[Effect of soil moisture on efficacy to eliminate dampness and relieve jaundice and flavonoid content of Sedum sarmentosum].

The growth environment of medicinal plants plays an important role in the formation of their medicinal quality. However, there is a lack of combined analysis studying the close relationship between the growth environment, chemical components, and related biological activities of medicinal plants. Therefore, this study investigated the effect of different soil moisture treatments on the efficacy to eliminate dampness and relieve jaundice and the flavonoid content of Sedum sarmentosum, and explored their correlation. The flavonoid content in the decoction of S. sarmentosum growing under field conditions with soil moisture levels of 35%-40%(T1), 55%-60%(T2), 75%-80%(T3), and 95%-100%(T4) was compared. The effects of these treatments on liver function parameters, liver inflammation, and oxidative damage in mice with dampness-heat jaundice were evaluated, and the correlation between pharmacological indicators and flavonoid content was analyzed. The results showed that the total flavonoid and total phenolic acid content in the decoction of S. sarmentosum were highest in the T1 treatment, followed by the T3 treatment. The content of quercetin, kaempferol, and isorhamnetin was highest in the T2, T1, and T3 treatments, respectively. Among the different moisture treatments, the T3 group of S. sarmentosum effectively reduced the levels of serum ALT, AKP, TBIL, DBIL, TBA, as well as hepatic TNF-α and IL-6 in mice with jaundice, followed by T2 treatment, especially in reducing AST level. The T4 treatment had the poorest effect. Correlation analysis showed a significant negative correlation between AST, ALT, AKP levels in mice and the total content of quercetin and the three flavonoids. MDA showed a significant negative correlation with the total flavonoid content and kaempferol. TNF-α exhibited a significant negative correlation with the content of isorhamnetin. In conclusion, S. sarmentosum growing under field conditions with a soil moisture level of 75%-80% exhibited the best efficacy to eliminate dampness and relieve jaundice. This study provides insights for optimizing the cultivation mode of medicinal plants guided by pharmacological experiments.

[Cloning and expression analysis of wpHSP90 gene from Whitmania pigra at different temperatures].

HSP90 is a widely distributed molecular chaperone that participates in a variety of cellular processes and plays an important role in the meiosis of germ cells. However, its role in the gonadal development of hermaphroditic Whitmania pigra is not yet clear. To explore the effect of HSP90 on the germ cell development of Wh. Pigra, this study cloned the wpHSP90 gene, performed bioinformatics analysis, and measured its expression levels. The results showed that the cloned wpHSP90 was 2 592 bp in length, with an open reading frame(ORF) of 2 373 bp, encoding 790 amino acids. Prediction analysis revealed 85 phosphorylation modification sites on serine, threonine, and tyrosine residues of the wpHSP90 protein. Structural domain prediction and multiple sequence alignment results showed that wpHSP90 contained two conserved domains of HSP90 and exhibited the highest homology with Helobdella robusta, with a sequence similarity of 80.72%. RT-qPCR results showed that the relative expression level of wpHSP90 in the gonads of 5-month-old Wh. pigra was positively correlated with temperature within the range of 12 ℃ to 28 ℃. The expression level in the female gonads was significantly higher than in the male gonads and correlated with the trend of germ cell development in the ovaries and testes. In conclusion, wpHSP90 may be involved in regulating the development of germ cells, particularly oocytes, in Wh. pigra. This study provides a reference for further research on the gonadal development mechanism in Wh. pigra.

[Effects of shading on photosynthetic physiology and energy metabolism of Asarum forbesii].

Light is the main source for plants to obtain energy.Asarum forbesii is a typical shade medicinal plant, which generally grows in the shady and wet place under the bushes or beside the ditches.It can grow and develop without too much light intensity.This experiment explores the effects of shading on the growth, physiological characteristics and energy metabolism of A.forbesii, which can provide reference and guidance for its artificial planting.In this experiment, A.forbesii was planted under 80%, 60%, 40%, 20% and no shade.During the vigorous growth period, the photosynthetic physiological characteristics such as fluorescence parameters, photosynthetic parameters, photosynthetic pigment content and ultrastructure, as well as the content of mitochondrial electron transport chain(ETC) synthase and nutrients were measured.The results showed that the photosynthetic pigment content, chlorophyll fluorescence parameters and net photosynthesis rate(P_n) decreased with the decrease of shading.Under 20%-40% shading treatment, the plants had damaged ultrastructure, expanded and disintegrated chloroplast, disordered stroma lamella and grana lamella, and increased osmiophi-lic granules and starch granules.The activities of nicotinamide adenine dinucleotide dehydrogenase(NADH), succinate dehydrogenase(SDH), cytochrome C oxidoreductase(CCO) and adenosine triphosphate(ATP) synthasewere positively related to light intensity.With the reduction of shading, the content of total sugar and protein in nutrients increased first and then decreased, and the content was the highest under 60% shade.In conclusion, under 60%-80% shading treatment, the chloroplast and mitochondria had more complete structure, faster energy metabolism, higher light energy-conversion efficiency, better absorption and utilization of light energy and more nutrient synthesis, which was more suitable for the growth and development of A.forbesii.

