| Anoectochilus roxburghii has a very high medicinal value,and its flavonoids,sterols,polysaccharides,triterpenoids and other ingredients have broad prospects for development.Among them,the medicinal value of sterols has been used to treat high cholesterol,atherosclerosis,etc.A.roxburghii has been proven to contain a variety of sterols,and studies have shown that its sterol extract has a certain anti-tumor effect.To find a certain correlation and regulation mechanism between the expression of key genes in A.roxburghii sterol synthesis and the change of sterol content,in order to improve the accumulation of tissue culture or artificial cultivation of A.roxburghii sterol compounds.In the future,cell engineering methods can be used to produce sterols.In this study,the molecular level regulation and product content of A.roxburghii were studied.The key enzyme gene on the synthetic pathway of A.roxburghii sterol was cloned with sterol as the core,and the relative expression of related genes was used as an aid to analyze by bioinformatics.And subcellular localization,the A.roxburghii sterol biosynthesis pathway is clarified to obtain the key to the A.roxburghii sterol biosynthetic pathway.The results of this study are as follows.1.Ultrasound-assisted extraction and High performance liquid chromatography were used to explore the extraction and detection methods of A.roxburghii sterol.Three sterols?Ergosterol,stigmasterol,?-sitosterol?were extracted and content measured from A.roxburghii induced by 100 mmol·L-1 salt?NaCl?at three different time points?0h?blank control?,6h,12h?.It was found that A.roxburghii sterol was extracted with95%ethanol as the extractant,and the extraction ratio of 15:1 was superior.The content of three sterols increased at 6h,and was significantly different from 0h.At 12h,the content of ergosterol and stigmasterol decreased,while the content of?-sitosterol still increased.By summing the contents of the three sterols measured at three time points,at 12h,although?-sitosterol showed an increase,overall,the total sterol content decreased,and the A.roxburghii sterol content was affected.The effect of salt,salt promotes the accumulation of sterols in a certain period of time,but this effect will weaken or recover after a certain period of time.2.The FPS?farnesyl pyrophaophate synthase,FPS?and GPPS?Geranyl pyrophosphate synthase,GPPS?genes of A.roxburghii were cloned based on the RNA-Seq.Sequence analysis indicated that the open reading frame of FPS gene was 1047 bp,which had the highest homology with Dendrobium huoshanense.After translation,the FPS gene encoded348 amino acid residues and the secondary structure of FPS protein was composed of?helix and random coil.The constitutive analysis indicated that its FPS protein was closely related to Dendrobium huoshanense and Anoectochilus formosanus.The open reading frame of GPPS gene is 1092bp,which has the highest homology with Phalaenopsis bellina.After translation,the secondary structure of GPPS protein composed of 363amino acid residues is mainly composed of?-helix and random coil.There is no transmembrane region,and phylogenetic analysis indicates that it is closely related to Phalaenopsis bellina and Gentiana rigescens.3.The relative expression of FPS gene of A.roxburghii at different time points under salt induction was detected and analyzed by real-time fluorescence quantification.The results showed that the relative expression of FPS gene increased at 6h and the difference was significant.At 12h,the relative expression decreased,and this trend was similar to that of the experimental results of sterol determination.It can be seen that the expression of FPS gene has a certain correlation with the content of A.roxburghii sterol.4.Subcellular localization of the FPS protein was carried out by inserting a relative cloning site containing an enhanced green fluorescent protein gene?eGFP?vector into the inner epidermis of the onion.Finally,observe with a microscope.Fluorescence signals were discoverd in the cytoplasm and nucleus of the inner epidermis of onions.This result confirms the prediction of cytoplasmic and nuclear localization of FPS proteins.The results indicate that FPS protein plays its role in the cytoplasm. |
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