Cloning Of Medicinal Gene And Promoter From Ganoderma

Ganoderma is one kind of Chinese medicine recorded in Pharmacopeia of the Peoples' Republic of China (first part), 2000. Ganoderma has many kinds of definite curative effect. This research work aims at the clone of medicinal genes and endogenous promoters. we hope our research work can provide technique support for the production of ganoderma medicinal component by zymosis. This research work is composed of four parts of work.The first part is clone of Ling Zhi immunomodulatory protein gene(LZ-8).In this part, we cloned 11 fragments using two pairs of primers (GimPFl-GimPRl, GimPF2-GimPR2), by genome PCR technique. The sequences we got are marked LY21 (about 620bp) , LY22 (about 620bp), LY23 (602bp), LY24 (671bp) , LY29 (658bp) , LY32 (about 750bp) , LY33 (about SOObp) , LY34 (about 750bp) ,LY35 (about 750bp) ,LY36 (about 750bp) and LY37 (about 750bp) . Four of these 11 fragments, LY32, LY35, LY36 and LY37, include the whole sequence of LZ-8 gene. The result of comparison within the four sequences shows us that LZ-8 gene from Tian Zhi is a little bigger different from the other three from Zi Zhi, Han Zhi and Chi Zhi. So we think maybe the divergence take place earlier between Tian Zhi and the other three kinds of Ling Zhi than among Zi Zhi, Han Zhi and Chi Zhi.The second part is clone of , 3-Glucan synthase gene.In this part we designed two pairs of primers (LgluFl-LgluRl, LgluF2-LgluR2), and got one sequence marked LY28 by genome PCR technique. But LY28 isn't the interesting gene, there is no high similarity sequences in Genebank neither.The third part is clone of tyrosinase gene.In this part we design two pairs of primers (TyragaFl-TyragaRl, TyrlenF2-TyrlenR2), and got 8 sequences marked LY1.1 (about 1500bp) , LY2.4 (about 750bp) , LY3.1 (1552bp ) ,LY3.2 (about 800bp) ,LY5. 3 (about ISOObp) .LY9.2 (about lOOObp) .LY10.2 (584bp) .LY11.1 (about 21 OObp) by genome PCR technique. All the eight sequences find no high similarity sequences in Genebank, in nucleic acid analysis. But LY1.1 and LY3.1 have over 30% similarity with 26S protease regulatory subunit from many kinds of regions, in amino acid analysis.The forth part is clone of ras and gpd promoters.This part can be separated into 3 steps:(1) promoter clone. we designed 5 pairs of primers (rasF1-rasRl, gpdFl-gpdRl,. gpdF2-gpdR2, LgpdFl-LgpdR, LgpdF2- LgpdR) and got 12 sequences marked: LY14.1 (1373bp), LY15.1 (682bp), LY16.1 (404bp), LY17.1(724bp), LY18.3(1050bp), LY19. 3(1243bp), LY20. l(812bp), LY25(1093bp), LY26 (1179bp) , LY27 (740bp) , LY30 (about 1200bp) , LY31 (613bp) .(2) Sequence analysis. The result of BLASTn analysis shows that LY31 has 98% similarity with Lentinus edodes gpd promoter. The others has no high similarity sequences in Genebank. We also use "ProScan.7" and"Promoter 2.0 Prediction"to analysis the 12 sequences. The result is: LY26 LY14.1 and LY17.1 have high potential promoter activity. LY26 get 72. 28 scores in ProScan 7 analysis (cutoff score=53. 00). It's potential promoter zone locates in forward strand 569-819bp.LY17.1 get 69. 20 scores in Proscan 7 analysis. It's potential promoter zone locates in revert strand 519-269bp. LY14.1 is also regarded high possibility promoter in Promoter 2.0 Prediction analysis. It's transcription start site locates at 700bp. And LY3.1 also has potential promoter activity in promoter predict.(3) Test for promoter activity. In order to test the sequences we get really have promoter activity or not, we construct three expression vector through displacing 35S promoter by potential promoters (LY3.1, LY19. 3 and LY31). The expression vector are named as: "EPS. 1, EP19.3andEP31" . After transform them into straw mushroom, we examine the GUS activity of putative transformant. The results show us that LY3.1 has no promoter activity, LY19. 3 has weak promoter activity, LY31 has strong promoter activity.