Pseudogymnoascussp.OUCMDZ-4032生物合成基因簇分析及遺傳體系的構建

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Analysis of the biosynthetic gene cluster and establishment of the genetic system of Pseudogymnnosus sp. OUCMDZ-4032

An Chengze1.2.3，Du Rongtian1.2.3， Wang Weiyun1.2.3，and Wang Yi1，2，3 （1SchoolofMedicineandPharmacyOceanUniversityofChinaQingdao266o3;2LaboratoryforMarineDrugsandBioproducts， Qingdao Marine Science and Technology Center， Qingdao 266237; 3 Key Laboratory of Marine Drugs， Ministry ofEducation，Qingdao 266003）

Abstract Objective To analyze the secondary metabolite biosynthetic gene clusters of the psychrophilic fungus Pseudogymnoascus sp.OUCMDZ-4032 fromAntarctica，and establish the genetic transformation systemof this strain，layinga foundation foractivatingthe silent geneclusters in strains.MethodsThe biosynthetic geneclusters were analyzed via the antiSMASH website.High-quality protoplasts were prepared through antibiotic resistance test and the optimization of mycelium growth medium and enzymatic hydrolysis time. Based on the principle of protoplasts sweling upon water absorption，the protoplasts were detected and the regeneration rate was calculated.

The transformation system was validated by introducing the hph and neo drug-resistant fragments.ResultsIt was predicted that there were 50 secondary metabolite biosynthetic gene clusters in the genome of OUCMDZ-4032，among which 36 were orphan gene clusters.The strain was inhibited under the resistance concentrations of 300μg/mL G418 and 100μg/mL hygromycin B. Using PDB or SDYS as the mycelium growth medium， mixed enzymatic hydrolysis for 2h ，the protoplasts reached a maximum value of 6.1×106 protoplasts /mL ，with a regeneration rate of 47%±3% 主 Through PEG-mediated transformation，resistant plasmids were introduced into the protoplasts.Tenrandomlyselected transformants showed a 90% positive rate. Conclusion In the study， the secondary metabolite biosynthetic gene clusters of Pseudogymnoascus sp.OUCMDZ-4032 were analyzed，and a sophisticated genetic transformation system was established.This was conducive to studying the biosynthesis of the secondary metabolites of the strain and also provided a fundamental basis for the molecular biology research of psychrophilic microorganisms.

Key wordsAntarctic fungi; Biosynthetic gene clusters; Genetic transformation

南極長期暴露在惡劣的極端條件下，如低溫、強烈紫外線輻射、頻繁的凍融循環(huán)、周期性變化的光照時間、高度變化的光照強度、營養(yǎng)物質匱乏和高壓等[1-2]。（剩余10783字）