Transposon Mutagenesis Of Polar Psychrophilic Fungus And Screening Of Normal Temperature Adaptive Mutants

The unique environmental conditions of the polar regions make it impossible for most species to survive.But polar regions are also called treasure-houses which are rich in microbial resources.More and more studies have shown the diversity of microorganisms in the polar regions,especially in psychrophiles and psychrotrophs.However,most of the polar microbiological resources have not been well developed and utilized.Due to their special growth habitats,there are many difficulties in cultivating these microorganisms,such as low temperature,high salts,high pressure,and other special cultivations.Geomyces sp.WNF-15A,a cryophilic filamentous fungus isolated from Antarctic,can produce red pigments when cultured at low temperatures.This kind of red pigments have many advantages,such as high color value,good stability,simple separation,healthy and non-toxic.Geomyces sp.WNF-15A has a great potential to become an industrial strain of food pigment,which has received extensive attention.However,this strain is a typical cryophilic fungus,and the optimal culture temperature is 10?15?.The high pigment yield can only be obtained at low temperature.In this study,a complete genetic operation system was established for Geomyces sp.WNF-15A.By screening and constructing effective transposable systems,transposon insertion mutation method was developed.We aimed to build an efficient screening platform of psychrophiles to breed normal temperature adaptive mutants.It will help to promote the industrial application of this strain and provide some references for the development of other polar fungal products.Firstly,a complete genetic operation system of the Geomyces sp.WNF-15A was established.Two effective screening markers were identified from a variety of antibiotics.Both 200 ?g/ml of G418(spores number less than 1.1×105)and 30 ?g/ml of hygromycin B(spores number less than 3.8×105)could effectively inhibit the growth of Geomyces sp.WNF-15A.Then,the protoplasm preparation process was optimized in detail from mycelium culture temperature,medium,culture time,enzyme content,enzymolysis temperature,enzymolysis time,pH and the other parameters.Finally the optimal conditions were determined:The fungus were cultured in YPD medium at 20? for 36 h to obtain the mycelia.The enzyme solutions contained 3%cellulase,1.5%snailase and the pH was adjusted to 7.5.After the mycelia of Geomyces sp.WNF-15A were digested by enzyme solutions at 28? for 1.5?2 h,3×1010/ml protoplasts can be obtained.Furthemore,the effects of incubation time,exogenous vector concentration and the PEG concentration on protoplast transformation were investigated.It was found that 2 ?g vector transformed under the condition of 60%PEG,36 h incubation time,achieved the highest transformation efficiency of?50%.At the same time,the method of spore PCR was also optimized to improve the experimental efficiency.Secondly,three fungal transposable systems of Impala,Fot1,and Helitron were selected.The conserved amino acid sequences of transposases were aligned to verify whether there is influence of endogenous transposases in Geomyces sp.WNF-15A.It can ensure the controllability and stability of exogenous transposable system.Based on the results of amino acid alignment,it can be preliminarily inferred that the endogenous transposable enzyme of Geomyces sp.WNF-15A does not affect the three transposable systems.On this basis,using filamentous fungus generic plasmid pFC332 as the start,the vector backbone pFC000 was constructed.Based on pFC000,the Impala transposable vector pFC001 as well as the Fot1 transposable vector pFC002 were constructed respectively.Similarly,the construction of Helitron transposon binary vectors were also completed.So far,three kinds of transposable vectors with common elements of filamentous fungi have been successfully obtained.Finally,the transposable vectors were transformed into Geomyces sp.WNF-15A.After multiple rounds of transformation,screening and identification,13 mutants in pigment production and 16 mutants in growth were screened.At 14?,the mutant MP1 reached the highest yield of all strains(OD520?39),which was 40%higher than the wild strain.Under the condition of 20?,the mutant MP14 reached a maximum yield(OD520=14.8),which was nearly twice that of the wild type strain.Under the condition of 25?,the red pigments yield of mutant MP2 was OD520=2.2,and that of mutant MP10 was OD520=3.2,while the wild type strain had no obvious red pigments synthesis.The efficiency analysis shows the transformation efficiency of the three systems is basically the same(?10%).By comparing the insertion efficiency of the three transposable systems,we found that the activity of Helitron transposable system in Geomyces sp.WNF-15A was better than Impala and Fotl.And the positive mutation rate of Helitron transposable system was also the highest.