【作者】 丁一新；

【导师】 赵明文；

【作者基本信息】 南京农业大学 ， 微生物学， 2008， 硕士

【摘要】 灵芝(Ganoderma lucidum)在我国已有悠久的应用历史,可用于多种疾病的治疗。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药效高低的重要指标。作为次生代谢产物,三萜类化合物是通过甲羟戊酸途径,由法呢酯焦磷酸产生鲨烯、再转化成2,3-氧化鲨烯,最后经过一系列的氧化、还原和环化反应形成的。法呢基焦磷酸合酶(FPS)处于甲羟戊酸途径中的第一个分支点,是类异戊二烯生物合成过程中的一个关键酶,它可以催化两分子异戊烯基焦磷酸与二甲基烯丙基焦磷酸的相继性头尾缩合,形成法呢基焦磷酸(FPP)。FPS的含量与活性高低会影响三萜化合物产量的多少。根据已发表的其它物种FPS氨基酸序列的保守区,设计简并引物,以灵芝cDNA文库为模板,经PCR扩增,获得了一个456 bp的灵芝法呢基焦磷酸合酶基因特异cDNA片段。利用获得的灵芝法呢基焦磷酸合酶基因特异片段,设计特异性引物,结合cDNA文库的通用引物,扩增得到灵芝FPS基因的5′端cDNA片段。提取灵芝原基时期的RNA,经反转录后的cDNA作为模板,通过3′RACE,获得灵芝FPS基因的3′端cDNA片段。根据扩增获得的序列信息设计引物,以反转录的原基cDNA为模板,得到灵芝FPS基因的全长cDNA序列,该序列全长为1083 bp,编码360个氨基酸残基。其推导的蛋白分子量为42.27kDa。根据灵芝FPS基因全长cDNA序列设计引物,扩增灵芝的基因组DNA,得到灵芝FPS基因的全长基因组序列,该序列全长为1388 bp,FPS基因由4个外显子和3个内含子构成。灵芝FPS蛋白序列比对发现,灵芝FPS与另一种担子菌黄汁乳菇的FPS表现出最高的相似性(59%),并且包含有不同物种FPS序列中的四个保守区。在FPS的分型中,灵芝FPS属于Ⅰ型(真核状态)法呢基焦磷酸合酶,这与灵芝的物种分类相吻合。将灵芝FPS基因全长cDNA序列克隆到酵母表达载体pYF1845中,转入因缺失FPS活性而依赖麦角甾醇的酵母突变株CC25。结果转化子可以在36℃、不含麦角甾醇的培养基平板上生长,证实了所获得FPS基因cDNA序列的功能。根据灵芝FPS基因DNA序列设计引物,以灵芝基因组DNA为模板,扩增FPS基因上游启动子序列,该序列长约1288 bp,用Softberry软件对启动子序列进行分析,发现灵芝FPS基因启动子中包含有明显的TATA-box和CAAT-box,并发现了多个与固醇、激素和环境压力等调节相关的顺式作用元件。通过竞争性RT-PCR的方法,检测了不同发育阶段灵芝中FPS在mRNA水平的表达差异。结果证明,在灵芝原基中,FPS mRNA的转录水平要明显高于菌丝体中的转录水平。这种差异与Hirotani等所报道三萜含量的变化呈正相关。在培养基中添加植物激素茉莉酸甲酯(methyl jasmonate,MeJA),对灵芝三萜的生物合成有很好的诱导效果。基于此,对由不同浓度茉莉酸甲酯处理的菌丝体进行了灵芝FPS基因mRNA转录水平的检测。50～200μm茉莉酸甲酯作用下,灵芝FPS基因的转录本表现出持续性增加,150μm的浓度是最佳诱导浓度。同时,在灵芝FPS基因的启动子中发现了两个茉莉酸甲酯响应元件,这样的结果综合表明,灵芝FPS基因可能是经由茉莉酸甲酯响应元件而参与了由茉莉酸甲酯信号调控的灵芝三萜生物合成途径。更多还原

