| Letinous edodes(Lentinula edodes)is one of the most famous edible fungi in the world,and it is the world's second largest medicinal fungus production after Agaricus bisporus(Agaricus bisporus),it has nearly a thousand years of cultivation history in China.Due to its outstanding role and value of food and medicine,the letinous edodes' s demand increased significantly letinous edodes,which promoted the rapid development of industry,letinous edodes total output of more than Japan since 1987.Chinese has maintained the world's first letinous edodes superpower.But with the rapid development of the letinous edodes industry,the genetic transformation of letinous edodes is lagging behind.With two letinous edodes whole genome sequencing completed and published,genetic studies on functional genes of letinous edodes will become hot.Genetic transformation technology is the use of molecular biology and genetic engineering methods such as exogenous gene fragment inserted into the recipient genome through replication,transcription,translation,and to change the physiological biochemical characteristics of directional receptor strain purpose.Therefore,stability,construction has important significance and value of efficient genetic transformation system.This paper will explore the Agrobacterium mediated genetic transformation system for mutation of random insertion in letinous edodes protoplast monokaryon strains.Through letinous edodes mycelium on hygromycin resistance test,determine the Agrobacterium transformation screening concentration 10mg/L,screening concentration by the toxicity test of cefotaxime sodium for letinous edodes mycelium12mg/L,determined to build the best concentration of Cef 600mg/L.Plasmid pYN6982 and GPiE with the hygromycin resistance gene hyg as marker gene,enhanced by type egfp fluorescent protein gene as a report gene by Agrobacterium EHA105 And LBA4404 is mediated,at the same time the transformation of 3 letinous edodes monokaryon strains.Letinous edodes innovative use of rice receptor Mycelium Culture medium and transformation,using small grain medium culture does not need to select the blockwith the same diameter from the punch,every grain of rice is of uniform size of the individual,it can be directly out of use,does not need to be inoculated in sterile glass covered with paper MYG fixed medium,not pollution bacteria.Agrobacterium infection hyphae after rice were compared to the commonly used method is convenient,PDA pieces of bacteria or pellets,this method can be more efficient and more easy to disperse the receptor material,and can be more convenient and efficient statistical conversion efficiency.Receptor after Agrobacterium infection hyphae,cultivation stage were cultured by hygromycin resistance screening by PCR and sequencing,transformants containing hygromycin resistant gene fragment.Through fluorescent microscope,the hyphae of letinous edodes transformation can be observed in green fluorescence,and without control strains has no fluorescence conversion signal generation,show enhanced green fluorescent protein in transformed letinous edodes mycelium was expressed,after the transformants have transformed the mitotic stability test.The genetic stability of transformants was carried out on the part of the letinous edodes based on colony morphology test in cultured PDA,and untransformed control letinous edodes mycelium than,found a small part of transformants of colony morphology and growth rate difference may be associated with the insertion of T-DNA destroyed the colony morphology of the transformants functional gene in the genome,which leads to the difference of transformants and control hyphae.This study explores a new method of culture and transformation of culture medium with small grain,and small grain growth from mycelium in time based on Agrobacterium type,type of promoter plasmid,ultrasound time of Agrobacterium infection process,total incubation time and temperature were studied and optimized.The conversion efficiency at the same time the plasmid containing the promoter in the same Agrobacterium transformation efficiency and the same plasmid in different Agrobacterium.The results show that the mycelium in rice culture medium for 15~20days when optimal Agrobacterium infection transformation;different Agrobacterium EHA105 and LBA4404 infection hyphae no big difference after conversion efficiency;conversion efficiency with conversion efficiency of Agaricus bisporus is higher than that of Agpd promoter containing ganoderma Gl-gpd promoter;Agrobacterium infection in the process of ultrasonic vibration is the best time for 5 minutes;add acetosyringone in the process to improve the conversion efficiency of co culture;co culture time is 7 days for the best the incubation time,the optimum temperature is25? co culture.A stable transformation of Agrobacterium mediated genetic letinous edodes system,and obtain the stability and the expression of genetic transformation of letinous edodes strains. |
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