[Analysis and suggestions on harvest period standards of plant medicinal materials in 2020 edition of Chinese Pharmacopoeia].

The improvement of the harvest period standards is critical in the quality control of Chinese medicinal materials. The present study statistically analyzed the harvest period standards of plant medicinal materials in the 2020 edition of Chinese Pharmacopoeia(Vol.Ⅰ) and put forward the existing problems and suggestions based on herbal records and modern research to provide references for the improvement of the standards. According to the statistical analysis, in 499 types of plant medicinal materials, harvest period standards are recorded under 486 types, accounting for 97.4%, and are lacking in the remaining. Only one medicinal material(Stellariae Radix) is recorded with the standard of the harvest year. The standards of the harvest season and phenological period are recorded under 233 types, accounting for 46.7%. For 237 types, only harvest season is specified, accounting for 47.5%, and for 15 types, only harvest phenological period is specified, accounting for 3.0%. Among 222 types mainly derived from cultivation and 51 types from wild resources and cultivation, only 11 types are recorded with harvest period of cultivated products. Only Stellariae Radix is recorded with the harvest period standards for the wild and cultivated products separately. The harvest period standards of plant medicinal materials with different medicinal parts have certain rules to follow. The main problems about the harvest period standards are discovered. Specifically, no harvest period standards are recorded under 13 types of plant medicinal materials. Almost all perennial cultivated medicinal materials are not recorded with harvest year standard. No phenological period standard is found under 250 types of plant medicinal materials. There is no clear distinction between the harvest period standards of cultivated and wild products. The evidence for harvest period standards of 26 types of plant medicinal materials that can be harvested all year round is insufficient. As a result, it is proposed to strengthen basic research in response to the above-mentioned problems and improve the harvest period standards as soon as possible to ensure the quality of Chinese medicinal materials.

[Screening and validation of RT-qPCR reference genes of Viola yedoensis under different light intensities].

Screening the reference genes that were stably expressed under different light intensities for Viola yedoensis could provide reference for the related molecular research. In this study, 11 candidate reference genes were detected by RT-qPCR for tissues of V. yedoensis treated with different light intensities. Ge Norm, Norm Finder, Best Keeper, and Ref Finder website were used to comprehensively evaluate the reference genes, and verify the stability of the reference gene based on CAT1. Finally, the ideal reference gene was determined. The results showed that CYP, Actin, and SAMDC had small Ct value ranges and stable expression. Ge Norm demonstrated that CYP, SAMDC, and Actin were suitable reference genes. Norm Finder showed that the expression of α-TUB was the most stable. Best Keeper recommended CYP, Actin, and SAMDC as reference genes. Ref Finder assessed that SAMDC, CYP, α-TUB, and Actin had better stability, while GAPDH had the worst stability. The expression trend of CAT1 gene was consistent when calibrated with SAMDC, CYP, and Actin, while it was quite different from that calibrated with GAPDH. In summary, SAMDC, CYP, and Actin can be used as ideal reference genes for the gene expression profiling of V. yedoensis under different light intensities.

[Effects of natural organic soil amendments on quality of Panax notoginseng].

