The Response Law Of Taxoids In Taxus Cuspidata To UV-B Radiation And Analysis Of Molecular Mechanism Of Metabolic Regulation

Paclitaxel,which contained in Taxus cuspidata is a diterpenoid compound with extremely high medicinal value recognized in the world.Because of its unique anti-cancer mechanism,it has been used as a broad-spectrum anti-cancer drug in the clinic of various cancers.There is a huge demand in the medical market for treatment.Limited by other sources and production methods of paclitaxel,there are technical bottlenecks and it is difficult to apply on a large scale.Therefore,the current supply of paclitaxel is still directly or indirectly derived from natural resources of Taxus.However,Taxus cuspidata grows slowly and the paclitaxel content is extremely low.The Taxus is on the verge of extinction due to human over-deforestation.Studies have shown that the biosynthetic pathway of paclitaxel is affected and regulated by a variety of physiological and ecological factors.As an important photoecological factor,UV-B has become a research hotspot in related fields for its application in the regulation of secondary metabolism of medicinal plants and the induction of increase in active ingredients.Therefore,based on the establishment of an accurate and reliable quality control analysis method for secondary metabolites,this article explored the effect of UV-B induction on the content of taxoids in Taxus cuspidata leaves,combined with physiological and biochemical indicators and transcriptomes.Sequencing analysis initially explored the mechanism of induction and regulation.The candidate key enzyme genes and regulatory factors of paclitaxel biosynthesis pathway in Taxus cuspidata induced by UV-B were excavated and expression verification.The main contents and results of this study are as follows:1.Established a UPLC-MS/MS quality control analysis methods for the simultaneous determination of 14 major taxoids and flavonoids in the leaves of Taxus cuspidata(10-deacetyl baccatin ?,baccatin ?,10-deacetyl paclitaxel,cephalomannine,paclitaxel,7-epitaxel,7-xylosyl-10-deacetyltaxol,quercitrin,isoquercitrin,kaempferol-3-O-rutinoside,rutin,bilobetin,ginkgetin,sciadopitysin).Methodological verification shows that each target component presents a good linear relationship within the detection range,and the sensitivity,precision,reproducibility,and accuracy of the analysis method are good.In addition,We optimized and determined the sample preparation process using high-speed homogenization combined with ultrasonic-assisted extraction,which can provide the necessary technical basis for the high-throughput and accurate detection and analysis of target active ingredients in subsequent samples.The results showed that the quality control analysis method used in the detection and analysis of true and complex Taxus leaf samples can achieve the expected qualitative and quantitative effects.The need for high-throughput detection and analysis of targeted metabolites with low content and similar structure in Taxus provides necessary detection methods and technical support for quality control and follow-up related research.2.Exploring the metabolic response of taxoids and flavonoids of Taxus cuspidata to UV-B.Firstly,the seedlings of Taxus cuspidata were treated with different intensities of UV-B radiation,and the content of taxoids and flavonoids in Taxus cuspidata after UV-B radiation induction was monitored for 0 h-96 h.Monitoring to investigate the effect of UV-B on the content of taxoids in the leaves of Taxus cuspidata.The results showed that the accumulation of major taxoids in the leaves of Taxus cuspidata generally increased and then decreased after UV-B radiation.And the higher the radiation intensity,the more significant the effect of inducing increment.The content of each taxoid component mainly reached the maximum at 48 h or 72 h,and the taxoid component generally showed a significant positive correlation.The results showed that most of the flavonoids in the leaves of Taxus cuspidata were significantly increased and then decreased after UV-B radiation.And the greater the stress intensity,the more significant the induction effect.Most of the flavonoids were concentrated in 48h to reach maximum value.In the secondary metabolic response induced by UV-B,the flavonoids are more sensitive and faster than the taxoids,and the maximum increase of the flavonoids reached 1.81-3.21 folds of the taxoids.And there is a significant positive correlation between the content of taxoids and flavonoids,indicating that the biosynthesis of taxoids and flavonoids in Taxus cuspidata may have a certain synergistic effect during self-defense.It can provide important data reference and theoretical basis for the UV-B targeted high-efficiency cultivation of Taxus cuspidata,the analysis of the synthesis and regulation mechanism of taxoid components at the gene level,and the efficient use of the renewable leave resources of Taxus cuspidata.3.In order to explore the physiological and biochemical response and regulation of Taxus cuspidata and its regulatory mechanism,different UV-B radiation time was used to induce Taxus cuspidata seedlings.UV-B radiation could reduce the leaf biomass and relative water content of Taxus cuspidata.The greater the radiation intensity,the more unfavorable the accumulation of leaf biomass and more significant impact the cell water physiology.The gas exchange parameters of Taxus cuspidata leaves were significantly reduced by UV-B radiation.Under high-intensity radiation,the net photosynthetic rate,stomatal conductance,intercellular CO2 concentration and transpiration rate decreased by 56.53%-77.68%reduced by UV-B induced.The content of carotene and chlorophyll generally showed a trend of increasing and then decreasing.The greater the radiation intensity,the greater the impact on the chlorophyll content,while the carotenoid content had a more significant impact at low intensity.Taxus cuspidata activates the antioxidant enzyme system to respond and defend against UV-B radiation.The activities of SOD,CAT,and POD show a significant upward trend in the early stage of UV-B induction,and then decrease with the increase of radiation time.The greater the intensity,the greater the impact on enzyme activity.The content of MDA will gradually increase with the extension of UV-B treatment time,and the cell membrane will gradually be damaged.Correlation and cluster analysis results show that there is a significant positive correlation between gas exchange parameters and photosynthetic pigments(p<0.05)during UV-B radiation,while there is a very significant negative correlation with malondialdehyde content.