Preliminary Research On The CRISPR/Cas9 Genome Editing System In Poria Cocos And The Creation Of Germplasm Based On The PcTor1

Poria cocos(Schw.)Wolf grows in a parasitic manner on pine.Its sclerotium is one of the earliest Chinese herbs used in China.P.cocos has various pharmacological activities and good compatibility.P.cocos plays special roles in invigorating spleen for diuresis,tranquillization and so on.However,the degradation of germplasm species,the mixed germplasm resources and the poor ability to resist pathogenic bacteria which affect the development of P.cocos industry.TOR(target of rapamycin)is a relatively conserved serine/threonine protein kinase.It is essential in regulating the growth and metabolism of organisms and responsing to environmental stresses.In this study,it was explored the CRISPR/Cas9 gene editing technology of P.cocos.Meanwhile,it was used the transgenic technology to create new germplasm targeting the PcTor1.It is expected to provide a new method,new gene and new material for molecular breeding of P.cocos,and also propose new ideas to breed medicinal plants.The main results are as follows:1.The CRISPR/Cas9 gene editing system in P.cocos was constructed based on the p EExpco Cas9-2Targets B vector.The sequence of Cas9 was optimized based on the P.cocos’ codon bias.Its promoter was replaced with Pgpd.In this study,the GIM5.219 strains was used as the test wild-type strain.To knock out the targeted editing PcTor1’sequences,2 specific sg RNA sequences need to be synthesized respectively.Using the Gibson reaction,2 CRISPR/Cas9 gene editing systems could be easily constructed.The plasmid was introduced into the strains by Agrobacterium tumefaciens GV3101 mediation.And the GIM5.219/Cas9 strains were successfully obtained.Compared with the Golden Gate CRISPR/Cas9 vector construction strategy,this method is more convenient and efficient.Although transformants were identified,no mutants were obtained in this study,which may due to the specific genetic background of P.cocos,or the target PcTor1 may be crucial to its development.2.The overexpression and silent expression vectors of the PcTor1 were successfully constructed by using the vector of p Blue Script SK.The PEG-mediated protoplast transformation method was used to obtain the stable genetic transformation strains.The Real-time quantitative PCR was used to detect the expression level of overexpressing and silencing strains.The results showed that the expression leval of PcTor1 was significantly up-regulated in three overexpression strains compared with wild-type and psk-type strains,and 1 silent transformant strains of PcTor1 was significantly down-regulated compared with the wild-type strain.It was found that the expression leval of overexpression strains OE-1,OE-2 and OE-3 were 3.9,1.7 and 1.6 times than that of the wild type strain,respectively.The silencing efficiency of silencing transformed strain reached 51%.3.Compared with the wild type strains,the growth rate of the overexpressed strains were 1.23 times than the wild strains which was significantly accelerated.However,the growth rate of silent strains were 0.63 times that of wild type,which was significantly reduced.The total triterpenoids content of the overexpressed strains OE-1 and OE-2 were38.6 mg/g and 34.95 mg/g respectively,1.5 and 1.4 times than that of the wild type,and the total triterpene content of the silent strains were significantly reduced to 22.0 mg/g,0.8 times than that of the wild type.By adopting the dual culture for test,the inhibitory rate of Trichoderma Harziana against wild-type strains was 37%.The inhibitory rate of overexpression OE-1 and OE-2 was significantly reduced,which were 13.12% and16.79%,respectively,indicating that these two overexpression strains had a stronger antagonism than the wild type strains.