| Taxus chinensis var.mairei is a national first-class protected plant and a variety of yew tree distributed in China.It is mainly distributed in the south of the Yangtze River basin,is currently recognized as the world’s endangered natural rare anti-cancer plant.Mycorrhizal fungi are beneficial microorganisms that can establish reciprocal symbionts with most plant roots.The hyphae infects the root of the host plant,which velamen hasn’t been suberized,forming a hyphal sheath,epitaxial hyphae and other characteristic structures on the surface of the root tip,and can also expand the absorption area of plant root system,improve the water utilization,promote the absorption and utilization of mineral nutrients,improve the carbon cycle in the body,and affect the primary and secondary metabolism of plants by forming dense mycelium net between the rhizosphere soil and root cortex cells.Research found,the original root tissue in the mycorrhiza of T.chinensis var.mairei had higher paclitaxel accumulation than other organ tissues,to reveal the biological mechanisms,in this article,the technologies and methods of HPLC,microscopic observation,tissue culture,morphological observation combined with biological analysis of fungal ribosomal transcription spacer(r DNA-ITS)sequence,liquid suspension culture of fungi were used to research on the microscopic observations of morphological anatomy and paclitaxel content in important tissues of T.chinensis var.mairei mycorrhiza,the isolation,purification and species identification of mycorrhizal fungi,single bacterial strain pure culture,multiple strains mixed culture,the interacted culture using the strains with the callus of T.chinensis var.mairei to produce paclitaxel.The result as follows:Through microscopic observation of mycorrhizal morphology anatomy,it was found to be ectomycorrhizas and mainly composed of mycorrhizal sheath and mycorrhizal primitive root tissue.The red area in the center of the mycorrhizal cross section is the primitive root tissue,which consisted of a red layer of nearly suberized epidermal cells and vascular bundles surrounded by them,and the outer layer of which is the casparian strips and mycorrhizal sheath parenchyma cells.The two structures were separated and the paclitaxel content was detected.The results showed that it’s content in the primitive root tissue was 0.2086%,which was 0.1891% higher than that in mycorrhizal sheath tissue,and 0.0654% higher than that in fibrous root,and one order of magnitude higher than that in other organ tissues.Therefore,it was inferred that the higher paclitaxel accumulation in mycorrhizal primitive root tissue is the effect of symbiotic interaction between mycorrhizal fungi and T.chinensis var.mairei roots.In order to verify the above inference,the mycorrhiza frome the mycorrhizal sample of T.chinensis var.mairei collected in the same sample plot were isolated and purified by tissue culture,and the species identification was carried out by morphological observation analogy combined with molecular biological methods,26 strains obtained belong to 3 phyla,6 classes,9 orders,11 families and 14 genera,including Aspergillus,Trichoderma,Fusarium,Penicillium,Cylindrodendrum,Cylindrocladiella,Clonostachys,Xylaria,Cladosporium,Corynespora,Paraboeremia,Pezicula,Colletotrichum and Bjerkandera,in which the first two genera are dominant fungi.It demonstrated that the mycorrhizal fungi of T.chinensis var.mairei have high species diversity,which was significantly different with endophytic fungi of it’s root,bark,bast,wood,leaves and other organ tissues reported in the literature,which had preliminarily verified the above conclusion.To further verify,following closely that,the production paclitaxel was studied by liquid pure fermentation of 26 mycorrhizal fungi,the result showed that 11 strains produced 10-DABⅢ or paclitaxel.Among these strains,the yield of 5 strains of10-DABⅢ were respectively 209.93,282.70,507.14,108.88,274.01 μg/L,but no paclitaxel was detected,the yield of 6 strains of paclitaxel were respectively 154.16,277.21,459.61,276.70,326.78,117.81 μg/L,but no 10-DABⅢ was detected.However,the productivity of paclitaxel or 10-DABⅢ gradually decreased to no detection after nine generations of pure culture of single strain.The results showed that the ability to produce paclitaxel or 10-DABIII of a single strain was acquired from the secondary metabolic antagonism adaptive interaction relationship established in the biologic system of mutualistic symbiosis between mycorrhizal fungi and T.chinensis var.mairei root cells for biochemical protection,when separated from this system,the biochemical properties on that aspect of the strain are gradually lost,which makes the above inference more deeply verified.According to the above results,there should also be secondary metabolic antagonistic adaptation interaction relationship between strains.In order to find a biotechnological system to maintain the ability of paclitaxel production or 10-DABⅢ in vitro,the mixed single-stage and multi-stage linked co-culture of 4 strains combinations was carried out.In single-stage mixed culture system,the 10-DABⅢ productivity of the first,second and third combinations was 2247.96,2235.65,480.29 μg/L respectively,but didn’t produced paclitaxel.The productivity of the fourth combination 10-DABⅢand paclitaxel was 150.70 and 2194.61 μg/L respectively,and both of which were much higher than that of the single strain pure culture system,and reversed from no10-DABⅢ or paclitaxel to high yield.In multi-stage linked co-culture system,the10-DABⅢ productivity of the second stage is 4809.83 μg/L,and was 2.15 times that of the first stage,but it still didn’t produced paclitaxel.However,the 10-DABⅢproductivity of the third stage is only 690.80 μg/L,which is significantly lower than that of the second stage and didn’t produced paclitaxel too.The paclitaxel productivity of the stage 4 was up to 5683.53 μg/L,and was 2.59 times that of stage 1 mixed culture,and that of 10-DABⅢ is 953.22 μg/L,and was 6.33 times that of stage 1 mixed culture.It showed that 10-DABⅢ was significantly reduced after the third stage mixed culture,which provided the biochemical basis for the fourth stage mixed culture biosynthesis of paclitaxel.The results fully demonstrated that there was a secondary metabolic antagonistic adaptation interaction relationship between strains,which greatly promoted the biosynthesis of 10-DABⅢ or paclitaxel in multi-stage linked co-culture system,thus confirming the conclusion.The paclitaxel content in callus of Taxus chinensis var.mairei stem segment was only 0.0373%(dry weight),after which co-cultured with mycorrhizal fungi in the same culture system,the growth rate of the strain was much faster than that of the callus,resulting in the rapid inactivation of the callus cells,resulting in the decrease of the productivity of 10-DABⅢ and paclitaxel.However,the paclitaxel yield in callus was significantly increased after mutural beneficial cooperation co-culture,and it was up to0.1045%(dry weight),which was 2.8 times that of the original callus.The 10-DABⅢcontent(0.0196%,dry weight)is reduced because of chemical equilibrium.In conclusion,this study revealed that the biological mechanism of high paclitaxel content in the primitive root tissue of T.chinensis var.mairei mycorrhizae is the biological effect of symbiotic interaction between strains and root cells or strains each other.And found that only in this symbiotic interaction system,mycorrhizal fungi only had the ability of continuous biosynthesis paclitaxel or 10-DABⅢ.The results of this study have very important reference and application value for the research of the useful secondary metabolites by plant mycorrhizal fungi and the genuineness of herbal medicine derived from the roots. |
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