Construction Of Carboxin Resistance Gene Carrier With The Site Directed Mutagenesis And The Application Research In Genetic Transformation In Lentinula Edodes

Lentinula edodes is a kind of edible fungi with abundant production and extensive cultivation area.It tastes delicious,and it has special fragrance and loved by the people;It has rich nutrients and high value,so it is also the object of many researchers.The genetic and functional gene research of Letinous edodes has become a hot spot with the completed and publication of the whole genome sequencing of Letinous edodes.The basis of genetic and gene function research is the perfect genetic transformation system.It is necessary to use easily identified marker genes in many genetic transformation methods,which can easily and quickly screen the transformants.At present,the commonly used selectable marker in edible mushrooms is hygromycin resistance gene(Hyg~R),an exogenous antibiotic resistance marker gene,which is unstable in transformation process and will cause certain toxicity to transformants.When screening is completed,the selection of marker genes is of no value or even side effects.Especially for Letinous edodes,which is used to eat food,it is risky to transfer to any non endogenous gene fragment.The homologous marker gene can be avoided from the receptor or its own genus.At the same time,homologous gene transformation can lead the target vector to integrate at the homologous part of the receptor chromosome,avoid methylation,facilitate gene expression,and effectively improve the transformation rate and integration rate.The purpose of this study is to establish a transformation method with the carboxin resistance gene as the selectable marker in Letinous edodes,and apply homologous selection marker genes to the study of genetic and breeding of edible fungi.Carboxin is a kind of succinate dehydrogenase fungicides,has a specific inhibitory effect on basidiomycetes.Succinate dehydrogenase is an important functional part of the three carboxylic acid cycle.It consists of four subunits,namely,flavin protein(Fp,SdhA),iron sulfur protein(Ip,SdhB)and two kinds of membrane proteins(SdhC and SdhD).Among them,SdhA and SdhB of succinate dehydrogenase in peripheral membrane region,is a kind of conservative,with the activity of succinate dehydrogenase;two block membrane protein SdhC and SdhD domains of lower homology between species,mainly is the role of the SdhA and SdhB fixed in endometriosis,with ubiquinone reductase activity.In this study,we found that most of the mutations occurred on the B subunit of succinate dehydrogenase,mostly from Histidine(His)to Leucine(Leu).The protein sequence of Letinous edodes succinate dehydrogenase B subunit was compared with the sequence of B subunit protein of carboxin resistant fungus.It was found that there are many homologous domains with others resistant fungi near the 243 Histidine(His)of Letinous edodes B subunit,and this region is quite conservative.Then,turn the Letinous edodes B subunit 243 Histidine(CAC)into Leucine(CTC)and make a gene fragment of Letinous edodes succinate dehydrogenase B subunit with pseudo carboxin resistance.Linked GPiE plasmid that was cuted by restriction enzyme SmaI and DNA fragments that has carboxin resistance used In-Fusion method,Construction of G-cbx vector with constructed Cbx~R gene as the homologous selection marker and the green fluorescent protein EGFP as the reporter gene.Through the carboxin resistance experiment in Letinous edodes mycelium,determined that the initial screening concentration of monokaryon"207"is 0.5mg/L,1mg/L is the second screening concentration;the initial screening concentration of dikaryon"Hu Xiang F2"is 1mg/L,second screening concentration is 2mg/L.It shows that the mycelium of Letinous edodes is very sensitive to carboxin,and the growth of mycelia can be inhibited only in a very small amount.It avoids the damage caused by the high drug concentration on the growth and development of mycelium cells.Using two Agrobacterium tumefaciens EHA105 and LBA4404 mediated Letinous edodes receptor of monokaryons"207"and dikaryon“Hu Xiang F2”.The results show that Letinous edodes indeed access to carboxin resistance by turn Letinous edodes B subunit 243 His into Leu;The vector with carboxin resistance can transforme Letinous edodes mycelium successfully by two Agrobacterium tumefaciens strains;The transformation efficiency were more higher than the Hygromycin resistance gene of laboratory transformation with the same method,in Letinous edodes monocaryons;and the transformation efficiency in the dikaryon were both very low;After 5 generations of passage,the resistance phenotype of monokaryons transformants remained stable,while the dikaryon transformants resistance phenotype basically lost;PCR detection of transformants carboxin resistance(Cbx~R)gene fragment the band,size and position is the same as the control plasmid;detected the expression of green fluorescent protein of EGFP in transformants,found that under the fluorescence microscope,the transformants can detect green fluorescent protein,non transformants are not.The result show that Cbx~R fragment leading-in genomic DNA of Letinous edodes monocaryons successful and expression efficiently,shows the corresponding resistance phenotype and can stable inheritance.Therefore,in terms of conversion rate,stability,integration rate and safety,it is a more suitable and applied selectable marker gene in Letinous edodes.