Research On Metabolic Pathway Of The Alkaloid Biosynthesis And Key Gene In Aconitum Vilmorinianum Kom.

Aconitum vilmorinianum Kom.is Aconitum of Ranunculaceae.There are 350 species of Aconitum in the world,mainly distributed in temperate areas of the northern hemisphere.There are more than 200 species of Aconitum in China,and 76 species are used as drugs.Yunnan is the distribution center of Aconitum,with 66 species,25 varieties and 4 forms.A.vilmorinianum is mainly produced in Yunnan and is a genuine traditional Chinese medicine in Yunnan.A.vilmorinianum contains diterpenoid alkaloids such as yunaconitine,vilmorrianine A,and bulleyaconitine A,.It has the effects of dispelling wind and dispersing cold,promoting blood circulation and relieving pain,anti-tumor,dehumidification and detoxification.It is the main raw material of famous Chinese patent medicines such as Yunnan Baiyao and bulleyaconitine A in Southwest China.With the in-depth study on the pharmacological activity and mechanism of A.vilmorinianum,it will show a wider range of pharmacological effects and medicinal value.Due to the overexploitation of wild A.vilmorinianum,the wild resources are on the verge of depletion,so it is necessary to artificially cultivate A.vilmorinianum to meet the demand.At present,there are many studies on the influence of environmental factors,cultivation,active components and pharmacological effects of A.vilmorinianum,but the research on the biosynthetic pathway and regulation of alkaloids of A.vilmorinianum is still blank.Therefore,this study will master the biological characteristics of A.vilmorinianum,establish the genetic transformation system,reveal the alkaloid synthesis pathway and regulation mechanism,and provide support and theoretical guidance for the study of gene function,alkaloid synthesis and regulation.The following results were obtained:1.By observing the characters of A.vilmorinianum and analyzing the content of effective components in various organs,the roots of A.vilmorinianum were divided into three types:straight root type,carrot root type and oval type.The content of aconitine in carrot root was the highest,up to 174.9μg/g;The color of roots can be divided into white heart,yellow heart and red heart.With the color of roots turning red,the content of aconitine increased;The stem branches are single stem,double stem and multi stem.The stem colors are divided into purple,purple green and green.The content of aconitine alkaloids in flowers was the highest,reaching154.18μg/g,followed by roots and tubers,and the content of stems was the lowest,only 4.95μg/g;The content in roots was higher at the initial stage of root formation,26,53μg/g,the highest at the middle stage,70.01μg/g,and decreased at the end,21.55μg/g;The content of cortex and center was similar,higher than that of bud head.2.QRT PCR was used to screen the internal reference genes of A.vilmorinianum root,stem,leaf and flower tissue.GADPH and MDH can be used as the internal reference genes of root group,MDH and 18 S r RNA can be used as the reference genes of stem tissue,MDH,18 S r RNA and ACTcan be used as the internal reference genes of leaf tissue,GAPDH β-TUB gene can be used as an endofloral gene.3.According to the transcriptome and metabolic analysis of A.vilmorinianum,119660 unigenes and 69978 annotated uingene were obtained.The metabolic pathway of diterpene alkaloids in A.vilmorinianum was revealed.It was identified that the metabolic pathway had 27 genes,transcription factors regulating the synthesis of diterpene alkaloids,miRNA and CYP450 genes.4.356 target genes of 54 known miRNA families and 48 target genes of 151 new miRNA were identified by miRNA sequencing analysis.miR6300 and its target gene HMGR were identified to participate in the formation of terpene skeleton and regulate the synthesis of diterpene alkaloids.5.The sequences of Av HMGR,Av DXS,Av DXR,Av KS and avi-miR6300,as well as the plant overexpression and inhibition expression vectors,as well as the activity characteristics of these enzymes and related biological information were obtained through the cloning of key genes of alkaloid anabolic pathway of Aconitum Aconitum.6.The regeneration system and genetic transformation system of A.vilmorinianum were established.Av HMGR and avi-miR6300 were introduced into A.vilmorinianum to obtain the transient expression system;The EGFP fluorescent marker was detected by vacuum penetration tuber soaking method,seed ultrasonic soaking method and flower dipping method.The target genes of Av HMGR and avi-miR6300 were successfully introduced into A.vilmorinianum,and its the genetic transformation system was established.Through this study,the biological characteristics of A.vilmorinianum were mastered,the regeneration system and genetic transformation system were established,the alkaloid biosynthesis pathway and the transcription factors,miRNA and CYP450 regulatory factors regulating the target genes of this pathway were preliminarily revealed,and the plant overexpression vector and inhibitory expression vector of the key genes of this pathway were obtained,which provided the basis for the functional gene identification,gene resource development,molecular breeding Genetic improvement research has laid a solid foundation.