Studies On The Preparation Of Hericium Erinaceus And A Preliminary Analysis On Genome Squence

Objective:Tosequence the Whole-genome sequence of monokaryon whic h regenerates from the protoplast of Hericium erinaceus(Rull ex F.)Pers., a nd identifiy the key gene by genes analysis of diterpenoids biosynthesis; Tr y to establish a genetic transformation system by ATMT.Methods: Single factor experimental method has been used for the rese arch of preparation of protoplast. The factors include: Three types of enzy me systems, Concentration oflywallzyme, Types of carbon source and additi ve ingredients in synthetic medium, Mycelial age.The Orthogonal test of fo ur factors andthree different levels has been used for the research of regen eration of protoplast. The factors include: concentration of lywallzyme, enz ymolysis time, enzymolysis tempreture and mycelial age. The factors in sin gle factor test include: types and concentration of osmotic pressure stabilize r and ingredients in regeneration medium. Whole-genome of H.erinaceus m onokaryons has been sequenced bynext-genration sequencing platform. The key gene has been researched by genes analysis of diterpenoids biosynthesi s. The genetic transformation system has been established by ATMT.Results: The higest yield of protoplast can be acquired by using the li quid fermentation mycelium which cultured in potato medium for 72 h. Theoptimal treatment conditions is2% lywallzyme, 0.6mol/LKCl, p H5.5 and 30℃.The highest regenration were obtained from 72 h old mycelium after trea tment with 2.0%(w/v) lywallzyme for 3h at 30℃.The best osmoticstabilizer s for regeneration of H.erinaceus protoplasts is 0.6mol/L mannitol. The gen ome size of H.erinaceus is greater than 50 M, and GC% is greater than 40%.Phylogenetic tree of GGPPs gene indicate that the relationship between H.e rinaceusand Penicillium aurantiogriseum is closed. Two transformation has been obtained which doesn’t integrate with hygromycin resistance gene.Conclusion: The conditions for preparation and regeneration of protopla st are very important to the sequecnce of the whole-genome sequence of H.erinacers. The identification of the key gene in the biosynthesis of diterpen oid can lay a foundation for the regulation of secondary metabolism resear ch. The test steps of ATMT are fussy, however, conversion efficiency is hi gh and destructivepower is lower than other methods.