforme,mainly distributed in arid and semi-arid,desert and semidesert regions,is a first-class national protective wild plant.It has important ecological and edible value.Due to the special nature of its habitat,it has strong adaptability to drought and ultraviolet environmental stress.Early researches in my lab found that low dose UV-B induced formation in their natural form in Nostoc flagelliforme cultivation process.In this process,the content of mycosporine amino acids(MAAs)was significantly increased.However,it is not clear what molecular structure of MAAs is,in addition,the synthetic route and molecular regulation mechanism hasn't been researched in Nostoc flagelliforme.Thus,the present study is based on preliminary laboratory work.We identify the molecular structure of MAAs,explain MAAs synthetic route and study transcription factor which regulated MAAs synthesis.There are the following research results:1 The maximum ultraviolet absorption wavelength of MAAs extracting from Nostoc flagelliforme by using methanol is 330 nm.By the early identification of MAAs synthesis gene cluster containing five genes in Nostoc flagelliforme,respectively,ORF1,ORF2,ORF3,ORF4 and ORF5,we got these five genes into Anabaena PCC 7120.Then,we identified the synthetic substances of ORF1-5 transgenic strains was the same to MAAs.Its synthetic substances were purified by molecular sieve and then used through LC-MS for separation and identification of molecular weight.The molecular weight of MAAs is 756 Da,which is different from reported MAAs.MAAs obtainted by molecular sieve and preparative HPLC had high purity.Ultimately,the molecular structure as follows was confirmed by 1H NMR.It is different from the MAAs of other species,namely Nostoc flagelliforme has synthesized a new type of MAAs.Anabaena PCC 7120 can't synthesize MAAs.In order to further study MAAsbiosynthetic pathway of Nostoc flagelliforme,the MAAs biosynthetic genes ORF1-2,ORF1-3,ORF1-5 were transferred to Anabaena PCC 7120.The synthesis product of eachgene was studied.As a result,ORF1 and ORF2 involved in the synthesis of cyclic skeleton,then,ORF3,ORF4 and ORF5 involved in processing amino acid to side chain of MAAs.2 This study verified interaction between transcription factor ORF4223 regulated MAAs synthesis which was found in early laboratory reaserch and promoter element probe-5 by yeast one-hybrid analysis.While taking advantage of the establishment of transgenic system in our lab,ORF4223 over-expressing transgenic Nostoc flagelliforme strains were obtained.It confirmed ORF4223 overexpression Nostoc flagelliforme strain increased the synthesis of MAAs and enhanced tolerance to UV-B radiation.In ORF4223 as bait by yeast two-hybrid screening cDNA library of Nostoc flagelliforme,we got some ORF4223 potentially interacting protein-coding genes,which were conducive to a variety of metabolic pathways for further study of Nostoc flagelliforme.