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 on Leaf Quality of Different Artemisia argyi Germplasm Resources / 中国实验方剂学杂志

Objective:To analyze the quality differences among different germplasm resources of <italic>Artemisia argyi </italic>and to screen out the specific germplasm by comprehensively evaluating 14 quality traits of 100 germplasm resources. Method:Germplasm resources of <italic>A. argyi </italic>were collected from all over the country. The output rate of moxa and the content of total volatile oil in <italic>A. argyi</italic> leaves were determined，and the contents of 12 flavonoids and phenolic acids in<italic> A. argyi </italic>leaves were detected by ultra performance liquid chromatography（UPLC）. The correlation analysis，principal component analysis and clustering analysis were used to comprehensively evaluate the quality of <italic>A. argyi</italic>. Result:There was rich genetic diversity of<italic> A. argyi</italic> germplasm resources，and the variation coefficients of 14 quality traits ranged from 25.67% to 127.34%，among which the coefficient of variation of chlorogenic acid，cryptochlorogenic acid，isoxiafotaside，isochlorogenic acid B and isochlorogenic acid A was more than 70%，with high variation. The output rate of moxa was negatively correlated with 9 quality traits，while the content of total volatile oil was positively correlated with 10 quality traits，and most of the flavonoids and phenolic acids had synergistic effects. 12 flavonoids and phenolic acids were analyzed by principal component analysis，and 4 principal components could be extracted. The highest contents of flavonoids and phenolic acids were found in S98（Hangzhou，Zhejiang province），S84（Longhui county，Shaoyang city，Hunan province），S66（Futian river town，Macheng city，Hubei province），S35 （Balihu town，Qichun county，Huanggang city，Hubei province），and S15 （Fudao town，Tangyin county，Anyang city，Henan province）. The systematic clustering analysis showed that the 100 germplasm could be divided into four groups when the euclidean distance was 8.0，with 90，3，3，3 and 4 accessions in group Ⅰ，Ⅱ，Ⅲ and Ⅳ，respectively. The germplasm resources in group Ⅱ contained the highest content of flavonoids and phenolic acids，the group Ⅲ contained the highest content of total volatile oil and the group Ⅳ contained the highest output rate of moxa. Conclusion:The leaf quality of different <italic>A. argyi </italic>germplasms is different. This study can provide the basis for the quality evaluation and variety breeding of <italic>A. argyi</italic> germplasm resources.

Effects of Combined Application Organic Fertilizer and Chemical Fertilizer on Agronomic Characters，Yield and Quality of Artemisiae Argyi Folium / 中国实验方剂学杂志

Objective:To explore the effect of different proportions of organic fertilizer and chemical fertilizer on the agronomic traits， yield and quality of Artemisiae Argyi Folium in Qichun county， and provide a theoretical basis for scientific fertilization of its planting. Method:Field plot experiment was carried out to set up 5 treatment methods with different proportions of organic fertilizers and chemical fertilizers［OM₀ （no combined application of biological organic fertilizer）， OM₁₇ （combined application of 17% biological organic fertilizer）， OM₃₃ （combined application of 33% biological organic fertilizer）， OM₆₇ （combined application of 67% biological organic fertilizer）， OM₁₀₀ （combined application of 100% biological organic fertilizer）］. The effects of different treatment methods on the agronomic characters， leaf yield， output rate of moxa， volatile oil content， flavonoid and phenolic acid contents and mineral element contents of Artemisiae Argyi Folium in Qichun county were determined. Result:With the increase of the proportion of organic fertilizer in application， the seedling number per unit area， plant height， stem diameter， leaf number， leaf width， leaf length， height of dead leaves and leaf yield of Artemisiae Argyi Folium were increased at first and then decreased. Among them， the yield of Artemisiae Argyi Folium in OM₃₃ treatment was 61.37% higher than that in OM₀ treatment. With the increase of the proportion of organic fertilizer， the output rate of moxa of Artemisiae Argyi Folium showed continuously increasing trend， contents of volatile oil and volatile components （eucalyptol， <italic>α</italic>-thujone， borneol， camphor and caryophyllene oxide） increased at first and then decreased， while the contents of <italic>α</italic>-caryophyllene and <italic>β</italic>-syringene decreased gradually， the contents of phenolic acids （neochlorogenic acid， chlorogenic acid， isochlorogenic acid B， A and C） increased at first and then decreased， while the contents of flavonoids （jaceosidin and eupatilin） increased continuously， and the contents of mineral elements （Ca， Cu and Zn） continued to increase， but the content of K decreased significantly at the high proportion of organic fertilizer. After treated with principal component analysis （PCA）， it was found that OM₁₇ treatment had the highest quality， while OM₁₀₀ and OM₀ treatment had low quality. Conclusion:Based on comprehensive analysis of agronomic traits， yield and quality indexes of Artemisiae Argyi Folium in Qichun county， it is suggested that 17%-33% proportion of organic fertilizer should be used in its production， in order to improve the quality and efficiency of Artemisiae Argyi Folium industry in Qichun county.

