【作者】 徐飞；

【导师】 赵明文；

【作者基本信息】 南京农业大学 ， 微生物学， 2007， 硕士

【摘要】 灵芝[（Ganoderma lucidum（Leyss.ex.Fr.）Karst）]属于真菌界（Mycota）,担子菌亚门（Basidiomycotina）,多孔菌目（Aphyllophprates）,灵芝菌科（Ganodermataceae）,是我国著名的药用真菌,具有重要的药理作用,作为药物已经有2000多年的历史。三萜类化合物是灵芝的重要药用成分,具有保肝、抗肿瘤、抑制组织胺释放、抑制胆固醇的合成和吸收以及抗HIV和抗HIV蛋白酶的作用等功能。因此,灵芝中三萜含量的高低已经成为灵芝质量高低的重要指标。目前要通过分子生物学技术培育优良的灵芝菌株、提高灵芝菌株的药用价值,就必须构建灵芝的高效表达载体。在一些报道的其他真菌中,提高转化效率的一种方法就是使用同源强启动子,而在灵芝中,就目前为止,还没有这样的启动子被报道。在酿酒酵母中,三磷酸甘油醛脱氢酶被报道是大量表达的,在酵母中,god基因占总mRNA 2%-5%,因此三磷酸甘油醛脱氢酶基因启动子是一个强启动子,而且在很多不同的真菌中,gpd基因启动子已经被成功地用来构建转化系统。根据已报道的GPD氨基酸序列,针对GPD的保守区域设计简并引物,以灵芝基因组DNA为模板,经PCR扩增,获得一个长624bp的灵芝三磷酸甘油醛脱氢酶基因特异DNA片段。利用已经获得的灵芝gpd基因特异片段,设计一对专一引物,结合cDNA文库两臂的通用引物,分别扩增得到灵芝god基因的5′和3′端DNA片段,根据基因的5′和3′端DNA序列设计引物,扩增灵芝的cDNA文库,得到灵芝gpd基因的全长cDNA序列,该序列全长1014bp,编码337个氨基酸。将灵芝gpd基因全长cDNA序列进行Blast分析,发现与基因具有较高同源性的物种是Pycnoporus coccineus。根据基因的5′和3′端DNA序列设计引物,扩增灵芝的基因组DNA,得到灵芝gpd基因的全长基因组序列,该序列全长1401bp。灵芝gpd基因中,由7个外显子和6个内含子构成。对灵芝gpd基因进行一系列生物信息学分析,发现gpd基因内存在1个保守的活性位点,利用Clust X和PUAP4.0建了灵芝gpd基因的系统进化树,发现其与真菌界中担子菌亲缘关系最为密切,进化关系符合经典的物种分类的结果。通过RT-PCR的方法,证实了在灵芝中,gpd基因表达量很大。根据灵芝gpd基因组DNA序列,设计引物,以总基因组DNA为模板,采用TaKaRaLA PCR^TMin vitro Cloning Kit试剂盒扩增了其上游启动子序列,该序列长约1.2Kb,使用了启动子分析软件,在5′的启动子区域发现有两个可能的启动子区域,启动子区域包含有2个CAATboxes（CCAAT）,还有一个TATAbox（TATAAAA）,没有发现在A.nidulans and A.niger gpd gene中发现的gpd,pgk,qut,and qa boxes。以pCAMBIA-1300质粒为母板,构建双元载体GLP-1300,PCR扩增潮霉素基因以及烟草花叶病毒35S终止子,然后将克隆的片段插入pCAMBIA-1300质粒母板中,1.2kb的灵芝gpd启动子序列用来调控潮霉素基因的表达,并使用这个质粒进行PEG转化灵芝的原生质体,转化效率为约为20个/μg plasmid DNA+10⁷个原生质体,转化子在不含潮霉素的培养基上经五代以上的继代培养后,仍然能稳定保持潮霉素抗性。PCR验证潮霉素基因已经整合到灵芝的基因组DNA中。更多还原

