| Flammulina velutipes,a very important edible fungi,which is also used in medicine in Southeast Asia as well as the world.As its unique taste,good nutritional and medicinal value,F.velutipes was appreciated in East Asia,especially in China,Japan and South Korea.Because lacking of genetic transformation system,the molecular biology of F.velutipes develops very slowly.In this study,we first introduced the Liposome-mediated transformation method in F.velutipes,and constructed a simple and effective genetic transformation system.Using Liposome-mediated transformation,we could get 60-80 transformants per 104 protoplasts at the 5th-6th days after transformation,however,electroporation-mediated transformation could get 10-20 transformants per 104 protoplasts at 10th day after transformation,what’s more,Agrobacterium-mediaied transformation could only get 5-10 transformants per 104 protoplasts at the 14th day after transformation.Compared to electroporation-mediated transformation and Agrobacterium-mediated transformation,the time of Liposome-mediated transformation was shorter and the efficiency was higher in F.velutipes.In order to study the role of laccase genes in F.velutipes,we screened laccase gene silencing transformations and overexpression transformations using Liposome-mediated transformation.In F.velutipes,we got nine laccase sequences by comparison.We used RT-PCR to detect the transcription of the nine laccase genes,and the results showed that:compared to Lac5,Lac6,Lac7,Lac8,Lac9,the transcription of Lac1,Lac2,Lac3,Lac4 were higher.The results of the identity of the nine laccase genes showed that the identity of Lac2,Lac3 and Lac4 are higher.As the transcription of Lac2 is highest,we constructed the gene silencing vector using the higher identity sequence of Lac2,besides,we constructed the overexpression vectors of Lac2 in F,velutipes.Using Liposome-mediated transformation,we screened Lac2 silencing transformations and overexpression transformations by Hygromycin flat.The silencing efficiency reached 75 percent and the transcription of Lac2 in overexpression transformants was 1.6 times.To further study the impact of laccase silence transformants and overexpression transformants,laccase,mycelial growth rate and biomass,as well as mushroom growth rate in situation were observed in silencing transformations and overexpression transformations.The results showed that:silence laccase transformants,mycelium biomass was less than the wild-type,primordia eat feed rate and speed slower than the wild type;overexpression laccase transformants,mycelial biomass was higher than wild-type,primordia eat feed rate and germination also faster than the wild type.Taken together,these results indicated that:increase laccase gene transcript levels could be increased paint activity,mycelia biomass,accelerate mycelial primordia eat feed rate and promote germination.In summary,it was the first time to establish in F.velutipes then we got an efficient liposomal genetic transformation system,it laid the foundation for subsequent research gene mushroom paint;through the study of mushroom enzyme found that increasing laccase gene transcript levels could be increased paint activity,mycelia biomass,speed up the mycelium and primordia eat feed speed germination rate. |