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 miRNA-21 on paclitaxel-resistance in human breast cancer cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of miR-21 on paclitaxel-resistance in human breast cancer MCF-7/PR and SKBR-3/PR cells.</p><p><b>METHODS</b>Paclitaxel-resistant human breast cancer cell lines MCF-7/PR and SKBR-3/PR were established by stepwise selection in increasing concentration of paclitaxel. Cellular morphology, mRNA and protein level of MDR1, BCRP and MRP1 in MCF-7/PR and SKBR-3/PR cells were determined. The expression of Bax, Bcl-2 and miR-21 in parental and paclitaxel-resistant cells was detected by RT-PCR and Western blotting. The synthetic miR-21 inhibitor or miR-21 mimic were transfected into MCF-7/PR, SKBR-3/PR and MCF-7, SKBR-3 cells with Lipofectamine 2000. The miR-21 levels were determined by RT-PCR, and P-gp, Bcl-2 and Bax protein levels were examined by Western blotting. MTT assay was used to measure the cell viability, and flow cytometry was performed to analyze the cell cycle and apoptosis.</p><p><b>RESULTS</b>The levels of MDR1, BCRP, MRP1, Bcl-2/Bax and miR-21 in MCF-7/PR and SKBR-3/PR cells were significantly higher than those in MCF-7 and SKBR-3 cells. The protein levels of P-gp, Bcl-2 were up-regulated, and Bax was down-regulated compared with parental cells. MiR-21 was significantly down-regulated after miR-21 inhibitor was transfected; and the levels of MDR1, BCRP, MRP1 and Bcl-2/Bax (P <0.05) were also down-regulated. MiR-21 inhibitors significantly suppressed G0/G1 transition of the cell cycle, and induced cell apoptosis in MCF-7/PR and SKBR-3/PR cells. MTT results showed that miR-21 inhibitors induced sensitivity of MCF-7/PR and SKBR-3/PR cells to paclitaxel. And miR-21 mimic can increase the expression of MDR1, Bcl-2/Bax and change cell morphology from parental cells to resistant cells.</p><p><b>RESULTS</b>The established MCF-7/PR and SKBR-3/PR breast cancer cells show typical multidrug resistance characteristics, which can be used as the model for drug resistance study. Down-regulated miR-21 expression in MCF-7/PR and SKBR-3/PR breast cancer cells can enhance cell sensitivity to paclitaxel.</p>

Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.</p><p><b>METHODS</b>Total RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.</p><p><b>CONCLUSION</b>This strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.</p>

Construction of pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and their interaction in living cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP eukaryotic expression plasmids and to investigate the interaction of chemokine receptor 4 (CXCR4) and viral macrophage inflammatory protein-II(vMIP-II) N terminal 21 peptides (NT21MP) in living cells.</p><p><b>METHODS</b>DNA fragment encoding NT21MP was chemically synthesized and inserted into BiFC eukaryotic expression vector pBIFC-VC155. The full length of CXCR4 DNA fragment was amplified by RT-PCR from SKBR (3) cells and inserted into BiFC eukaryotic expression plasmid pBIFC-VN173. Two recombinant vectors were identified by restriction enzyme digestion and DNA sequencing. The recombinant vectors were cotransfected into Africa green monkey kidney fibroblast COS-7 cells by using Lipofectamine 2000. The interaction of NT21MP and CXCR4 was detected by bimolecular fluorescence complementation (BiFC) assay.</p><p><b>RESULTS</b>The restriction enzyme digestion and DNA sequences and open read frames of two vectors were consistent with experiment design. The BiFC plasmids were successfully cotransfected into the target cells and expressed. The strong BiFC signals were detected in pBIFC-VN173-CXCR4 and pBIFC-VC155-NT21MP cotransfected cells and the fluorescence signal was located in the cytoplasm.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmids for BiFC assay are successfully constructed. The interaction of NT21MP and CXCR4 in living cells can be detected by using this technology.</p>

Effect of interfering hepatocyte nuclear factor-1 alfa in HepG2 on the expressions of apoM, apoA-I and the correlative key enzyme of cholesterol metabolism / 中华肝脏病杂志

To determine wether there were connections among hepatocyte nuclear factor-1 alfa (HNF-1a), liver receptor homolog-1 (LRH-1), apolipoprotein M (apoM) and to investigate the effects of HNF-1a in HepG2 on the expressions of apoM, apolipoprotein A-I (apoA-I) and the key enzymes in cholesterol metabolism and biotransformation. The mRNA expressions of apoM, LRH-1 and HNF-1a were detected by RT-PCR. HNF-1a was interfered and RT-PCR was used to detect the changes of apo M, apo A-I, Cyp7A1, farnesoid X receptor (FXR) and small heterodimer partner-1 (SHP-1). Western blot was used to detect the change of apo M protein. The expressions of apoM, LRH-1 and HNF-1amRNA were obviously higher in HCC tissue than that in para-cancer tissue (the vaule of t is -7.167, -7.075, -8.803, P less than 0.01 respectively). HNF-1a and LRH-1 positively correlated with the expression of apoM (r=0.353, P less than 0.01; r=0.523, P less than 0.01 respectively); RT-PCR and western blot results showed that the expressions of apoM, FXR and SHP-1 mRNA, could be obviously suppressed by HNF-1a interfering as compared to the negative controls by 47.4%, 47.9%and 65.2% (P less than 0.01) respectively, and the expression of apoM protein also decreased by 54.3% (F = 43.482, P less than 0.01). The expressions of HMGCR and CYP7A mRNA increased by 101.1% and 138.5% (P less than 0.01) respectively as compared to the negative control. But there is no effect on expression of apoA-I mRNA (F = 0.170, P more than 0.05). HNF-1a could promote cholesterol biotransformation by increasing the expression of apoM and the key enzymes in cholesterol metabolism and decreasing inhibiting factor. So HNF-1a provided protection against cardiovascular disease.