Differences In Phenotypic Identificarion、Coumarin Content And Expression Of Key Enzyme Genes In Glehnia Littoralis From Different Origins

As a traditional Chinese medicinal herb with a long history of cultivation in China,Glehnia littoralis grows in sandy coastal soil,but in recent years,due to beach development and utilization,the growth environment of G.littoralis has been destroyed,and the number of wild species of G.littoralis is becoming increasingly scarce.In recent years,due to the exploitation of the beach,the growing environment of G.littoralis has been destroyed,and the number of wild species of G.littoralis is becoming increasingly scarce.At present,the main cultivation areas of G.littoralis are Chifeng in Inner Mongolia,Anguo in Hebei and Laiyang in Shandong.It is rich in active substances,including flavonoids,polysaccharides,coumarins,etc.Among them,coumarins are important chemical components in G.littoralis,and their pharmacological effects include anti-inflammatory,anti-tumor,free radical elimination,analgesic,cough relief,etc.,which are commonly used in clinical treatment and drug development.The synthesis process of coumarins in G.littoralis is subject to extremely complex and precise metabolic regulation,and some studies have hypothesized that the IPT gene is a rate-limiting enzyme gene at a key step in the coumarin synthesis pathway,controlling the synthesis of coumarins such as psoralen.Therefore,the role of IPT gene in coumarin synthesis pathway could be verified at a certain level by detecting the expression of IPT gene and performing statistical analysis with coumarin content.In addition,a comprehensive and systematic comparative analysis of coumarin-like compounds in G.littoralis is beneficial to gain insight into the differences of G.littoralis germplasm resources from various origins.However,so far,the research on the phenotypic evaluation of germplasm resources and the comparative identification of superior germplasm resources of G.littoralis is relatively weak,so there is still great research space and value for the comparison and exploitation of superior germplasm resources of G.littoralis.In the experiment,eight species of G.littoralis were selected as raw materials,including two cultivars: Chifeng(CF)and Laiyang(LY),and six wild species: Jinshitan(JST),Haiyang(HY),Tianhengdao(THD),Qingrendao(QRD),Baxiandao(BXD)and Rizhao(RZ),and the leaf phenotypic indexes,coumarin content and gene expression of the eight species were measured.The correlation of each index among the various sources of B.pinnatifida was statistically analyzed,and the scores of B.pinnatifida were statistically ranked.Through the comprehensive evaluation of phenotypic,compositional and molecular biological levels of different wild and cultivated species of G.littoralis,it can provide theoretical support for the development and utilization of germplasm resources of G.littoralis,and is also important for the development of high quality standards and plant protection of authentic medicinal herbs of G.littoralis.The main results of this study are as follows:(1)The leaf phenotypes of wild species and cultivated species of G.littoralis were observed and the results showed that: the leaf area was larger for CF,LY,smaller for BXD,JST;according to the leaf length,the larger ones were LY,CF,smaller ones were HY,JST;according to the leaf circumference,the larger ones were: CF,LY,smaller ones were BXD,JST.the largest stomatal density was HY,the smallest was CF,the larger stomatal length was BXD,JST.The smallest was CF,and the stomatal length was larger for CF,THD,smaller for BXD,HY.The results of chlorophyll content showed that: HY,JST and chlorophyll content were more,and BXD and CF were the least.The results of Fv/Fm of chlorophyll fluorescence measurement showed that the photosynthetic performance of HY was the highest and that of THD was the weakest.The correlation analysis showed that the number of stomata was highly significantly and positively correlated with stomatal density and total chlorophyll content,positively and negatively correlated with leaf surface,and the leaf length was highly and positively correlated with petiole length and serration number.Principal component analysis was performed on leaf phenotypes and chlorophyll-related indexes,thus ranking the leaf indexes of G.littoralis from the source of eight species,and the results showed that CF and LY scored higher and the comprehensive leaf indexes were evaluated more excellent,while JST scored the lowest and the comprehensive leaf indexes were evaluated more average.(2)Quantification of coumarin content in wild and cultivated species of G.littoralis by HPLC showed that: scopolin content was higher in BXD and JST species sources;xanthotoxol content was higher in LY and BXD species,and the two origins with higher xanthotoxin content were CF and HY;the highest content of imperatorin was in LY species;the species with higher content of esculetin were BXD,followed by JST;the highest content of osthole was CF and LY;the variety with the highest content of isopimpinellin was BXD;the highest content of isoimperatorin was HY;the two highest contents of psoralen were LY and JST.(3)Quantification of IPT gene expression in wild species and cultivated species of G.littoralis showed that IPT gene expression was higher in HY,CF and LY varieties,and the relative expression was lower in QRD,BXD and RZ.(4)Statistical analysis of coumarin content and IPT gene expression in wild species and cultivated species of G.littoralis showed that IPT gene expression was positively correlated with psoralen and isoanisole content,respectively,which laterally verified that IPT gene had some influence on coumarin synthesis;the scores of G.littoralis species were obtained by principal component analysis and ranked according to the scores.The results showed that HY,LY,JST,CF,THD,RZ,QRD and BXD were ranked from 1 to 8,among which HY and LY had high content of active ingredients and relatively higher medicinal value.