Identification and expression analysis of flavonoid O-methyltransferases gene family in Artemisia argyi / 药学学报

italic>Artemisia argyi (A. argyi) is a Chinese herbal medicine in China. The main active components are volatile oils, flavonoids, and other compounds, which have various pharmacological activities. Methoxylated flavonoids are the main active ingredients in A. argyi. Flavonoid O-methyltransferase (FOMT) is a key enzyme in the O-methylation of flavonoids. In order to further understand the function and characteristics of FOMT proteins, this paper carried out the whole genome mining and identification of FOMT genes in A. argyi and performed phylogenetic, chromosomal localization, gene sequence characterization, subcellular localization prediction, protein structure, gene structure analysis, and expression pattern analysis. The results showed that a total of 83 FOMT genes were identified in the genome of A. argyi. The phylogenetic tree shows that FOMT genes are divided into two subgroups, CCoAOMT (caffeoyl CoA O-methyltransferase) subfamily (32 genes) and COMT (caffeic acid O-methyltransferase) subfamily (51 genes). Gene sequence analysis showed that the number of amino acids encoded by FOMT was 70-734 aa, the molecular weight was 25 296.55-34 241.3 Da, and the isoelectric point was 4.51-9.99. Compared with 32 members of the CCoAOMT subfamily, nearly 1/3 of the 51 members of the COMT subfamily were hydrophobic proteins and 2/3 were hydrophilic proteins. Subcellular localization prediction showed that more than 80% of CCoAOMT subfamily members were located in the cytoplasm, and 96% of COMT subfamily members were located in the chloroplast. COMT subfamily members have more motifs than CCoAOMT subfamily members. The N-terminal motifs of COMT subfamily proteins are relatively variable, while the C-terminal motifs are relatively conserved. Expression pattern analysis showed that CCoAOMT subfamily members were mainly expressed in roots, while COMT members were mainly expressed in leaves. Some FOMTs showed the tissue expression specificity by real-time quantitative PCR analysis, especially in leaves. In this study, we identified and analyzed the FOMT gene family in A. argyi, and provided a theoretical basis for further research on the function of FOMTs and the biosynthesis of methylated flavonoids in A. argyi.

Content of mineral elements in different Artemisia argyi germplasms and their relationship with quality properties / 中国中药杂志

To clarify the content characteristics of mineral elements in different Artemisia argyi germplasm resources and their relationship with the quality properties of Artemisiae Argyi Folium, this study measured the content of 10 mineral elements including nitrogen(N), phosphorus(P), potassium(K), calcium(Ca), magnesium(Mg), aluminum(Al), manganese(Mn), iron(Fe), copper(Cu), and zinc(Zn) in 100 Artemisia argyi germplasm samples. Besides, their relationship with the quality properties of Artemisiae Argyi Folium was explored by correlation analysis, path analysis, and cluster analysis. The results demonstrated that the variation coefficient of the 10 mineral elements in Artemisiae Argyi Folium ranged from 12.23% to 64.38%, and the genetic diversity index from 0.97 to 3.09. The genetic diversities of N, P, and Zn were obvious. As revealed by the correlation analysis, N, P, and K showed strong positive correlations with each other. Except that Mg and Al were negatively correlated, Ca, Mg, Al, Mn, Fe, Cu, and Zn were positively correlated. The correlation analysis of mineral elements with the quality properties of Artemisiae Argyi Folium proved the significant correlations of 17 pairs of characters. According to the path analysis, P, K, Ca, and Mn greatly affected the yield of Artemisiae Argyi Folium, P, K, and Mg the output rate of moxa, N, P, and K the content of total volatile oil, P and K the content of eucalyptol, and P, K, and Ca the content of eupatilin. The 100 germplasm samples were clustered into three groups. Specifically, in cluster Ⅰ, the enrichment capacity of P, K, and Mg elements was strong, and the comprehensive properties of mineral elements were better, implying good development potential. Ca, Mn, Fe, and Zn elements in cluster Ⅱ and N and Al in cluster Ⅲ displayed strong enrichment capacities. This study has provided new ideas for resource evaluation and variety breeding of A. argyi and also reference for fertilizer application.

Analysis and evaluation of volatile oil content in leaves of different Artemisia argyi germplasm resources / 中国中药杂志

Volatile oil is the main effective component and an important quality indicator of Artemisia argyi leaves. In this study, 100 germplasm resources of A. argyi were collected from all the related habitats in China. The total volatile oils in A. argyi leaves were extracted by steam distillation and the content was determined by GC-MS. The result demonstrated that the content of total volatile oils was in the range of 0.53%-2.55%, with the average of 1.43%. A total of 39 chemical constituents were identified from the volatile oils, including 13 shared by the 100 germplasm resources. Clustering analysis of the 39 constituents showed that the 100 A. argyi samples were categorized into groups Ⅰ(9), Ⅱ(2), Ⅲ(66) and Ⅳ(23), and group Ⅲ had the most volatile medicinal components, with the highest content. Five principal components(PCs) were extracted from 13 shared constituents, which explained 73.454% of the total variance. PC1, PC2, and PC3 mainly reflected the pharmacological activity of volatile oils and the rest two the aroma information. The volatile oils identified in this study lay a foundation for variety breeding of and rational utilization of volatile oils in A. argyi leaves.