The effects of four natural organic soil amendments on the quality and pesticide residues of Panax notoginseng were investigated through field experiments and the suitable dosage ratio of each soil amendment was selected to provide a new idea for the pollution-free cultivation of P. notoginseng. The four natural organic soil amendments used in this study were Jishibao, Jihuo, Fudujing, and omnipotent nutrients, which were produced by mixed fermentation of aboveground parts of different plants, biological waste residue, and biochar. During the experiments, only four soil amendments were applied to P. notoginseng instead of any pesticides and fertilizers. The experiment was designed as four factors and three levels. There were three dosage gradients(low, medium, and high) for Jishibao(A), Jihuo(B), Fudujing(C), and omnipotent nutrients(D). When the dosage of one soil amendment changed, the do-sage of the other soil amendments remained medium. There were 10 groups in addition to the soil amendment-free group as control(CK). The results showed that the four soil amendments could significantly improve the growth environment of P. notoginseng and increase the seedling survival rate and saponin content of P. notoginseng. The seedling survival rates of the treatment groups increased by 8.24%-30.05% as compared with the control group. Furthermore, the content of pesticide residues in P. notoginseng was too low to be detected, and that of heavy metals in P. notoginseng was far lower than the specified content in the Chinese Pharmacopoeia(2020). The optimal effect was achieved at medium dosage for all the soil amendments with the highest content of saponins, high seedling survival rate, and significantly reduced heavy metals, such as lead, cadmium, arsenic, and mercury.

[Cloning and expression of AeMYB4 gene related to stolon development of Amana edulis].

Stolon is an important organ for reproduction and regeneration of Amana edulis. Previous analysis of transcriptome showed that MYB was one of the most active transcription factor families during the development of A. edulis stolon. In order to study the possible role of MYB transcription factors in stolon development, the authors screened out an up-regulated MYB gene named AeMYB4 was by analyzing the expression profile of MYB transcription factors. In the present study, sequence analysis demonstrated that AeMYB4 contained an open reading frame of 756 bp encoding 251 amino acids, and domain analysis revealed that the predicted amino acids sequence contained two highly conserved SANT domains and binding sites for cold stress factor CBF. By multiple sequence alignment and phylogenetic analysis, it is indicated that AeMYB4 clustered with AtMYB15 from Arabidopsis thaliana, belonging to subgroup S2 of R2 R3-MYB. And most of the transcription factors in this subfamily are related to low temperature stress. The GFP-AeMYB4 fusion protein expression vector for subcellular localization was constructed and transferred into Agrobacterium tumefaciens to infect the leaves of Nicotiana benthamiana, and the results showed the protein was located in the nucleus. To investigate the transcriptional activation, the constructed pGBKT7-AeMYB4 fusion expression vector was transferred into Y2 H Gold yeast cells, which proved that AeMYB4 was a transcription activator with strong transcriptional activity. Real-time quantitative PCR was used to detect the expression of AeMYB4 gene in three different development stages of stolon and in leaves, flowers, and bulbs of A. edulis, which indicated that AeMYB4 transcription factor was tissue-specific in expression, mainly in the stolon development stage, and that the expression was the most active in the middle stage of stolon development, suggesting that AeMYB4 gene may play an important role in stolon development. This study contributes to the further research on the function of AeMYB4 transcription factor in stolon development of A. edulis.

[Protective effects of aqueous extracts from Sedum sarmentosum growing under different light intensity on CCl_4-induced damage in HepG2 cells].

To explore the effect of light intensity in cultivating environment on the hepetoprotective activity of Sedum sarmentosum, S. sarmentosum were planted under five water treatments for 60 days, namely 100% full sunlight(G1), 77% full sunlight(G2), 60% full sunlight(G3), 38% full sunlight(G4), and 16% full sunlight(G5) and CCl_4 drug-induced liver injury model in vitro was used. Cell viability, cell cycle, and cell apoptosis were individually detected by MTT, PI single staining, and Annexin-V FITC/PI double staining assays. Additionally, ALT, AST and antioxidant index in supernatant were determined by colorimetry. And the relationship among the protective effects, chemical composition and antioxidant activity were also analyzed. The results showed that S. sarmentosum aqueous extract could significantly improve the HepG2 cell viability. Among the five S. sarmentosum groups, the cell viability of G1(100% full sunlight) treatment was the highest, and the cell apoptosis was the least. Meanwhile, the level of ALT, AST, and MDA in G1 was the lowest, but it achieved the highest level of SOD and GSH. Moderate light shading(60% full light) also improved the effect of protecting liver and reducing the enzyme. It was found that cell viability was positively correlated with ferricion reducing capacity. ALT activity was positively correlated with isorhamnetin content. Taken together, different light intensity had great influence on hepatoprotective effect of S. sarmentosum, which may be related to its antioxidant capacity. From the perspective of hepetoprotective activity, S. sarmentosum should be planted under full light.

