Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis.

BACKGROUND: Kazal-type serine protease inhibitors play a role in physiological processes such as blood coagulation and fibrinolysis. The amino acid residues at the P1 site are different, and they inhibit different types of proteases. The inhibitory mechanism of the protease in the salivary glands of Poecilobdella manillensis is still unclear. METHODS AND RESULTS: Based on cloning, prokaryotic expression and bioinformatics analysis, we studied the role of Kazal-type serine protease inhibitors in P. manillensis and analyzed their expression by quantitative real-time PCR. The results suggested that the recombinant protein was successfully expressed in the supernatant when a prokaryotic expression vector was constructed and induced with 0.2 mmol/L IPTG at 37 °C for 4 h, and the enzymatic activity was determined. The mature protein encodes 91 amino acids and has a relative molecular weight of 9929.32 Da, and after removing the signal peptide, the theoretical isoelectric point was 8.79. It is an unstable protein without a transmembrane domain. The mature protein contains two Kazal-type domains, in which all P1 residues are Lys, consisting of an α helix and three antiparallel ß sheets. The upregulated expression of the mRNA was induced after a meal was provided, and the results showed an increasing and then decreasing trend. CONCLUSIONS: Taken together, the results indicate that mature proteins from P. manillensis inhibit thrombin activity, laying the foundation for the subsequent in-depth study of the function of genes encoding Kazal-type serine protease inhibitors.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

[Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju'].

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using ß-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

[Analysis on character of mineral elements in Changium smyrnioides and rhizosphere soil].

The contents of 22 kinds of mineral elements in different parts of Changium smyrnioides and in the rhizosphere soil of 10 different populations were determined by microwave digestion-inductively coupled plasma atomic emission spectrometry (ICP-AES). The characteristics of mineral elements in the plants and the soil of main distribution area of Ch. smyrnioides was analyzed and the mechanism of the quality formation of Ch. smyrnioides was explored to provide the basis for the quality evaluation and cultivation regulation of Ch. smyrnioides.The results showed that the quality of soil environment was better in the main distribution area of Ch. smyrnioides, the content of trace elements was higher, the contents of P and K were lower and the contents of mineral elements in the soil of each distribution area was significantly different. The three elements of Se, P and K are significantly accumulated in the root of Ch. smyrnioides. There were significant differences in the total contents of mineral elements in the roots of Ch. smyrnioides in different producing areas. The contents of mineral elements in different parts of Ch. smyrnioides were significantly different. Ch.smyrnioides of the main distribution area of Ch. smyrnioides belonged to the safety level, the distribution of mineral elements in the plants can be used as an indicator of the quality of medicinal herbs.

[Studies on effects of water temperature, stocking density and feeding cycle on growth and feeding in Hirudo nipponica].

Effects of different water temperature, stocking density and feeding cycle on growth, feeding and survival of Hirudo nipponica have been studied, six temperature gradients were set: 18, 22, 26, 30, 34 and 38 ℃, five stocking density gradients were set: 30, 60, 120, 180 and 240 leech/L, four feeding cycle gradients were set: 2, 5, 10 and 20 d, respectively. The results showed that there exists a significant regression relationship between water temperature and specific growth rate: y=-0.016 5x²+0.836 9x-6.847 5（R²=0.990 8）（P<0.05）, a regression analysis indicated that specific growth rate reached the maximum (3.76) at 25.36 ℃. When water temperature was beyond 30 ℃, the survival rate significantly decreased as water temperature increased （P<0.05）. The specific growth rate and survival rate decreased as stocking density increased. A linear relationship exists between the feeding cycle and the SGR: y=-0.094 1x+3.832 9（R²=0.992 7）. From this study, it can be concluded that the optimal water temperature and stocking density for the growth of H. nipponica is 22-26 ℃ and 30-120 leech/L, respectively.

Physiological characteristics, dry matter, and active component accumulation patterns of Changium smyrnioides in response to a light intensity gradient.

CONTEXT: Changium smyrnioides Wolff (Apiaceae) is an endangered medicinal plant with numerous pharmacological uses. OBJECTIVE: To investigate the effect of light intensity levels on the growth and accumulation of secondary metabolites of C. smyrnioides, cultivated seedlings were subjected to different relative light intensities via sun-shading. MATERIALS AND METHODS: Changium smyrnioides seedlings were subjected to five irradiance treatments (100, 60.54, 44.84, 31.39, and 10.56% sunlight) in glasshouse for 9 months. Enzymatic and non-enzymatic antioxidants with spectrophotometric method, photosynthetic parameters with Li-6400XT, dry matter accumulation and active component contents in the root with spectrophotometric and HPLC method were analyzed. RESULTS: With an increase in relative light intensity levels, activities of enzymatic and non-enzymatic antioxidants, and malondialdehyde (MDA) contents were increased overall, while net photosynthetic rate (Pn) and dry matter accumulation patter first increased and then declined. The highest net photosynthetic rate (30.68 µmol/m2·s) and dry root weight (5.07 g) were achieved under 60.54% sunlight. Lower relative light intensity levels stimulated the accumulation levels of bioactive compounds in the roots so that the highest contents of mannitol (1.35%) and choline (405.58 µg/g) were recorded under 31.39% sunlight, and the highest polysaccharide content (10.80%) were achieved under 44.84% sunlight. With a decrease in the relative light intensity levels, the water-soluble component content increased first and then decreased. DISCUSSION AND CONCLUSION: The results revealed that 31.39-60.54% sunlight serve as appropriate relative light intensity conditions for cultivated C. smyrnioides.