Diversity of Artemisia argyi germplasm resources based on agronomic and leaf phenotypic traits / 中国中药杂志

In this study, in order to evaluate the phenotypic diversity of Artemisia argyi germplasm resources and improve its efficiency of cultivation and breeding, 100 accessions of A. argyi germplasm resources from 58 regions in China were collected, 20 agronomic traits and leaf phenotypic traits were observed and described. The data were used for phenotypic diversity analysis, correlation analysis, principal component analysis and cluster analysis. The result showed that the genetic diversity index of 20 traits ranged from 0.82 to 4.37, among which the largest was the base depth and the smallest was the leaf width; the coefficient of variation of the 12 quantitative traits ranged from 10.55% to 41.47%. the highest coefficient of variation was the height of dead leaves, and the smallest was the content of chlorophyll, except for the angle of branches, all the quantitative characters tended to be normal distribution. The correlation analysis showed that 28 pairs of traits had significant correlation(P<0.01), and 13 pairs had significant correlation(P<0.05). According to principal component analysis, 20 traits were simplified into 9 principal components, and the cumulative contribution rate was 73.414%, nine traits including plant height, dead leaves heigh, stem diameter, symmetry of leave base, stipule, leaf tip shape, depth of the first pair of lobes, depth of the second pair of lobes and leaf yield were selected as key indexes for evaluating agronomic traits and leaf phenotypic traits of A.argyi germplasm resources. With cluster analysis, 100 accessions of A.argyi were classified into 3 groups, the groupⅠincluded the dwarf plants with thick stem and large leaf, the groupⅡincluded high plants with wide leaf and high yield, the group Ⅲ included dwarf plants with thin stem and flat bottom shape of leaf, which could provide the basis for cultivation identification and variety breeding of A.argyi germplasm resources.

[Effects of different planting density, leaf position and leaf age on growth and quality of Artemisia argyi var. argyi 'Qiai'].

In order to guide the standardized planting and scientific harvesting of Artemisia argyi var. argyi 'Qiai', effects of diffe-rent planting density, leaf position and leaf age on the growth and quality of A. argyi var. argyi 'Qiai' were studied in this paper. The results showed that appropriate sparse planting could increase stem diameter, compact leaf spacing, increase the leaf size, reduce the rate of withered leaves and increase the number of effective leaves. Dense planting can significantly increase the yield of A. argyi var. argyi 'Qiai' per Mu(1 Mu≈667 m~2) and the output rate of moxa, but reduce the yield per plant and the number of effective leaves. With the decrease of leaf position and the increase of leaf age, the leaf size and the weight of one hundred leaves increased at first and then decreased, the density of non-glandular hair field of the lower epidermis and the output rate of moxa decreased. With the increase of planting density, the contents of eucalyptus oleoresin, camphor, α-platyclone, and cyanidin decreased gradually, the contents of chlorogenic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in the leaves of A. argyi var. argyi 'Qiai' increased gradually, while the contents of borneol, bornyl acetate and isozelanin increased at first and then decreased significantly. With the decrease of leaf position and the increase of leaf age, the contents of volatile oil, phenolic acid and flavonoid in A. argyi var. argyi 'Qiai' decreased gradually. PCA analysis can divide the leaf quality characteristics of A. argyi var. argyi 'Qiai' into two parts: the middle and upper 10-30 days leaf age, the middle and lower 40-50 days above leaf age. Based on the above factors, the planting density of 28 000 plants/Mu(row spacing of 10 cm×20 cm) can be selected for cultivation of A. argyi var. argyi 'Qiai' rhizome, and the effective plants in the field are about 142 800 plants/Mu. In terms of harvesting, it is suggested that the A. argyi var. argyi 'Qiai' should be harvested on "March 3 rd" and "May 5 th" of the lunar calendar. If it is the traditionally harvested at one time in May, the A. argyi var. argyi 'Qiai' should be divided into two parts: the middle and upper leaves, the middle and lower leaves, so as to achieve high quality and high price of A. argyi var. argyi 'Qiai' leaves and improve the economic benefits of A. argyi var. argyi 'Qiai' planting.

[Effect of planting period and ridge pattern on yield and quality of Artemisia argyi var.argyi 'Qiai'].

To solve the problems of the unclear planting period and the traditional flat planting of Artemisia argyi var.argyi 'Qiai', such as the serious dead leaves in the middle and lower parts of the plant and the low yield and low quality in the field, the effects of different planting period and ridge pattern on yield and quality of A. argyi var.argyi 'Qiai' were studied. The results showed that the growth and development of A. argyi var.argyi 'Qiai' was better when planted in autumn and winter, and the ridge directions had little effect on the growth and yield of A. argyi var.argyi 'Qiai', while the ridge width had a significant effect. Compared the traditional wide ridge planting mode(the width of the ridge is 160 cm) with the ridge width of 80 cm, 60 cm and 40 cm, the results showed that the density per unit area, the number of effective plants, and the rate of dead leaves were decreased, while the number of productive leaves per plant, the number of lateral branches, and the total yield per plant were increased, the total yield was decreased. The output rates of moxa in the leaves of A. argyi var.argyi 'Qiai' decreased with the planting time postponed and the width of planting ridge increased. The contents of volatile oil, 1,8-oxido-p-menthane, camphor and borneol in A. argyi var.argyi 'Qiai' reach to the highest when planted in autumn and winter and the ridge width was 80 cm. In addition, the contents of total flavonoids, kaempferol, jeceosidin and eupatilin flavonoids in the leaves of A. argyi var.argyi 'Qiai' gradually decreased as the planting period postponed and the ridge width increased. The results show that the A. argyi var.argyi 'Qiai' should be planted in autumn and winter, and the double row ridge planting mode with width of 60-80 cm is more suitable for the cultivation and production of A. argyi var.argyi 'Qiai' in Qichun County of Hubei Province. This study will provide a theoretical basis and guidance for higher yield and quality in cultivation of A. argyi var.argyi 'Qiai' in Qichun county.