Cloning Of GPD Promoter And Related Medicinal Component Gene From Grifola Frondosa

Maitake (grifola frondosa) is the Japanease name, for an edible fungi with a large fruiting body characterized by overlapping waves.Maitake is increasingly being recogonized as an advanced healthy food in North American and Japanease market.In this study , we designed 3 pairs of specific primers according to GPD(glyceraldehyde-3-phosphate dehydrogenase gene) promoter sequence of Lentinus edodes that is reported with strong promoter activity. Using PCR technique, we have got 8 fragments from maitake DNA, six of these(gpd11 gpd23 gpd24 gpd25 gpd31 gpd43) is amplified by primer gpdFl/gpdRl that are little similar with sequences in GenBank .The frangment(Lgpd114) by LgpdFl/R and one(Lgpd211) of these by LgpdF2/LgpdR are high similar with the upstream sequence of gLeGPD gene of Lentinus edodes , respectively 96% and 98%.By promoter prediction software, wo have got the conclusion that gpd23,gpd24,gpd25,Lgpd114, Lgpd211 maybe have strong promoter activity.They have many important cis-acting element such as TATAbox, Ibox, Ibox, CAATbox, GATAbox, GCCcore. All these elements play an important role in transcription and translation. The above sequences that have promoter activity forcastedly are inserted into the expressed vector pCAMBIA1301, to displace 35S promoter that make GUS expressed. Then the new constructed expressed vectors were transformed into straw mushroom. By GUS dyeing, we can get the result that Lgpd114 has stronger promoter activity.In this research, we are to isolate some medicinal gene, with maitake mycelium as the trial material, so mat to know about maitake medicinal component deeply. Based on reported tyrosinease gene,immunoregulatory protein gene and glucan synthase gene sequence ,primers are designed. By PCR technique, we have got 12 fragment from maitake DNA, six (01,115,122,3114,44,46)of these are amplified by tyrosinease primers, and three(314,413,422) of these amplified by immunoregulatory protein primer, and three (513,521,613)of these amplified by -glucan synthase primer as well.All these fragments are presented to NCBI and analysed by BLASTN and BLASTX. The resultis that in GeneBank, there have no sequence that is high similar with the 12 fragments. Among these ,by BLASTX, tyr01 and tyr115 are a little similar with Monoblepharella sp.JEL15 and hypothetical protein UM01332.1 [Ustilago maydis 521],respectively 39% and 34%,and 513 is similar with Protein KIAA 1404,40%. 314 is similar with hypothetical protein [Neurospora crassa], 50%.