[Correlations between content of linarin in Chrysanthemum indicum and climatic factors in habitats].

Chrysanthemi Indici Flos(CIF), the capitulum of Chrysanthemum indicum, is widely used in proprietary Chinese medicine and daily chemical products. At present, CIF is mainly produced from wild resources and rarely cultivated. This study aims to reveal the correlations between linarin content in CIF and climatic factors in different habitats, and provide a theoretical basis for suitable zoning and rational production of medicinal materials. The content of linarin in CIF was determined by HPLC. Grey relational analysis and Pearson correlation analysis were carried out for linarin content with climatic factors. The results showed that the content of linarin in CIF was significantly different among different habitats. The grey relational degrees of climatic factors with linarin content was in an order of average annual precipitation>annual average sunshine hours>annual average temperature>longitude>annual frost-free period>latitude>altitude. Longitude, annual average temperature and average annual precipitation had significantly positive correlations with the content of linarin in CIF, whereas latitude and altitude showed negative correlations with it. The annual frost-free period and annual average sunshine hours had no significant correlation with the content of linarin in CIF. The content of linarin in CIF varied significantly in different habitats. High longitude, low latitude, low altitude, high annual average temperature and high annual average precipitation could be used as indicators for the habitats of high-quality Ch. indicum. This study provides a reference for selecting suitable producing areas of Ch. indicum and establishing artificial cultivation system.

[Cloning and expression analysis of protein kinase C gene from Whitmania pigra].

Protein kinase C(PKC) is a type of protein kinase widely involved in cell proliferation and development, but the developmental mechanism in the gonads of androgynous animals is still unclear. In order to explore the role of protein kinase C in the development of Whitmania pigra germ cells, the Wh. pigra PKC(Wp-PKC) gene was cloned, bioinformatics analysis was conducted, and fluorescent quantitative PCR was used to analyze the expression of female and male gonads. The results showed that:(1)The cloned Wp-PKC had a full length of 2 580 bp, a relative molecular weight of 76 555.19, and contains an open reading frame encoding 670 amino acids, Wp-PKC was closely related to Danio rerio PKC-α and rat PKC-γ. The similarity of amino acid sequence was 55% and 58%.(2)The protein encoded by Wp-PKC had no signal peptide and was a hydrophilic protein. The secondary structure is mainly composed of random coils, α-helices, extended chains, folds and folds, with the largest proportion of random coils and α-helices. Wp-PKC protein does not contain a transmembrane domain. Multiple sequence alignment and domain prediction analysis show that Wp-PKC contains 4 conserved domains of classical protein kinase C.(3)Fluorescence quantitative results showed that the expression of Wp-PKC in Wh. pigra gonads was positively correlated with the development of germ cells, and the expression in male gonads was significantly higher than that in female gonads. In summary, Wp-PKC is a classic PKC, and Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads, and provide references for further research on the developmental mechanisms of Wh. pigra.

[Comprehensive antioxidant and anti-inflammatory activity of alcohol extracts from Chrysanthemum indicum in different areas based on entropy weight and TOPSIS methodology].

The study is aiming at investigating the application of entropy weight TOPSIS method in the comparison of the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) of Chrysanthemum indicum. The DPPH, ABTS, salicylic acid and spectrophotometry were used to determine the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) of Ch. indicum from 31 different areas in vitro. Take the half inhibition rate of as the evaluation index, two principal components were extracted by the principal component analysis, and their cumulative contribution rate reached at 92.4%. The different areas of Ch. indicum could be divided into Dabei Mountain and Qinling-Taihang Mountain by use principal component to analysis. The entropy weight TOPSIS method was used to objectively assign weights to five indexes, calculate the weight of each index and set up the best and worst scheme of the evaluation object, and the relative proximity(C_i) was used as the measure to construct the multi-index comprehensive evaluation model of Ch. indicum. And then sort with the relative proximity value. The results showed that the relative proximity was between 0.098 and 0.983 which represents there were significant differences in the scavenging effect of DPPH, ABTS and hydroxyl radical and the inhibition effect of xanthine oxidase(XOD) and lipoxygenase(LOX) between extracts of Ch. indicum from different areas. The Ch. indicum from Dabie Mountain area have a relatively high relative degree of measurement and high-quality ranking. Taken together, the quality of Ch. indicum.from the Dabie Mountain area is better. The index weight coefficient and the classification result of producing area are basically consistent with the result of principal component analysis. The results show that the TOPSIS method based on entropy weight method can be used to evaluate the comprehensive quality of Ch. indicum.

