Functional Study And Potential Distribution Prediction Of The Mevalonate Kinase Gene Of Fritillaria Unibracteata

Fritillaria cirrhosa D.Don is derived from the dried bulbs of some plants of the genus Fritillaria in the lily family,and has a long history of use as a traditional Sichuan medicinal herb.The 2020 edition of Pharmacopoeia of the People’s Republic of China stipulates that the herbs of Sichuan mussels include“Songbei”,“Qingbei”,“Lubei”and“cultivated mussels”,among which“Songbei”are of the best quality.The bulbs of Fritillaria unibracteata are often used as the mainstay of“Songbei”and are in great clinical and market demand.However,due to the special ecological environment of Fritillaria unibracteata and the over-harvesting by human,the population of Fritillaria unibracteata,which has not yet been solved for domestic cultivation,has been drastically reduced and is on the verge of extinction.Therefore,the author believes that it is urgent to use genetic engineering and artificial intelligence to conserve and utilize the resources of Fritillaria unibracteata.This thesis focuses on two issues related to the conservation and utilization of Fritillaria unibracteata resources:Issue 1,the index components of Fritillaria unibracteata are components such as sipeimine,but the content is low（not less than 0.05%according to Pharmacopoeia of the People’s Republic of China）.At present,there are very few studies on the biosynthetic pathway of sipeimine,and research on the enzyme genes related to sipeimine biosynthesis will provide important candidate target genes for improving the yield of sipeimine and other components using metabolic engineering,thus reducing the dependence on the wild resources of Fritillaria unibracteata and achieving the goal of Fritillaria unibracteata resource conservation.In this thesis,the mevalonate kinase gene of Fritillaria unibracteata（FuMK）was investigated and the high expression of FuMK gene in leaves was determined by quantitative PCR method.The pET28a-FuMK recombinant vector was constructed and transformed into E.coli-BL21（DE3）.The recombinant strain successfully expressed FuMK recombinant protein under Isopropyl-thiogalactopyranoside（IPTG）induction,and the highest soluble expression of FuMK recombinant protein was achieved at IPTG concentration of 1mM,induction temperature of 28℃ and induction time of 8 h.The purified soluble FuMK protein was subjected to enzymatic activity assay under RS-mevalonate（MVA）and ATP substrate conditions using Mg²⁺as a metal catalyst,and the results showed that when c（MVA）=0.8 mmol/L,c（ATP）=1.0 mmol/L and c(Mg²⁺)=0.8 mmol/L the activity of FuMK was the highest.Based on the results of comparative homology analysis,it was hypothesized that two amino acid residues,Gly142 and Ser147,were involved in the binding of ATP by the enzyme;the results of the enzyme activity assay showed that the substrate affinity of the mutant recombinant protein was reduced and the reaction efficiency and specificity were decreased,but the maximum reaction rate was increased,demonstrating that the conformational change of the amino acid site affects the binding of the protein to the substrate.Using the flower dip method to obtain the trans-FuMK gene in Arabidopsis,preliminary studies revealed that the transgenic seeds had a reduced germination rate and a smaller biomass but more developed root system under drought and salt stress（using wild-type Arabidopsis as a control）,and it was hypothesized that overexpression of the FuMK gene might affect the synthesis of some secondary metabolites and/or phytohormones in the transgenic plants.In question 2,the wild resources of the genus Phyllanthus,represented by Fritillaria unibracteata and Fritillaria taipaiensis,etc.,have declined dramatically.To alleviate the conflict between the demand for Fritillaria unibracteata and Fritillaria taipaiensis,etc.and the scarcity of wild resources,the introduction and artificial cultivation of Fritillaria unibracteata and Fritillaria taipaiensis,etc.is a feasible alternative method.This thesis uses artificial intelligence techniques such as the maximum entropy model to predict the potential distribution areas of Fritillaria unibracteata and Fritillaria taipaiensis,which will help to provide a basis for the wild nurturing and cultivation of Fritillaria unibracteata and Fritillaria taipaiensis,and expand the scope of artificial cultivation sites.At the same time,this part of the research results will also provide theoretical guidance for the analysis of the habitat of other mollusks.The prediction results showed that the environmental factors that contributed more to the distribution of the Fritillaria unibracteata were annual precipitation（26.12%）and altitude（20.11%）,and the most suitable potential distribution area is mainly concentrated in southwestern Gansu,southeastern Qinghai,and northern and northwestern Sichuan;the environmental factors that contributed more to the distribution of the Fritillaria taipaiensis were mean diurnal temperature difference（41.21%）and altitude（26.42%）,and the most suitable potential distribution areas are concentrated in central and northeastern Sichuan,northern Chongqing,southern Gansu,northwestern Guizhou,southwestern Shaanxi,and western Hubei.