| Wolfiporia cocos is an edible and medicinal fungus,whose sclerotia is known as Fulin in China with high medicinal and health care value.Functional genomics studies of sclerotia or active ingredient formation are important for the development of W.cocos' s industry.In recent years,the completion of the genomic sequencing work has provided much molecular information for the study of sclerotia development and active components accumulation.However,a simple and effective genetic function identification system is lacked in the study of W.cocos.Therefore,this study aims to construct a technical system suitable for the study of gene function in W.cocos,and the main research contents and results are as follows:(1)Cloning and analysing endogenous orotidine-5'-phosphate decarboxylase gene(URA3),glycerol triphosphate glyceraldehyde dehydrogenase gene(GAPDH)promoter and NADPH oxidase related genes,and clarifying of the sequence and structural characteristics of these genes.(2)Selecting pCAMBIA1300-g with hygromycin resistance gene as the vector backbone,and use the endogenous orotidine-5'-phosphate decarboxylase gene(URA3)as the reverse screening marker.Antisense fragment of the conserved domain in URA3 was selected as our interference fragment,and two silencing vectors were constructed with Legpd and Leactin promotors from Lentinula edodes,and the Wcgpd promoter from W.cocos respectively to drive the express of it.In this study,Agrobacterium-mediated method was first used in W.cocos with that method used in W.cocos commercial strain Jingzhou 28.Eleven RNAi transformants with dual exogenous promoters and ten RNAi transformants with Wcgpd endogenous promotor were obtained respectively after screening on CYM with hygromycin for five generations.Real-time quantitative PCR analysis of these transformants showed that the expression of ura3 gene was obviously down-regulated in ten out of eleven RNAi transformants with dual exogenous promoters and three out of ten RNAi transformants with Wcgpd endogenous promotor respectively.Then some transformants which the expression of URA3 were significantly down-regulated were selected randomly in above two types for fruther study.These transformants which were cultured on CYM with 10 ?g/mL of 5'-FOA,it was showed that these transformants were able to grow and form colonies,but the wild-type strain could not grow.Therefore,it is considered that the expression of the gene URA3 has been successfully interfered.(3)By comparing the RNAi transformants of two different promoters,it was found that two promoters of Lentinus edodes is also suitable for the transformation,and the silencing effect of the RNAi transformant of the two promoter is somewhat better than that of the one promoter.In addition,the effects of different culture methods of the receptor materials on the transformation efficiency was discussed in the study,but the results showed a difference among our selected mediums.(4)Choosing the antisense fragment of the conservative functional area of the NADPH oxidase-related gene NoxR as an interference fragment,and the RNAi vector was constructed with dual promoter of L.edodes' s Legpd and Leactin.Five stably growing RNAi transformants were obtained after screening on CYM with hygromycin for five generations.Real-time quantitative PCR analysis showed that the expression level of three of the five transformants was significantly down-regulated compared with the wild-type strain.The above results indicated that the Agrobacterium-mediated RNA interference gene silencing system in W.cocos was successfully constructed in this study.This study laid the foundation for the study of gene functions such as gene recombination,overexpression and insertion mutation. |
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