[Selection and validation of reference genes for quantitative Real-time PCR analysis in Amana edulis].

Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.

[Effects of Wp-PKC on gonadal development of Whitmania pigra].

Protein kinase C(PKC) is a kind of kinase which is widely involved in cell proliferation and development. PKC(Wp-PKC) in Whitmania pigra body belongs to classic PKC. In order to investigate the effect of Wp-PKC on the development of Wh. pigra germ cells, 17ß-estradiol(17ß-E2)(100 ng·mL~(-1)) and methyltestosterone(MT)(150 µg·L~(-1)), 150 µg·L~(-1)(MT)+0.5 mg·L~(-1) PKC, 0.5 mg·L~(-1) PKC inhibitor were added to Wh. pigra culture water, and no addition group(control group) was added, and the effects on the development of Wh. pigra germ cells and the expression of Wp-PKC were observed. The results showed that: Wp-PKC in male gonads was always higher than that in female gonads; MT promoted the development of male gonads in Wh. pigra, while the expression of Wp-PKC was significantly higher than that in the control; 17ß-E2 promoted the development of female gonads in Wh. pigra and Wp-PKC expression significantly lower than that of the control; while the development of the female and male gonads in the PKC inhibitor group was inhibited, the expression of Wp-PKC was significantly lower than that of the control. In summary, Wp-PKC may promote the development of Wh. pigra, especially the development of male gonads.

[Effects of light intensity on cleaning away heat property of Viola yedoensis].

The current study was conducted to explore the effects of light intensity in cultivating environment on the cleaning away heat property of Viola yedoensis. In the present study, we established the acute inflammation model of ICR mice by injecting carrageenan. We compared the effects of V. yedoensis grown under different light intensities(100%, 80%, 50%, 35% and 5% of full sunlight) on mice body temperature, thermal radiation and the swelling degree of foot tissue before and after modeling observing by thermal infrared imaging technique and weighing method. The changes of energy metabolism related enzymes in liver were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the effects of V. yedoensis grown under different light intensities on human lung cancer cell A549 proliferation were explored with MTT method. The results showed that the body temperature of all groups of mice in V. yedoensis group were significantly lower than that of the blank group, except 5% full sunlight group, and the body temperature declined in positive proportion to light intensity. V. yedoensis group could alleviate foot swelling, reduce SDH activity in liver tissue(especially 100% full sunlight group and 80% full sunlight group were significantly lower than model group), and the degree of alleviating and reducing was positively correlated with light intensity. There was no significant difference in the activity of Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase in liver tissue among treatments. The contents of IL-1ß, IL-6, TNF-α, PGE_2 in foot tissue of mice in V. yedoensis groups were significantly lower than those in model group. Among them, the lowest levels of IL-1ß, IL-6, TNF-α, PGE_2 were found in 80% full sunlight group, and there was no significant difference in TNF-α among different groups. The effects of V. yedoensis aqueous extract on A549 cell line proliferation inhibition rate increased with the light intensities of V. yedoensis cultivating environment. And the effects of V. yedoensis grown under 100% of full sunlight showed significantly higher A549 cell line proliferation inhibition rate compared with other groups(P<0.05). In summary, the light intensity of V. yedoensis cultivating environment is positively correlated with the cleaning away heat property of V. yedoensis, which conforms to the "light-cold and heat property" hypothesis,The V. yedoensis should be planted under full light according.

[Effects of light intensity on physio-biochemical characteristics of Chrysanthemum indicum].

