[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 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 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.

[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".

Unveiling the Identity of Wenwan Walnuts and Phylogenetic Relationships of Asian Juglans Species Using Restriction Site-Associated DNA-Sequencing.

Juglans species have considerable ecological and economic value worldwide. In China, Wenwan walnuts have been collected by aristocrats and noblemen for more than 2000 years. As a diversity center of Asian Juglans, five species are widely distributed in China. The most famous of these is Mahetao (J. hopeiensis), which is an uncharacterized species that is mostly cultivated. Wild J. hopeiensis individuals are very rare and are endemic to Hebei Province. Because of the minimal variations in previously used molecular markers and the heterogeneity between chloroplast and nuclear genomes, determining the phylogenetic relationships among the Juglans species has been challenging, and has hindered subsequent evolutionary inferences. In this study, we collected enough materials for both cultivated and wild Mahetao to construct well-resolved phylogenetic trees for Asian Juglans species. We used a high-throughput genome-wide restriction site-associated DNA sequencing method. Consequently, the identity of J. hopeiensis has been clearly resolved. Our results indicate that J. hopeiensis is a hybrid of J. regia and J. mandshurica. However, J. hopeiensis, J. regia and J. sigillata should be considered as a single species from section Juglans. Additionally, J. ailantifolia, J. cathayensis, and J. mandshurica likely represent one species from section Cardiocaryon according to morphological and molecular studies. These results are supported by population structure analysis and morphological comparison. We propose that J. hopeiensis trees growing in the wild should be conserved because of the economic value of their nuts. These trees may be of particular importance to impoverished communities. Furthermore, they may serve as a valuable genetic resource relevant for enhancing the production of edible walnuts. The 2b-RAD method is a viable option for future phylogenetic studies of Juglans species as well as other plant species.