Gene Cloning And Expression Analysis Of P5CR And SwnH2 In Endophytic Fungus Alternaria Oxytropis OW7.8

Alternaria Oxytropis is a swainsonine(SW)-produced endophytic fungus isolated from Oxytropis glabra by our group.SW is an indolizidine alkaloid which poisons animals by inhibiting Golgi?-mannosidase II and lysosomal acid?-mannosidase.SW was produced from other endophytic fungi,Slafractonia leguminicola,Metarhizium anisopiae and M.robertsii in previous studies.Many reports were published in S.leguminicola,M.anisopliae and M.robertsii.However,there are few studies on endophytic fungi.In SW synthesis pathways,it was speculated that the protein encoded by pyrroline-5-carboxylate reductase gene(P5CR)catalyzed the formation of pipecolic acid(PA)from?1-piperidine-6-carboxylic acid(P6C).The swnH2 geng encoded protein 2-oxoglutarate-dependent nonheme-iron dioxygenases which catalyzed formation of 1,2-dihydroxyindolizine from 1-hydroxyindolizine.In this study,qRT-PCR was used to analyze the expression levels of P5CR gene in fungi both from experimental group(supplemented with L-PA in PDA media)and control group at different culture time(23d,26d,29d,32d).The SW levels in mycelia were detected by HPLC-MS.The swnH2 gene and its cDNA were identified,and the amino acid sequence was predicted by bioinformatics.The knockout and overexpression vectors of swnH2 gene and P5CR gene were constructed and verified by PCR,restriction enzyme digestion and DNA sequencing.The results are as follows:1.The total RNA was extracted from mycelia of OW7.8 cultured at23d,26d,29d and 32d,respectively.The expression levels of P5CR gene were analyzed by qRT-PCR.The expression levels of P5CR gene in the mycelia ranged at 32d>23d>29d>26d in experimental group,while they ranged at 23d>26d>29d>32d in control group.The expression levels of P5CR gene in experimental group were lower than that in control group on 23d and 26d,while they were higher than that in control on 29d and32d,which inferred that the exogenous L-PA was going to be exhausted,therefore,the expression of P5CR gene was increased to maintain the demand of L-PA in fungal cells.The levels of SW from mycelia determined by HPLC-MS in experimental group were higher than that in the control group,which suggested that synthesis of SW was promoted after the addition of L-PA.2.The swnH2 gene sequence was cloned by PCR with specific primers designed according to A.oxytropis OW7.8 genome sequence,which was 1016 bp from ATG to TAA.There was one intron in accordance with GT-AG rule,and the ORF was 957bp which was predicted as 318 amino acids.Compered to the sequence of A.oxytropis(KY365741.1)submitted by Cook,a nucleotide at 588 was different(T?C),but this variation did not change encoded amino acid glycine.The swnH2 gene encoded a stable hydrophilic protein,whose molecular formula was C₁₅₇₈H₂₅₀₉N₄₂₅O₄₈₁S₁₄with 35579.68D of MW and5.16 of p I.There was no transmembrane region and signal peptide.The secondary structure was mainly random curling.The protein was likely to be located in cytoplasm.3.The 5?terminal of swnH2 gene and part of upstream sequences(-329bp?57bp,386bp in length,containing restriction sites of Xma?and Age?),the hygromycin phosphotransferase gene(hph,containing promoter,terminator,restriction sites of Age?and Sbf?),the 3?terminal of swnH2 geng and part of downstream sequences(601bp?1016bp and downstream 42bp,457bp in length,containing restriction sites of Sbf?and Eco R?)were amplified,after they were digested by corresponding restriction enzymes,the digested products were ligated to form a swnH2gene knockout fragment.The swnH2 gene knockout fragment and p UC19were digested by Xma?and Eco R?respectively,and ligated to form a swnH2 gene knockout vector(4913bp).The ORF of swnH2 gene cDNA was amplified with Eco R?and Eco R?lingkers at two 5?terminals.This sequence and p BARGPE1-Hygro were digested by Eco R?and Eco R?respectively,than they ligated to construct a swnH2 gene overexpression vector(6949bp),which contained gpd A promoter,trp C terminator,hph and Amp^r.4.The 5?terminal of P5CR gene(1bp?405bp,405bp in length,containing restriction sites of Bam H?and Age?),the hph gene(containing promoter,terminator,restriction sites of Age?and Sbf?),the3?terminal of P5CR gene(760bp?1179bp,420bp in length,containing restriction sites of Sbf?and Eco R?)were amplified,after they were digested by corresponding restriction enzymes,the digested products were ligated to form a P5CR gene knockout fragment.The P5CR gene knockout fragment and p UC19 were digested by Xma?and Eco R?respectively,and ligated to form a P5CR gene knockout vector(4890bp).The ORF of P5CR gene cDNA was amplified with Xma?and Eco R?lingkers at two 5?terminals.This sequence and p BARGPE1-Hygro were digested by Xma?and Eco R?respectively,than they ligated to construct a P5CR gene overexpression vector(6940bp),which contained gpd A promoter,trp C terminator,hph and Amp^r.5.The protoplast preparation technique of OW7.8 endophytic fungi was optimized,and the yield was increased to 2.85×10⁷cells/m L,which was 100 times higher than that in previous research.