Response Of Medicinal Constituents On Shading And Bagging Techniques And Transcriptionomic Analysis Of Chaenomeles Speciosa Germplasms

Chaenomeles speciosa is a Rosaceae plant.Chaenomeles speciosa has very high medicinal nutritional value and it is the important materials of health food and traditional Chinese medicine.This study analyzed the contents of oleanolic acid,ursolic acid,chlorogenic acid,protocatechuic acid and some trace elements in different Germplasms of Chaenomeles speciosa,comprehensively analyzed and evaluated the medicinal value of Chaenomeles speciosa,and explored the high-quality germplasm resources of Chaenomeles speciosa;secondly,the shading and bagging technolgy were used to explore its effects on Chaenomeles speciosa.The regulatory effects of Chinese medicinal ingredients in melon fruits provide experimental basis for improving the medicinal value of Chaenomeles speciosa.Furthermore,the key genes in chlorogenic acid biosynthesis pathway were excavated through transcriptome sequencing data of Chaenomeles speciosa,which provided an important scientific basis for further development of Chaenomeles speciosa genes,and accumulated experimental materials for further research and resource development and utilization of Chaenomeles speciosa.The main results of this study are as follows:1.The chemical constituents of oleanolic acid,ursolic acid,chlorogenic acid and protocatechuic acid in Chaenomeles speciosa fruits were determined by high performance liquid chromatography.The results showed that the content of oleanolic acid was the highest,up to 8.024 mg·g^-1,followed by chlorogenic acid,up to 5.662 mg·g^-1,and there were significant differences among different plasmids.The variation coefficients of oleanolic acid,ursolic acid,chlorogenic acid and protocatechuic acid were 44.30%?28.67%?32.27%and39.58%respectively.Among the 14 germplasms,the highest content of four components was M₁₃(15.530 mg·g^-1),and the lowest was M₆(4.281 mg·g^-1).2.The contents of six trace elements were detected by Flame Atomic Absorption Spectrometry:Zn,Fe,Mn,Cu,Ca and Mg.The results showed that 14 germplasms contained more essential trace elements for human body,among which Ca was much higher than other elements,followed by Mg,Fe,Mn and Zn,and Cu was the lowest.Among them,the highest content of six elements was M₄,with Ca content as high as 1606.80?g.g^-1.The variation coefficients of Zn,Fe,Mn,Cu,Ca and Mg were 27.12%,28.26%,8.28%,18.33%,23.17%and 12.66%respectively.3.According to the determination results of oleanolic acid,ursolic acid,chlorogenic acid,protocatechuic acid and six trace elements,correlation analysis and principal component analysis were carried out.The correlation results showed that Fe was negatively correlated with oleanolic acid and chlorogenic acid,with correlation coefficients of-0.881 and-0.753,and it was positively correlated with protocatechuic acid,with correlation coefficient of 0.725.The negative correlation coefficient between zinc and ursolic acid was 0.555,and that the positive zinc and protocatechuic acid was 0.617.Ca was negatively correlated with oleanolic acid,and the correlation coefficient was-0.579.Fe was positively correlated with Zn,Mg and Ca,while oleanolic acid was positively correlated with chlorogenic acid,negatively correlated with protocatechuic acid,but not with other contents.We can infer the contents of four by measuring the contents of these six trace elements inChaenomeles speciosa.4.Shading and bagging methods were used to test the fruits of Chaenomeles speciosa,in order to explore whether cultivation technologies could improve the content of its medicinal ingredients.The results showed that oleanolic acid content and ursolic acid content increased significantly after 30 days of light transmission and 70%shading treatment,while chlorogenic acid and protocatechuic acid content decreased.After 30 days of bagging treatment,oleanolic acid,ursolic acid,chlorogenic acid and protocatechuic acid content decreased in different degrees,indicating that bagging method reduced pests and pesticide residues.Partly it goes against the synthesis and accumulation of medicinal ingredients.Generally speaking,in the daily cultivation of Chaenomeles speciosa,shading can be carried out to a certain extent,so that the plants can make full use of light and promote the accumulation of organic substances,so as to achieve the purpose of improving some medicinal ingredients.5.In this experiment,Illumina sequencing platform was used to sequence the transcriptome of the fruits of Chaenomeles speciosa.A total of 105236920 reads were obtained.After filtering,102531308 clean reads were obtained,accounting for 97.40%.The proportion of high-quality clean reads of both samples was over 97%.The clean reads were spliced with Trinity to obtain non-redundant bases.Because of 109211 UniGene data,GC content was 46.95%,Q30 was more than 93%,the average length was 1492 bp,and N50 was1963 bp.These unigenes sequences are compared and annotated with five databases,including Nr,Swiss-Prot,GO,KOG and KEGG.A total of 109211 Unigenes were provided with functional annotations.GO database annotations showed that 63658 unigenes?58.28%?were annotated into biological processes,cell components and molecular functions,with 25subclasses;27866 unigenes?25.51%?were annotated into 26 functional classifications of KOG database,among which the most annotated unigenes were post-translational modification,protein conversion and molecular chaperone function;KEGG database was used as the basis.According to the data,27478 unigenes were annotated to 129 metabolic pathways,of which carbon metabolism was the most common pathway.This provides some data support for the molecular biology research of Chaenomeles speciosa.6.In the analysis of M₆ vs.M₈ transcriptome,there were 4254 differentially expressed genes,including 2388 up-regulated genes and 1866 down-regulated genes.A total of 3313differentially expressed genes were assigned to three GO classifications and 41 functional subclasses of molecular functions,cell components and biological processes,of which 1405differentially expressed genes were up-regulated and 1908 differentially expressed genes were down-regulated.By comparing differentially expressed genes with metabolic pathways in KEGG database,1938 Unigenes were annotated to 114 metabolic pathways,of which the most abundant were found.Many KEGG pathways are plant hormone signal transduction.7.Chlorogenic acid is synthesized in the phenylpropane biosynthesis pathway.There are four enzymes involved in chlorogenic acid biosynthesis in the transcriptome of Chaenomeles speciosa.TheyarePAL?phenylalaninammonia-lyase??CYP73A?cinnamate4-hydroxylase??4CL?4-Coumarate:Coenzyme A Ligase??HCT?shikimate hydroxycinnamoyl transferase?There are six differentially expressed genes related to chlorogenic acid biosynthesis pathway in M₆ vs.M₈,four up-expressed genes?Cluster-4.63242;Cluster-4.1656;Cluster-4.15617;Cluster-4.9557?,and two down-expressed gene?Cluster-4.34315;Cluster-4.43160?,which laid a foundation for the study of chlorogenic acid biosynthesis,and provided a basis for improving chlorogenic acid content of Germplasm by genetic engineering.