| The H.marmoreus is a valuable large edible fungus from the northern temperate zone.Since it was introduced to China from Japan in the 1980 s,after decades of development,it has become an indispensable part of our edible fungus industry.At present,the construction system of the genetic transformation system of H.marmoreus has been preliminarily established,but there are still certain defects in the transformation system.The targeted receptors are mainly limited to the fruiting bodies and protoplasts,The protoplast preparation process is cumbersome,the cell wall of protoplasdts needs to be digested with lysozyme.Successfully prepared protoplasts need to be stored in a hypertonic solution to maintain a stable osmotic pressure.Since protoplasts are very fragile,they are easily broken by excessive centrifugal force during centrifugation,and the regeneration rate is low,which makes the efficiency of established H.marmoreus transformation system too low,besides,the efficiency of mycelium transformation can’t be calculated easily.The preparation method of arthrogenous spore is simple,counting is simple,the regeneration rate is higher,and the efficiency of transformation can be expressed more intuitively.Therefore,this experiment used Agrobacterium-mediated transformation method to transform the arthrogenous spore of H.marmoreus,hoping to build a stable and efficient transformation system of H.marmoreus.The research results of this article are as follows:1.Investigate the relevant factors of the number of spores produced by the mononuclear hyphae of H.marmoreus,including the Co-cultivation time and the type of strain.By comparison,it was finally determined that the number of spores produced by different strain types was slightly different,but the spores of the order of 106-108 were usually obtained through more than ten days of cultivation.The optimal mononuclear mycelium cultivation time determined by experiment is 20 days.2.Through the experiment,the plate screening conditions for transformation of Agrobacterium tumefaciens EHA105 into H.marmoreus spores were determined.The conditions were as follows: carboxin 2mg / L,Cefotaxime 300 mg / L.3.The experiment uses PCAMBIA2301 as the skeleton,cuts the kanamycin resistance screening gene and connects the new β-carotene synthesis gene crt IBY,constructs a intermediate carrier p001,and performs the enzyme digestion and connection after PCR amplification.The complete screening of Pleurotus eryngii resistance gene to obtain the recombinant plasmid p Sdi-crt IBY successfully constructed the expression carrier of H.marmoreus carrying Sdi screening gene and GUS gene.4.Transform the constructed recombinant plasmid pSdi-crt IBY into Agrobacterium strain EHA105,using the spores of H.marmoreus F4,F2,B5-15 mononuclear hyphae as the receptor material,using Agrobacterium tumefaciens-mediated Experiment with the conversion method.After screening of carboxin resistance,PCR identification and GUS staining experiment verification,it was proved that the exogenous gene which on transformate plasmid was successfully transferred into the H.marmoreus genome.The results showed that the experiment obtained several batches of stable genetic and expression transformation Agrobacterium tumefaciens-mediated genetic transformation system of H.marmoreus spores was initially constructed,and the transformation efficiency was higher than that of the currently reported spore transformation efficiency.5.Optimize the factors that may be related to the transformation efficiency in the transformation system,including the concentration of pre-cultured Agrobacterium bacteria solution,infection time,co-cultivation time and the role of phenols.According to the above four factors,the final optimization results are as follows: the best precultured Agrobacterium solution concentration is OD6000.5,the best spore infection time is 20 min,the best co-cultivation time is 48 h,and acetosyringone is necessary.6.The transformants were tested for growth rate,mycelium morphology and mushroom production.The results showed that the growth rate of transformants partially changed compared with wild-type hyphae,and the colony morphology of individual transformants slightly changed.This may be due to the random insertion of T-DNA during the transformation of Agrobacterium affecting the correlation between the DNA and the colony morphology gene.At the same time,the color of the mycelium changes,and it appears light yellow near the inoculation block and gradually spreads around.The color gradually fades with the growth of the mycelium.After the majority of transformants were paired through hybridization,mushrooms could be grown normally under suitable conditions.The observation of fruit body morphology showed that the transformant morphology was not significantly different from the wild type. |
