| Since Hypsizygus marmoreus was introduced to China in the 1980 s,it has been favoured by more and more people.Currently,H.marmoreus has become an indispensable part of the edible fungus industry with the gradual increase of its cultivation yield.Genetic transformation system for H.marmoreus has still been improving so as to obtain transgenic strains,and to study the gene function of H.marmoreus and the genetic improvement of new variaties.In this study,two genetic transformation plasmids denoted as either Ca MV35S-p012 and Hm GPD-p021 were constructed from the promoter of cauliflower mosaic virus 35 S RNA(Ca MV35S)combined with the castor bean catalase(CBC)gene intron and H.marmoreus glyceraldehyde-3-phosphate dehydrogenase(Hm GPD)gene combined with its first intron,respectively,to drive the expression of exogenous plant anthocyanin biosynthesis genes in H.marmoreus.The two plasmids were Agrobacterium-mediated transformed into monokaryotic hyphae obtained from the H.marmoreus,screening using transgenic carboxin resistance gene from Pleurotus eryngii.With transgenic verification of the pseudo-transformants with carboxin resistance by PCR,and using quantitative real-time PCR to positive transformation,the exogenous gene expression level were tested,and results showed that the promoters of both Ca MV35 S and Hm GPD genes successfully drove the transcription of anthocyanin biosynthesis genes in H.marmoreus,and the introns introduced to enhance gene expression were excised correctly during the transcription process.Moreover,the expression level of exogenous gene driven by Hm GPD promoter was 22-36 times stronger than that driven by Ca MV35 S promoter in transformants from H.marmoreus,indicating that the Hm GPD promoter was more capable to drive exogenous gene expression.At the same time,many traits of the positive transformants containing the anthocyanin synthesis gene by the above Agrobacterium-mediated genetic transformation system were assessed,including the biological characterization,cross-matching,cultivation and fruiting,and the anthocyanin content.The result showed that the carboxin resistance gene could be inherited stably in the transformants.Compared with the acceptor strain Fin C-W-247-F4,the addition of exogenous anthocyanin synthesis genes will have a certain effect on the growth of the mycelium of the H.marmoreus,but they didn’t influence the phenotype of the mycelium and the fruit body.Different pH would result in different anthocyanin content in transformants.In addition,the expression system of Cas9 gene in the genome of H.marmoreus was also preliminarily explored in the study.For the first time,the plasmid containing Cas9 gene was transferred into the mononuclear strain of H.marmoreus by Agrobacterium EHA105,and the transformant containing exogenous Cas9 gene was obtained.The results of this study will provide a basis for the efficient expression of foreign genes in H.marmoreus.The obtained transformants can provide materials for further study on the function of anthocyanin synthesis genes,and provide a reference for the establishment of CRISPR / Cas9 gene editing system in H.marmoreus. |