【Abstract】 Ganoderma lucidum（Leyss.:Fr.）Karst（G lucidum）is a species of basidomycetes that belongs to Ganodermaceae of Aphyllophorales,which has been popular as a healthy food and medicine for more than 2000 years in China.Ganoderic acid（GA）produced by this higher fungus has a number of important biological functions including cytotoxicity to hepatoma cells,inhibition of histamine release,inhibition of cholesterol synthesis and absorption,stimulation of platelet aggregation,and anti-HIV and anti-HIV-protease activities.The quality of G.lucidum products was determined by the content of GA.Attempts to enhance and extend the medicinal applications of G.lucidum through strain improvement have been hampered by the lack of an efficient transformation system. In other fungi,one approach to alleviating this shortfall has been the construction of transformation vectors in appropriate hosts using a strong endogenous promoter,however, to our knowledge,no homologous gene promoters have been reported in G.lucidum. Previous observations in the yeast,Saccharomyces cerevisiae,and in higher eukaryotes have suggested that the glyceraldehyde-3-phosphate dehydrogenase（gpd,EC 1.2.1.12） gene（gpd）is under the control of a highly active promoter.In such cases,the GPD protein is a tetramer composed of identical subunits and comprises up to 5%of the soluble cellular protein.Furthermore,2-5%of the poly（A）+ RNA in yeast is gpd mRNA.The flanking regulatory regions of these gpd genes have also been used successfully for the construction of transformation vectors in different fungi.Amino acid sequences conserved among a number of known gpd gene were used to design two degenerate oligonucleotide primers for PCR amplification of G.lucidum genomic DNA.Sequencing of the amplification product revealed a fragment of 624 bp encoding an amino acid sequence corresponding to gpd.Specific primers were designed according to the genomic DNA sequence.Collaborated with the universal primers of the cDNA library vector,the 5′and 3′end of G.lucidum gene was amplified.According to the sequence of the 5′and 3′end of G.lucidum gpd gene,two specific primers were designed, and the full length of cDNA was amplified from the cDNA library.The cDNA sequence was 1014bp,encoding 337 amino acid.Blast x program was used to search NCBI database and analyze the homology of the deduced G.lucidum GPD amino acid sequence.The sequence showed a relatively high homology with the sequence of Pycnoporus coccineus. Two specific primers were designed and synthesized based on the sequence of the gpd cDNA sequence,the full length genomic sequence（1401 bp）of G.lucidum gpd gene was amplified from G.Iucidum genomic DNA.Comparing the genomic DNA sequence with the cDNA sequence of gpd gene,7 exons and 6 introns,with a typical intron’s border "GT—AG" can be found.One conserved catalysis site were found using sequence alignment with some fungi gpd genes.A phylogentic tree of GPD was constructed with the program of Clustal X and PAUP 4.0.The results demonstrated that G.lucidum gpd gene is most closely related to basidiomycotina.The 5’-flanking region of G.lucidum was cloned by means of TaKaRa LA PCR^TMin vitro Cloning Kit according to the manufacturer’s instructions（TaKaRa）.Gene-specific primer（GSP）was designed from G.lucidum gpd sequence.There was one typical TATA box（TATAAAA）and two CAAT boxes（CCAAT）located in the 5’-flanking region.By the use of the promoter prediction software,two possible core promoter regions were found at the 5’-flanking regionThe gpd,pgk,qut,and qa boxes featured in the promoter of A. nidulans and A.niger gpd gene were not found in in the gpd promoter of G.lucidum.The hygromycin B phosphotransferase（hph）gene with cauliflower mosaic virus （CaMV）35Sterminator was amplified from pCAMBIA1300,then the amplified fragment was cloned into a pCAMBIA1300 mothboard and a 1.2kb promoter region of G.lucidum gpd gene was introduced to drive hph gene expression.The resulting plasmid,GLP-1300, was used for the transformation experiment.The transformation efficiency of this PEG-mediated method reached to 20 transformants/μg plasmid+10⁷protoplast.Transformants could still show HmB resistance after cultivating over 5 subcultures on media without HmB.An analysis of Ganoderma lucidum transformants using PCR revealed that foreign gene had integrated into Ganoderma lucidum’s genome.更多还原