Establishment Of Agrobacterium-mediated Silencing System Of Lentinula Edodes URA3 Gene

Lentinula edodes is the second largest edible medicinal fungus in the world,second only to the twin-cell mushroom.It has a unique aroma,delicious taste,thick meat and anti-tumor and other medicinal values,and has become one of the main edible fungi in large-scale production in China.At present,the research on the genetic law of important characters of Lentinula edodes and the in-depth research on the breeding of new varieties have always been the urgent needs of industrial development.Moreover,in recent years,with the completion of genome sequencing of schizoplectococcus,twinocytococcus,mushroom and lentinula edodes,a large number of edible fungus DNA sequences with unknown functions need to be further analyzed.And reverse genetic molecular biology methods can effectively verify the function of the gene,RNAi technology is one of the effective tools,its action mechanism and application of made a major breakthrough,which provides a new method and means for transgenic breeding,can reduce specificity or even shut down the expression of specific genes,so as to achieve specific breeding and the effect of improved varieties,is widely used to explore the study of gene function.However,the research on RNAi technology and its application in edible fungi is still at the initial stage,so this project aims to establish a RNA silencing system suitable for the study of Lentinula edodes gene function.The main work is as follows:(1)The endogenous URA3(whey 5 '-monophosphate decarboxylase),which can be used as a screening marker,was selected as the target gene for the establishment of Lentinula edodes RNAi system.The genomic DNA of mononuclear strain of Lentinula edodes was extracted,and the URA3 gene sequence of Lentinula edodes was analyzed to design relevant primers.At the same time,the primer design of 35 S and GPD promoter of Lentinula edodes was completed to amplify and purify the target fragment.(2)Use of mushroom and endogenous nucleoside-5 '-single phosphoric acid whey acid decarboxylase gene(URA3)as a reverse selection markers,and on the function of the gene structure of conservative area 451-884 bp reverse complementary pieces to interfere with the clips,the mushroom Lgpd and 35 S promoter,choose to have hygromycin resistance gene p CAMBIA1390 and GPi E as the carrier of the original skeleton,respectively,two kinds of structure of RNAi vector is constructed,That is,to transform p CAMBIA1390 into a carrier Hp V with a hairpin structure;GPi E was replaced by the dual promoter vector Dp V-2.(3)This study choose mushroom mononuclear strains 220 as test material,the first time using a small grain of rice as the medium of agrobacterium-mediated genetic variations infect mushroom mycelium,for the conversion of obtained child first by hygromycin resistance(Hyg)and uracil in early screening,early screening after transformation in use uracil and cef on PDA plate after five batches of Hyg gene PCR test,after sequencing directly compare respectively to obtain the stable growth of mushroom and bidirectional promoter RNAi into the son,and mushroom hairpin structure promoter of RNAi into children.Then the transformation obtained under 5-FOA and uracil medium on phenotypic observation,discovery into child than wild type strain can grow better in its tablet,for son of independent random samples in real time fluorescence quantitative PCR analysis URA3 gene expression quantity reduce,and received three expression quantity is significant transformation of the bidirectional promoter of RNAi and 12 express more significant amount of RNAi into sub hairpin structure promoter.The Hp V transformant with hairpin structure had a strong silencing interference efficiency,and the proportion of transformant with strong interference efficiency was up to 60%.The expression level of URA3 was reduced up to 0.1 of that of the original wild-type strain.Therefore,it is suggested that the established RNAi system can successfully interfere with the expression of endogenous gene URA3.(4)Using RNA interference system has been established,again to choose two different background of mononuclear strains Gsm207 and Bsm83 materials as receptors,respectively has built two RNAi plasmid genetic transformation experiment was carried out,and finally by comparing three different mononuclear strains and two different RNAi vector transformation,found Gsm207 and Bsm83 mushroom mononuclear strains can also be successful,and two different carrier interference efficiency were similar,However,the silencing effect of RNAi transformant obtained by strain Bsm83 was better than that obtained by strain Gsm207 to a certain extent.Meanwhile,this study also compared the transformation of different mycelium parts of the same transformant and the reduced expression of URA3 gene.The results showed that the transformation results of different mycelium parts of the same transformant were different,and the reduced expression level of URA3 gene was different in different parts of the same RNAi transformant.The above results indicated that this study completed the establishment of Agrobacterium-mediated endogenous silencing system of URA3 gene in Lentinula edodes,which laid a foundation for the future study of gene function of other genes in Lentinula edodes and the study of multi-gene silencing in Lentinula edodes.