Cloning And Characterization Of Genes In Different Developmental Forms Of Lentinula Edodes' Mycelium

Lentinula edodes (Berk.) Pegler is a kind of well-known large wood decaying fungus,with a cultivation history of more than 800 years in China.The fruiting body formation of L. edodes is the most complex developmental processin the life cycle of L. edodes. It is a highly organized process, which requires thecoordination between genetic, environmental, and physiological factors. In the initial stageof fruiting body development, upon nutritional depletion, hyphae form localized, highlybranched structures termed hyphal knots. Hyphal knots then develop into globosestructures, called sclerotia. The hyphae of the sclerotium differentiate further to formprimordium, that is essentially an embryonic fruiting body. This paper presents a simpleand inexpensive procedure for RNA extraction from L. edodes at various developmentalstages, especially from recalcitrant tissues of the fruiting body. When compared with otherroutine methods for RNA purification, the unique characteristic of this procedure isinclusion of a saturated NaCl solution treatment before continuing with organic solventextraction. This step promotes the removal of polysaccharides and proteins, and high yieldsof RNA can be readily obtained. Then the technique of mRNA differential display is usedto investigate the difference of gene expression between mycelium, hyphal knots, sclerotiaand primordium. Total RNA is firstly extracted from tissues at four different developmentalstages mentioned above, then cDNA is synthesized and RT-PCR performed using anchorprimers and arbitrary primers. After electrophoresis in agar gel, DNA fragments aredifferentially displayed. On the whole, the difference of gene expression betweenmycelium, hyphal knots, sclerotia and primordium is not obvious. By homology alignmentwith the database in Gene Bank, seventeen ESTs with altered expression involved inprotein synthesis, modification of cell wall, hyphal interaction, metabolism of C and Nsources, substrate transport, signal tranduction and cell rehabilitation are obtained. One ofthese ESTs proves to be the endopolygalacturonase gene, which shows an abundant expression pattern at the primordium stage after confirmation by Real-time PCR, while itsexpression at the three preceding stages is relatively low, demonstrating that it may beclosely invovled in primordim morphogenesis.