By analyzing the effects of light intensity on the growth, physiological and biochemical characteristics of Chrysanthemum indicum, the light intensity suitable for the growth of Ch. indicum was revealed, which provided a reference for the planting of Ch. indicum. There were five treatment groups of Ch. indicum, which was planted under 100%, 80%, 60%, 40% and 20% all natural light intensity conditions, respectively. In the meantime, these indicators of photosynthetic physiology, such as relative growth, photosynthetic pigment content, photosynthetic parameters, and chlorophyll fluorescence parameters etc. were measured in the peak period of growth of Ch. indicum as well as related indicators of the protective enzyme system, and the ultrastructure of chloroplast was observed with the use of transmission electron microscope technique. In our study, the results showed that the leaves of Ch. indicum occurred yellow phenomenon in different degrees when Ch. indicum was placed at more than 80% of the total natural light intensity condition, the height and stem diameter of plant reached the maximum at 60% of the total natural light intensity. More importantly, the number of branches of Ch. indicum was significantly increased under the total natural light intensity of more than 60%. Both the content of photosynthetic pigment and net photosynthetic rate were negatively correlated with light intensity, while photosynthetic parameters and chlorophyll fluorescence parameters showed a trend of rising first and then decreasing with the decrease of light intensity. The physiological indexes of Ch.indicum including stomatal conductivity, intracellular CO_2 concentration, transpiration rate, water use efficiency and actual photochemical quantum yield of PS Ⅱ had been determined, and the results showed that all of them were at the highest level under 60% total natural light intensity condition. The chloroplast structure of Ch. indicum was not obviously abnormal under 60% and 80% total natural light intensity treatments, but the stroma lamella was broken under 100% total natural light intensity, and not only the number of chloroplast, but also the number and volume of starch grains were decreased significantly under 20% and 40% total natural light intensity. With the decrease of light intensity, the enzymes activities of SOD and CAT decreased, the activity of POD increased in the early stage and decreased in the later stage, and the content of MDA showed a decreasing trend. The analysis of results indicated that the Ch. indicum can grow under 20%-100% total natural light intensity, but the best growth condition was under 60% total natural light intensity.

[Effects of constant temperature on growth and gonad development of overwintering Whitmania pigra].

The present study was conducted to explore the effect of constant temperature overwintering on the growth, gonadal development and internal quality of the Wh. pigra which were in overwintering period. Wh. pigra which in overwintering period were placed in light incubator at 21 ℃, and the Wh. pigra that overwintered under natural conditions were used as control. That the changes of growth performance, gonad index, internal quality of two groups of Wh. pigra were measured at the end of the overwintering. Simultaneously the tissue slice technique was used to observe the morphological structure of the spermary and ovary of the two groups. The results showed that the body weight of constant temperature overwintering Wh. pigra which were placed in light incubator at 21 ℃ was 2.35 times that of natural overwintering Wh. pigra which overwintered under natural conditions, and the weight of female gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 11.54% higher than that of Wh. pigra which overwintered under natural conditions, and the weight of male gonads of the Wh. pigra which were placed in light incubator at 21 ℃ was 48.33% higher than that of the Wh. pigra which overwintered under natural conditions. At the same time, that vitellogenesis cells and film forming cells which in ovarian vesicles were significantly higher than those of the Wh. pigra which overwintered under natural conditions, and that spermatogonia and primary spermatocytes which in seminal vesicle were significantly higher than those of Wh. pigra which overwintered under natural conditions. Most important of all, the anti-thrombin activity of Wh. pigra which were placed in light incubator at 21 ℃ increased by 27.85% compared with the Wh. pigra that overwintered under natural conditions. In conclusion, that constant temperature can promote the growth, the development of sperm and egg cells, and the increase of anti-thrombin activity of Wh. pigra which were in overwintering period.

[Screening of reference genes based on real-time quantitative PCR analysis in Monochasma savatieri].

Monochasma savatieri belongs to Scrophulariaceae family. It is a facultative parasitic plant distributed in southern China that has been found in Jiangxi, Guangdong, and Fujian provinces. Previous studies on this plant have focused on its chemical constituents and pharmacological effects. The research foundation of molecular biology and functional genomics is still very in-depth, and there is no report on the reference genes. In this study, 6 reference gene(UBQ, GAPDH, AP-2, ACT, TUB and CYP) from transcriptome database, were selected and analyzed in M. savatieri.The experiment mainly involve two variables, soil moisture content and arbuscular mycorrhizal symbiosis(Rhizophagus irregularis). And the different combination of the factors result in a total of 6 treatments.Statistical tools, including GeNorm, NormFinder and Bestkeeper were utilized to assess the suitability of reference genes based on their stability rankings for different treatment. The result showed that the stability of 6 reference genes were significantly different. TUB and GAPDH were the most stable gene, while ACT was the most instable one. The results would provide reliable and optional available reference genes in gene expression analysis of M. savatieri, which has important application value.

[Effects of light intensity on material and energy metabolism of Viola yedoensis].

