[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 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.

[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".