Optimizing Transformation Conditions And Gene Smk Research Of Wolfiporia Cocos

Wolfiporia cocos is one of our traditional Chinese medicine,has a very high medicinal value and dietary nutrition value.This topic selects Wolfiporia cocos strain GIM5.219 and systematically studies its conditions of protoplast preparation and regeneration and further optimizes protoplast-mediated transformation.Research Smk gene which involved in regulation of sclerotium forming using the modern molecular biology techniques and methods.To explore new cultivation technique does not rely on pine resources,precise regulation of Wolfiporia cocos sclerotium forming condition and increase yield,promote the development of industrialization of Wolfiporia cocos has important guiding significance.The results of the study are as follows:The results demonstrated that GIM5.219 strain grows very slowly on PDA between 0 to 2 days,from 2 to 6 days hyphae grow faster,but hypha growth tend to be stable after 7 days;In PDB,hypha balls grow more slowly in 0-2 days,from 2 to 7 days hypha balls grow faster,growth of hypha balls after 7 days tend to be stable;For subsequent experiment provides the basic information of characteristics of cultivation.8 factors that affected the formation and regeneration of the Wolfiporia cocos protoplasts were researched.In the condition of the mycelium cultivated for 6 days,for enzymolys in mixed enzyme solution containing 3.5% Lywallzym and 1% Driselase at 34-36?and pH5.6-6.2 for 2.5-3 hours to obtain the protoplasts,and regenerate on potato regeneration medium with double coating.The most was 39.20×107 protoplasts/g and the highest regeneration rate was 32.7%.For subsequent Wolfiporia cocos protoplast transformation efficiency provides important technical support.From six aspects to explore the protoplast-mediated transformation system,obtained the optimum condition:Adopt protoplasts that prepared from four-day iquid culture hypha balls and using MTC solution(0.6 mol/L mannitol,50mmol/L CaCl2,10mmol/L Tris-HCl?pH7.5)to regulate its concentration to 1 x 108 / ml,join 2 ug/DNA(100 ?l protoplast),with 45% PEG4000 to transform and get 27 transformants.The PCR tested all the transformants can amplify the hph specific fragment,explained foreign DNA has been inserted into the Wolfiporia cocos genome,and can be stable genetic with Wolfiporia cocos batches.Plasmid pCIT-Smk which silences the Smk gene of Wolfiporia cocos was successful transformed into the protoplasts of Wolfiporia cocos strain by PEG-mediated transformation.The optimized conditions was verified to be feasible which was impotant for studying the function of gene.Transformants which could silence Smk gene effectively and steadily were obtained.The selected transformants maintained silencing Smk gene even after subculturing for 5 times,which indicated the PEG-mediated transformation are mitotically stable.RT-PCR indicated the expression quantity of Smk gene in the one which not transformed was 28133.5 of all the transformants.The Smk gene was successful silenced,and genetic stability.Transformants were in the process of growth,there was obvious change in growth rate and morphological characteristics.It indicted that the Smk gene may be one of the important genes which regulated the hypha growth and sclerotium forming.The result of study has improved and enriched the protoplast preparation and regeneration of Wolfiporia cocos and further optimizes protoplast-mediated transformation.Study the Smk gene which regulated the hypha growth and sclerotium forming.It provides a theoretical basis of planting and breeding for Wolfiporia cocos.