Flammulina velutipes(Curt.ex Fr.)Sing,is a one of the most popular edible mushroom.In consumer market,people chasing it because of full of nutritional value and smooth taste.In addition,Flammulina velutipes also have the rapid development of production and wide cultivated in the world,especially in Asia.China as the birthplace of F.velutipes cultivation,the history of cultivation can traced back to ancient times.Now,China also is the biggest country of F.velutipes production with abundant germplasm resources of F.velutipes.With the passage of time and the development of technology,the researchers have made considerable progress in F.velutipes breeding.The way for breeding of F.velutipes has become diverse.Furthermore,the main purpose of development of in still focus on F.velutipes cultivation.However,there are also have a lot of exploring space and a lot of unknown to parsing in basic molecular biology research.F.velutipes is typical tetrapolar basidiomycetes and has clear stages in development.Therefore,the study of key gene in mating pathways has an important role to formation of fruiting body.After the F.velutipes genome data has published in 2014,our lab have construct the model of mating type gene structure.The reference of this study of Clp1 from Ustilago maydis that is similar to F.velutipes in evolution.We scan genome of L11 strain to located Clp1 gene that is a key gene of downstream of mating pathways by using local Blastp tools.The gene number of Clpl in L11 genome is gene4230.To confirm the gene structure by using zoom software,and then build 2 vector:overexpression and RNAi.The Agrobacterium tumefaciens-mediated transformation(ATMT)be used to transfer vector to F.velutipes.and mutants will be selected in medium with hygromycin.In this study,we analyzed the difference of mutants with overexpression and mutants with RNAi to exploring the function of Clpl in F.velutipes developmental stages.We cross the mutant with compatible strain to get dikaryon can grow the fruiting body.RT-qPCR be used to reveal the different of expression pattern between dikaryon with mutant and dikaryon with wild-type.The experiment main technical route and the results are as follows:(1)We download the amino acids sequence from NCBI(National Center of Biotechnology Information),and then to confirm the gene structure by using local Blastp and zoom software.The full length of Fv-Clp1gene in F.velutipes is 1011bp without introns.From the evolution analysis,Clp1 gene in F.velutipes is highly conserve to Clpl in Coprinus cinereus(2)We designed the primer of overexpression and RNAi as L11 genome be a template.PCR to get full length sequence of Fv-Clpl and fragment of RNAi,and then succeeded in construct the overexpression vector and RNAi vector by using homologous recombination.(3)Agrobacterium tumefaciens-mediated transformation be used to transfer the overexpression vector and RNAi vector to monokaryon of L11 strain and L22 strain.Hygromycin resistance maker be used to select the mutants and get 11postive mutants.(4)The measurement of expression.The expression of Fv-Clp1 in 14 positive mutants be measured by RT-qPCR.The result shows that the expression level of Fv-Clpl in CO1.CO2 and was 12and 14folds as many as that of wild type strain L11.While compared to wild type strain L11,expression level of Fv-Clp1 in CR4、CR5、CR6 and CR7 was down regulated by 6,24,6 and 12 folds,respectively.(5)Cultivation and growth rate determination experiment.The growth rate determination result shows the overexpression mutants are fast than wild type strain.And the mutants have the trend of thickening.While the growth rates of RNAi mutants relative to its wild type.We cross the mutants with compatible strain L22.And then cultivation and microscopy result shows Fv-Clp1 over expression mutants can forming clamps when cross with wild type strain.And its numbers of clamps as many as than wild type 1122 strain.Fv-Clp1 gene overexpression mutants can forming clamps when cross with the strain that same A mating type and different B mating type.it is showed similar to Coprinus cinereus.Fv-Clp1 RNAi mutants can forming clamps when cross with the compatible wild type strain,but the numbers of clamps not as many as than wild type 1122 strain.The time of fruiting body of overexpression mutants is early than wild type strain.And the stipe of mutants is thicker than wild type.The cap bigger than wild type.RNAi mutants also can form fruiting body but the forming time of primordium is later than wild type.Based on the above research and as a result,we suspect that,the overexpression of Fv-Clp1 gene can form clamps without compatible A mating type in F.velutipes.In this study,however,Fv-Clplgene be found play an imporetant role in primordium formation and fruiting body formation. |