Light energy is an important factor affecting plant growth. The hypothesis of "light-cold and heat property" holds that the original plants of traditional Chinese medicines(TCM) with cold property can obtain more energy to maintain growth in high light intensity environment, whereas the original plants of TCM with heat property prefer weak light environment. The current experiment investigated the effects of different light intensities on primary metabolites levels, energy levels, cell apoptosis, and leaves ultrastructure of Viola yedoensis, the original plants of TCM Violae Herba with cold property. There were five treatment groups of V. yedoensis, which was planted under Li1(8 500 lx),Li2(7 250 lx),Li3(6 000 lx),Li4(4 750 lx),Li5(3 500 lx)LEDs light intensity conditions, respectively. After harvest, primary metabolites levels, contents of ATP, ADP, AMP, activities of ATP synthesis and hydrolysis related enzyme, as well as cell apoptosis activation degree were measured, and transmission electron microscopy technique was used to observe leaves ultrastructure. The results showed that the total sugar, total protein, contents of ATP, ADP and AMP, activities of NADH dehydrogenase, cytochrome C reductase, ATP synthase and ATP hydrolase were positively correlated with light intensities(P<0.05). The crude fat content, activities of SDH and CCO enzyme showed a trend of increasing first and then decreasing, the highest value were found in Li2 group and Li3 group respectively(P<0.05). The vitality of caspase-3 and caspase-9 was negatively correlated with light intensities(P<0.05). The structure of chloroplast and mitochondria were normal and intact in Li1-Li3 groups, and the damage degree of Li4 and Li5 groups increased with the decrease of light intensities. The analysis of results indicated that the structure of chloroplast and mitochondria of V. yedoensis is normal under the light intensity of 6 000-8 500 lx, which can obtain more energy to maintain its growth and metabolism. When the light intensity is lower than 4 750 lx, the chloroplast morphology and mitochondrial membrane are damaged, affecting the metabolism of material and energy. There was no significant difference in energy charge of V. yedoensis in the light intensity range of 3 500～8 500 lx. The effect of light intensity on energy metabolism of V. yedoensis accords with the hypothesis of "light-cold and heat property".

[Application of ITS2 secondary structure phylogenetic information in DNA barcode identification of Chrysanthemum indicum and its related plants].

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.

[Effects of MT on growth and gonad development of natural overwintering Whitmania pigra].

The objectives of study were to explore the effects of exogenous methyltestosterone( MT) on the growth and gonadal development of overwintering Whitmania pigra. Before overwintering,0. 1,1. 0,10. 0,100. 0,150. 0 µg·L-1 of MT were added to the aquaculture water for 6 weeks. The changes of growth performance,gonad index,endogenous steroid hormones level and internal quality were measured after hibernate for 60 days. Then the tissue slice technique was used to observe the spermary( ovary) of Wh. pigra.The results showed that the body weight,survival rate and gonadal index increased first and then decreased with the increase of exogenous MT concentration; the male gland index was found the highest at the concentration of MT 10. 0 µg·L-1 and the female gland index was the highest at the concentration of MT 1. 0 µg·L-1. The survival rate of Wh. pigra peaked at the concentration of MT 10. 0 µg·L-1.The weight reaches a peak at a concentration of MT 100. 0 µg·L-1( P<0. 05). The number of primary spermatocytes in the testis was negatively correlated with the concentration of exogenous MT. The number of secondary spermatocytes and sperm cells increased first and then decreased. The concentration of secondary spermatocytes was the highest when the concentration of MT was 100. 0 µg·L-1.The number and volume of oocytes in the ovary and the yolk granules increased first and then decreased with the increase of exogenous MT concentration,and the highest was observed at the MT concentration of 100. 0 µg·L-1. The endogenous steroid hormone of Wh.pigra increased first and then decreased with the increase of exogenous MT concentration. The concentration of androgen and progesterone was the highest in MT 100. 0 µg·L-1 treatment( P<0. 05),and the concentration of estrogen was found the highest in MT 10. 0 µg·L-1 treatment( P<0. 05). After adding exogenous MT,Wh. pigra moisture content,acid-insoluble ash content,p H and anti-thrombin activity met the quality criterion of medicinal Wh. pigra in Chinese Pharmacopoeia( 2015 edition). In conclusion,the short-term addition of 1. 0-100. 0 µg·L-1 exogenous MT before hibernation can promote the growth,the development of sperm cells and the antithrombin activity of Wh. pigra.