| Cordyceps,parasitizing insects and Elaphomyces spp,are good model research systems for fungal genetics.Many species of Cordyceps are economically and medicinally valuable fungi.Ophiocordyceps xuefengensis,a recently described species of Ophiocordyceps associated with larvae of Phassus nodus(Hepialidae)in living root or trunk of Clerodendrum cyrtophyllum in the Xuefeng Mountains of Hunan Province in south China,is the world's largest known Cordyceps species and identified as the sister taxon of O.sinensis.In this study,the biological characteristics of the anamorphic stages,fermentation for production of mycelial biomass,the cultivation of stromata in artificial media,chemical composition,and the mating type genes of O.xuefengensis were systematically studied.(1)The anamorphic stages of O.xuefengensis were isolated from the stromata of the fungi.Cultural,morphological characteristics,the fungus bearing stromata successfully in artificial media,as well as molecular evidence(ITS sequence)have proved its identity.The mating-type gene MAT1-1-1 and MAT1-2-1 in different samples and tissues of O.xuefengensis were amplified by PCR,the results showed that only the mature ascus contained MAT1-1-1 and MAT1-2-1.Thus,O.xuefengensis is heterothallic species.(2)The optimization of flask-shaking batch fermentation medium for biomass production from 0.xuefengensis was studied.Single-factor experiments,Plackett-Burman design,the path of steepest ascent,Box-Behnken design,and response surface methodology(RSM)were employed to optimize the fermentation conditions,the statistical analysis was used to establish the mathematical model.Under optimized conditions,the biomass of mycelium in the fermentation broth reached 18.8 g/L,which was was 17.5%higher than that in single factor experiments,up to 16.0 g/L.In addition,the optimized conditions for the artificial cultivation of O.xuefengensis stromata were optimized by the L9(34)orthogonal test.The result revealed that artificial cultivation conditions for 0.xuefengensis stromata were as follows:Solid matrix 20 g,solid matrix,nutrient liquid 1:2(W/V),and the 3%inoculum size,was incubated in complete darkness for 15 d at 22?,then the tissue culture bottles were placed in a light intensity intensity of 20-40 lux,at 19? for 45 days.The production and the bioconversion ratio of stromata were 4.78 g/bottle and 22%,respectively.The improved production condition will be helpful for the development of O.xuefengensis in the fields of health foods and new drugs.(3)Further works such as the main nucleosides and nicleobases,the fatty acids,amino acids,etc.in natural and cultured O.xuefengensis were also performed.The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC.The concentrations of adenosine in the natural fruit body,the cultured stromata,and the cultured mycelium were 761.5 mg/kg,721.2 mg/kg and 623.9 mg/kg,respectively.The contents of adenine,guanosine,uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample.The concentration of guanosine in the cultured samples was also higher than in wild samples.The content of adenosine in cultivated 0.xuefengensis was over 0.06%,which was much higher than the content of adenosine in the 2015 edition of Chinese Pharmacopoeia(>0.01%).The contents of cordycepic acid in the three different tissues(sclerotium,stromata and mycelium)of O.xuefengensis were 7.8%,11.7%and 15.0%,respectively.The percent of unsaturated fatty acids(UFA)relative to total fatty acids in the three different tissues were 77.6%,66.1%and 65.4%,respectively.The fatty acids in the sclerotium of the natural O.xuefengensis was mainly oleic acid(monounsaturated fatty acids),and the fatty acids in the cultured O.xuefengensis was mainly linoleic acid(polyunsaturated fatty acids).The contents of amino acids in the three different tissues(sclerotium,stromata and mycelium)were 211.55 mg/g,191.48 mg/g and 282.26 mg/g,respectively.The contents of essential amino acids were 104.93 mg/g,60.79 mg/g and 108.49 mg/g,respectively.These results suggested that the cultured mycelium and cultured stromata of O.xuefengensis might be a promising substitute for natural O.xuefengensis.(4)The MAT1-1 and MAT1-2 locus of O.xuefengensis were cloned and analyzed.The High-Efficiency Thermal Asymmetric Interlaced PCR(hiTAIL-PCR)technique was employed to clone the mating type MAT1-1 and MAT1-2,and the genomic southern hybridization of O.xuefengensis were probed with the MAT1-1-1 and MAT1-2-1sequence.Comparison of the MAT1-1 and MAT1-2 sequence of O.xuefengensis,it can be concluded that:? the structures of the MAT1-1 and MAT1-2 locus in O.xuefengensis was similar to reported O.sinensis;? the MAT1-2 locus of O.xuefengensis contains a single gene(MAT1-2-1),which must be a single-copy gene;? the MAT1-1 locus contains three genes(MAT1-1-3,MAT1-1-2,and MAT1-1-1),which must be a single-copy gene;? 5'-flanking region of the idiomorphs(MAT1-1 and MAT1-2)are homologous,contain the conserved coding gene(DNA lyase);?The MAT1-1 locus,a single ORF of 728 bp with three putative introns coding for the complete MAT1-1-3 gene,the MAT1-1-2 ORF of 1318 bp with three putative introns,and the MAT1-1-1 ORF of 1220 bp also with two putative introns.The MAT1-2 locus contains complete MAT1-1-3 gene,a single ORF of 855 bp with two putative.In silico translation of MAT1-1-3,MAT1-1-2,MAT1-1-1 and MAT1-2-1 resulted in putative proteins of 192,382,371,and 249 amino acids,respectively.The MAT1-1-1,MAT1-1-2 and MAT1-1-3 protein similarities to O.sinensi were 89%?88%?84%and 90%,respectively.O.xuefengensis were identified as the sister taxon of O.sinensis.This study provides theoretical guidance for the artifcial cultivation of O.xuefengensis in the future;the characterization of the MAT1-1 and MAT 1-2 idiomorph in O.xuefengensis offer good model research systems for sexual reproduction and sexual selection,clue to better understand the genetics and biology of fungi. |
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