| "Cordyceps" is the general term of fungi in Cordyceps genus.The Cordyceps industry in China mainly involves in relatively rare fungal species such as Cordyceps sinensis and C.militaris.Among them,the composition and pharmacological effects of C.militaris are similar to those of C.sinensis,especially CMPs-4 polysaccharide that has important economic value.Moreover,C.militaris can be artificially cultivated,which is one of the hot spots in the research field of medicinal fungi.Artificial cultivation is an effective way to solve the serious shortage of wild source of C.militaris.The abundant rice and wheat resources in Xinjiang provide sufficient materials for the artificial cultivation of this mushroom.The fungal degradation is the crucial problem to restrict the development of the artificial cultivation.This research focuses on the breeding of high-quality and high-yielding strains and the condition optimization in order to provide theoretical and technical guidance for the large-scale production of C.militaris.In this study,the parent strains were selected from the collected and preserved strains and used to induce the fruiting bodies.The single spores were isolated for mating type identification.Thereafter,the new generated strains produced from different mating types was isolated and screened as the F1 generation strains with higher yield of fruiting bodies.In order to increase fruiting body production,a total of 60 single spore strains were obtained from different sources.Thereafter,the suitable substrates for the locally artificial cultivation were determined based on the analysis of fruiting body length,fresh and dry weight,biotransformation rate,yields and output value for the different culture substrates coupled with the local situation of the artificial cultivation in Xinjiang.Finally,the inhibitory effects of the polysaccharide CMPs-4 on the proliferation of human esophageal cancer cells by the MTT method;the effects of CMPs-4 on the cell cycle and apoptosis of Eca109 cells was investigated by the flow cytometry coupled with the scanning electron microscope for observing the apoptotic cells morphology;the effect of CMPs-4 on Eca109 cell apoptosis was detected by Hoechst33258 staining and Annexin V-FITC/PI double staining while the expression of Bcl-2,Bax,caspase-8 and caspase-3 were detected by western blot.Results indicated below:(1)A total obtained from the parent fruiting bodies were composed of 10 strains.After 10 representative strains were artificially cultured to form fruiting bodies,60 single ascospore strains were obtained by single spore isolation.PCR identification of mating types showed that 22 were MAT 1-1 and 15 were MAT 1-2.Meanwhile,37 strains can only amplify either MAT1-2 or MAT1-1,indicating that MAT1-1 and MAT1-2 cannot identify simultaneously and a obvious bipolar heterothallic system.Combination culture of the same mating type has slower growth and development,but higher compatibility.However,when MAT1-2 and MAT1-1 were cultured with different mating types,the developed better,but the compatibility was relatively weaker,indicating that different mating type strains with compatibility were more likely to form fruiting bodies.(2)15 strains with different genetic types(MAT1-1 and MAT1-2)were selected for mating,namely A2×B4,A2×B1,A2×A9,A2×A1,A5×B1,A5×A1,B3 ×B8,B3×B4,B3×B1,B9×B4,B9×A9,B9×A1,B11×B8,B11×B4,B11×B1,which provide an ideal strain combination for artificial cultivation.(3)A significant difference in the yield of fruiting bodies using different cultural media(P<0.05).Specifically,the yield of fruiting bodies induced in rice medium is higher,12.77 g of the fresh weight and 2.05 g of the dry weight.The bioconversion rate reached to 60.54% whilst the maximum output-to-input ratio was 4.45.Meanwhile,the output value was 2.05 Yuan/bottle and the net profit was 1.59 Yuan/bottle.The biological yield of its fruiting bodies was increased by 33.7% compared to the original.At the same time,the rice medium is more conducive to the accumulation of cordycepin and adenosine in fruiting bodies of C.militaris.(4)Polysaccharide CMPs-4 can inhibit the proliferation and induce apoptosis of human esophageal cancer Eca 109 cells,which anti-tumor effect is dependent on the dose and time.CMPs-4 significantly inhibited the growth and proliferation of Eca-109 cells.When the drug concentration was increased from 100 to 1000 ?g/m L,the growth inhibition rate increased from 11.70% to 66.54%,and the IC50 value of 24 h culture was 532.9 ?g/m L.Flow cytometry showed that the proportion of Eca109 cells in S phase gradually increased from 32.23% to 49.27%(P<0.05),which means that Eca109 cells were blocked in S phase after CMPs-4 treatment and could not transform to increases cells in the G2/M phase,and the cells in the G0/G1 phase cannot enter the S phase.The apoptosis rate increased from 3.12% to 14.44%(P<0.05),and the morphological characteristics of apoptosis can also be observed under the electron microscope.After treatment of Eca109 cells with CMPs-4,the expression of antiapoptotic protein Bcl-2 was significantly reduced,but the expression of pro-apoptotic proteins Caspase-3 and Caspase--8 showed increased.This study screened high-quality C.militaris strains and artificial mediums suitable for Xinjiang,optimized the culture system of C.militaris,and provided scientific basis and technical guidance for the large-scale development of C.militaris industry under the conditions of solar greenhouses and modern greenhouses,making full use of the abundant wheat and rice resources in Xinjiang.This study also conducted a research of the anti-tumor mechanism of C.militaris CMPs-4 polysaccharides,and provides scientific reference for further exploring the molecular mechanism of antitumor effect of C.militaris CMPs-4 polysaccharide. |
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