(p<0.01),the antioxidant enzymes SOD,CAT and POD are closely related.This study could provides theoretical basis and important guiding significance for the study of the regulatory effects of UV-B on plants,the UV-B high-quality cultivation of Taxus cuspidata,and the efficient utilize of regenerative coniferous resources of Taxus cuspidata.4.The transcriptome data of the leaves of Taxus cuspidata before and after UV-B induction were obtained by DNBSEQ sequencing technology.A total of 102301 unigenes with an average length of 1284 bp were obtained after assembly.A total of 5949 differentially expressed genes in the leaves of Taxus cuspidata after UV-B induction were excavated and screened.Among them,we can find that the number of up-regulated genes(4016)after UV-B induction was significantly higher than that down-regulate the number of expressed genes(1933).A total of 2119 unigenes were identified and annotated,distributed in 57 transcription factor families,mainly in the MYB,AP2-EREBP,C3H,bHLH,Trihelix,mTERF,WRKY and NAC families.In terms of biological processes that the GO classification and enrichment function analysis focuses on,the differential genes are mainly concentrated in cellular processes,metabolic processes,and responses,stimulus response,biological regulation and biological process regulation.A total of 3141 differentially expressed genes were mainly classified into 18 KEGG pathway types by KEGG analysis,and the metabolic pathway was the most annotated pathway.In addition,the expression of 12 key enzyme genes in the upstream pathway of paclitaxel biosynthesis was induced by UV-B.Among them,11 key enzyme genes of the MEP pathway were all up-regulated,and one of the MVA pathway genes(mevalonate phosphokinase)was regulated by a mixed mode.The results showed that the key enzyme genes in the upstream pathway of paclitaxel biosynthesis of Taxus cuspidata were significantly induced and regulated by UV-B,and the transcription level was significantly improved.5.The molecular mechanism of UV-B regulation of paclitaxel synthesis pathway in Taxus cuspidata was preliminarily analyzed.A total of 17 paclitaxel biosynthesis key enzyme genes and 24 differentially expressed sequence complete candidate WRKY protein sequences were excavated and screened.Bioinformatics analysis was performed on 24 TcWRKY transcription factors screened.Conserved domain analysis results show that they are divided into three categories:5 members of group ?;18 members of group ?;subgroup ?a have 4 members;subgroup ?b and ?e have 4 and 2 members respectively;subgroup ?c has 8 members;group? has only one member.Conserved motifs and phylogenetic tree analysis identified a total of 5 conserved motifs,and the motif types and number differences of each protein may be related to the specific functions of family members.Results showed that TcWRKY protein was relatively conserved during biological evolution.The qRT-PCR method was applied to analyze the expression patterns of key enzyme genes in paclitaxel biosynthesis.The results showed that the expression levels of the six key genes in the terpene backbone MEP pathway were positively regulated by UV-B.TcDXS,TcMCS and TcHDS genes are more sensitive to UV-B response.The expression of the 11 key enzyme genes of taxol biosynthesis screened generally showed an upward trend and then a downward trend with the increase of UV-B treatment time.TcT13H,TcTBT,TcPAM and TcDBTNBT genes were most sensitive to UV-B response at 48 h.The transcriptional expression levels of the 12 TcWRK Y transcription factor genes with the most significant differential expression under UV-B induction were measured.The results showed that UV-B could induce significant up-regulation of Tc WRKY gene expression levels in Taxus cuspidata,but there are differences in the specific expression patterns of each genes.TcWRKY1,TcWRKY4,TcWRKY11,TcWRKY12 genes are likely to be significantly more sensitive to UV-B induction at 48 h,and mainly focus on responding to UV-B in the middle and late stages to regulate related defense processes in Taxus cuspidata.The TcWRKY10,TcWRKY16,and TcWRKY17 genes may begin to exert their respective biological functions at 48 h for the stress defense process of Taxus cuspidata.The effective and reliable analysis method of paclitaxel and other target active components in the leaves of Taxus cuspidata established in this study can provide scientific basis and technical reference for the related research on the secondary metabolites of Taxus cuspidata,We systematically studied the content of secondary metabolites,physiological indicators and the response of key genes in Taxus cuspidata secondary metabolic pathways to UV-B radiation.We screened and analyzed expression pattern of the enzyme genes and transcription of paclitaxel synthesis pathway based on transcriptome information.This study provides data reference and theoretical basis for the establishment of the follow-up UV-B directed high-quality cultivation system of Taxus cuspidata,the analysis of the biosynthesis pathway and regulation mechanism of paclitaxel,and the utilization of the renewable coniferous resources of Taxus cuspidate.