| Tolypocladium guangdongense is a newly discovered Cordyceps resource in South China,having abundantly active ingredients,nutritional ingredients and obvious pharmacological effects.And,it has been approved as a new resource food.Ophiocordyceps sinensis,a traditional and precious Chinese herbal medicine,has clear and significant pharmacological function,and possesses a long application history.O.sinensis is a hypothermophilous fungus,whereas the optimum growth temperature of T.guangdongense is obviously higher than that of O.sinensis.So far,the development of large-scale artificial cultivation in T.guangdongense and O.sinensis has a different level technical hindrance.Temperature is an important environmental factor during the artificial cultivation process of T.guangdongense and O.sinensis.It is necessary to be explore and decipher how temperature affects the growth and metabolism of them,and what is the molecular regulation mode under different temperatures.This will provide the significant reference value for two Cordyceps strains,and promote the benign development of relevant industry.In this study,T.guangdongense and O.sinensis were selected as the materials.The effect of temperature on the mycelial growth in two Cordyceps strains and the primordial formation of T.guangdongense was analyzed.According to that screening results,we performed the transcriptome analysis of different temperature effect on the primordial formation of T.guangdongense and the growth of O.sinensis.In addition,we analyzed the expression pattern under different experimental conditions and primary research of gene function for trpE in T.guangdongense.The main results were as follows:1.Compared the mycelial growth of two Cordyceps strains in CYM medium.The mycelia of T.guangdongense grow best at 23℃ for ? d,and the mean of colony diameters is about 3.6 cm.The mycelia of O.sinensis grow best at 20℃ for ? d,and the mean of colony diameters is approximate 1.1 cm.2.Analyzed the difference of primordial formation of T.guangdongense in different temperature treatment.The growth of primordia at 23℃ was the best at the same period,and the numbers of primordia decreased with the increasement of days at 17 ℃ and 29 ℃,indicating that 17 ℃ and 29 ℃inhibited the formation of primordia,and were adverse to the formation of T.guangdongense primordia.3.Mycelial plugs of T.guangdongense were inoculated in the CYM plate,and were collected after about two month culture under diffentent tempetature conditions.Total RNAs were extracted by Trizol method and were sent to Beijing Genomics Institute for library construction and sequencing.According to the analysis of transcriptome data,2,495 differentially expressed genes(DEGs)were obtained.For the number of DEGs between the two groups,the most number of DEGs was 1,981,but the least number was 179.The analysis of GO annotation showed that DEGs were mainly involved in biological processes such as metabolism,biological regulation and stress;cellular components such as membrane,cells and organelles;molecular functions such as catalysis,transport and transcriptional regulation.The KEGG analysis showed that DEGs were mainly involved in regulatory pathways such as cell growth and apoptosis,cell transduction,cancer and carbohydrate metabolism,etc.4.The mycelial plugs of O.sinensis were inoculated on solid CYM medium and cultured at different temperature conditions.After approximate 45 d culture,the mycelial samples were collected.Total RNAs were extracted by Trizol method and were sent to Beijing Genomics Institute for library construction and sequencing.A total of 7,465 DEGs were identified in all comparisons.The most number of DEGs was 1,885,while the least was 545.The analysis of GO annotation showed that the DEGS were mainly involved in biological processes such as cell killing,metabolism,signal transduction and single biological processes;cellular components such as extracellular regions,membranes,cells and macromolecular complexes;molecular functions such as nucleic acid binding transcription factors,catalysis,transporters and binding.The analysis of KEGG showed that DEGs were mainly involved in regulatory pathways such as metabolism,biosynthesis of secondary metabolites,metabolism of microorganisms in different environment conditions,amino acid metabolism,etc.5.The coding sequence of trpE was cloned from T.guangdongense genome.And then,the bioinformatics analysis and phylogenetic analysis were carried out on the basis that sequence information.The expression pattern of trpE of T.guangdongense was analyzed at different developmental stages and temperature stress using real-time fluorescence quantitative PCR(RT-q PCR).The results showed that the coding sequence length of trpE in DNA and c DNA was 1,613bp(DNA)and 1,563bp(c DNA)respectively.It encoded 521 amino acids with about 56.86 k Da of molecular weight predicted by the software.Phylogenetic analysis showed that the TrpE protein sequence of T.guangdongense was clustered with those of ascomycetes,and was highly similar to those of T.paradoxum and T.capitatum.During the development process of primordia and young fruiting bodies,trpE was significantly up-regulated,but significantly down-regulated at mature fruiting stage.Meanwhile,trpE expression was significantly down-regulation during temperature stress process.6.The antisence of function conservative region from trpE,actin promotor and gpd promotor were amplified by PCR.Then,they were ligated and integrated into plasmid p CAMBIA1300-g by the homologous recombination enzyme.The recombinant plasmid p CAMBIA1300-i-trpE was introduced into Agrobacterium tumefaciens.And then,the mycelia of T.guangdongense were infected using the Agrobacterium-mediated transformation method.Five positive transformants were screened on the PDA plates containing 300 μg/m L hygromycin B.To sum up,this study preliminarily discussed the effect of temperature on the mycelial growth of T.guangdongense and O.sinensis,the primordial formation of T.guangdongense.Meanwhile,we analyzed the molecular mechanisms of temperature effect on the primordial formation of T.guangdongense and the molecular response mechanism of O.sinensis mycelia to temperature stress.In addition,the expression pattern and gene function of trpE in T.guangdongense were explored.This provides scientific significance and theoretical value for improving the artificial cultivation techniques of T.guangdongense and O.sinensis. |
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