【Abstract】 Ganoderma lucidum,called "Lingzhi" in China,has a long history of use for the treatment of many different diseases.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.As a secondary metabolite,ganoderic acid is synthesized via the mevalonate pathway, where farnesyl diphosphate is converted to squalene,then to 2,3-oxidosqualene,and finally undergoes a series of cyclization,oxidation,and reduction reactions.Farnesyl-diphosphate(FPP) synthase(FPS;EC 2.5.1.1./EC 2.5.1.10) catalyzes the sequential 1’-4 condensation of two molecules of IPP with dimethylallyl diphosphate (DMAPP) and the resulting 10-carbon compound geranyl diphosphate(GPP) to produce the 15-carbon compound FPP.FPS is located at the first multiple branch point in the isoprenoid biosynthetic pathway,suggesting that FPP biosynthesis is tightly regulated. Therefore,changes in FPS activity might alter the flux of GA.Based on a sequence comparison between the FPS genes from other species, degenerate oligonucleotides from the most conserved regions were synthesized for PCR amplification of cDNA library.A 456 bp putative cDNA fragment was obtained and sequenced to confirm that this fragment shared high similarity with known fungal FPS sequences.Specific primers were designed on the basis of the specific 456-bp fragment of FPS gene of G.lucidum.Collaborated with the universal primers of the cDNA library vector,the 5’ end of G.lucidum gene was amplified.Total RNA was prepared from G. lucidum primordium and reverse-transcribed to cDNA.We employed a 3’ RACE strategy to obtain the sequence of 3’ end of G.lucidum gene.Specific primers were designed according to the obtained sequence information and the full length of cDNA was amplified. Analysis of the GIFPS cDNA sequence indicated the presence of an open reading frame(ORF) of 1,083 bases.The ORF encoded a 360-amino acid polypeptide and a theoretical molecular mass of 41.27 kDa.The genomic sequence of G.lucidum FPS gene was amplified by the primes designed according to the 5’ and 3’ ends of FPS gene.The full-length genomic sequence was 1388 bp;the gene was consisted of 4 exons and 3 intronsProtein sequence comparison indicated that GlFPS is closely related(59%identity) to the FPS of Lactarius chrysorrheus,another basidiomycetous fungus.The alignment of GlFPS with orthologs available in the databases showed four highly conserved regions. GlFPS contains the typeⅠ(eukaryotic) FPS signature,which is consistent with traditional evolutionary affinities.The cDNA sequence of FPS gene was cloned into yeast expression vector pYF1845. The recombinant plasmid was introduced into a FPS-defective yeast mutant strain CC25, whose growth requires ergosterol.Transformants could grow on media lacking ergosterol at 36℃.The results indicate that GlFPS is a functional fungal gene encoding FPS in G. lucidum.The promoter sequence of FPS was amplified from G.lucidum genomic DNA by the SEFA-PCR method.Using Softberry software,predicted TATA boxes and CAAT boxes were found.The potential regulatory elements associated with sterol,hormone,and stress-related responses were also found in the GlFPS promoter region.We monitored the levels of the GlFPS mRNA at different developmental stages by competitive PCR.The GlFPS mRNA level was relatively low in mycelia.However,the fruiting process increased the GlFPS mRNA level.The higher GlFPS mRNA level was probably correlated with the increased triterpene synthesis as described by Hirotani et al..Treatment of mycelia with exogenous methyl jasmonate(MeJA) also caused a large accumulation of triterpene biosynthesis.Thus,we monitored the effect of MeJA on the GlFPS mRNA level in the mycelia.GlFPS transcripts accumulated following the addition of 50-150μm MeJA to the culture medium,whereas mycelia treated with 200μm MeJA showed reduced promotion of GlFPS mRNA.The highest mRNA level was observed at a concentration of 150μm.Interestingly,sequence analysis of the GlFPS promoter identified two potential MeJA responsive elements.Our results suggest that GlFPS is probably involved in the triterpene biosynthesis regulation pathways of MeJA signalling via the putative MeJA-response elements.更多还原