Genetic variation in Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis, three endemic and endangered species in China using SSR and TRAP markers.

Leeches are not only important medicinal animals worldwide but also are endangered. We aimed to (i) explore the level of genetic diversity within/among populations of three leeches, (ii) assess genetic differentiation among these three leeches, and (iii) discuss an appropriate strategy for conserving leech germplasm. A total of 315 individuals of Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis from 21 populations were collected in China and Vietnam. The genetic structure and genetic diversity among and within the 21 populations were evaluated using target region amplified polymorphism (TRAP) and simple sequence repeat (SSR) markers. Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Shannon's index (I) and Nei's gene diversity index (H) for the three leeches were high at the species level (I=0.4980 and H=0.3323 for TRAPs, I=0.4487 and H=0.2969 for SSRs in W. pigra; I=0.4147/0.3769, H=0.2788/0.2566 for H. nipponica; and I=0.4616/0.4717, H=0.3099/0.3203 for P. manillensis). However, low genetic diversity was determined at the population level; the average genetic diversity measures within populations were H=0.1767/0.1376, I=0.2589/0.2043 for W. pigra, H=0.2149/0.2021, I=0.3184/0.3000 for H. nipponica and H=0.2850/0.2724, I=0.4152/0.3967 for P. manillensis. We conclude that there was limited gene exchange within/among populations and species, as the gene flow number (Nm) was 0.5493/0.5807. However, for all three species, the genetic diversity was different at the population level. Gene differentiation (Gst) and Nm were 0.4682 /0.5364 and 0.5678/0.4321 for W. pigra, 0.2294/0.2127 and 1.6797/1.8512 for H. nipponica and 0.1214/0.1496 and 3.6202/2.8412 for P. manillensis. STRUCTURE analysis, Unweighted Pair-Group Method with Arithmetic means (UPGMA) cluster analysis and Principal Coordinates Analysis (PCOA) all yielded similar results. The isolation-by-distance pattern was not significant for any of the three species by the Mantel test. These data emphasize the need for management, conservation, and rehabilitation of this animal species. Finally, an appropriate strategy for conserving leech is proposed.

[Effects of harvest and different processing methods on anti-thrombin activity of Poecilobdella manillensis].

The effects of harvest and different processing methods on the anti-thrombin activity of Poecilobdella manillensis were respectively studied. The indicators included processing methods (vacuum freeze drying, fresh homogenate, drying under sunlight, freezing, scalding, baking under different temperatures), different parts (entire body, cephalon, pygidium, exudate) and body weights (≤10, 10-20, 20-30, 30-40, ≥40 g). The anti-thrombin activities of P. manillensis with different processing methods were evaluated by direct anti-thrombin titration. The results indicated that the processing methods significantly affected the anti-thrombin activities of P. manillensis. Among the 11 groups, the anti-thrombin activity of P. manillensis processed with vacuum freeze drying (1 303.56 U•g⁻¹) was significantly highest than the other groups (P<0.05), and that processed with baking under 90 ℃ (15.44 U•g⁻¹) was the lowest. The anti-thrombin activity of the cephalon of P. manillensis (226.42 U•g⁻¹) was the highest, and that of the pygidium (102.12 U•g⁻¹) was lowest; the anti-thrombin activities for different body weights were significantly different (P<0.05); and among the five groups, the body weight of ≤10 g (328.86 U•g⁻¹) was the highest (P<0.05), and the body weight of ≥40 g (87.71 U•g⁻¹) was the lowest. In conclusion, harvest and different processing methods had a significant impact on the anti-thrombin activities of P. manillensis. In the study, for the optimal processing method for P. manillensis, the body weight between 20-30 g is recommended, and the vacuum freeze drying is preferred, which is followed by the drying under sunlight.

[Effect of water temperature, stocking density and feeding cycle on growth of juvenile Poecilobdella manillensis].

The effect of water temperature, stocking density and feeding cycle on the growth of Poecilobdella manillensis juvenile was conducted P. manillensis was conducted respectively under different conditions: water temperatures(18, 22, 26, 30,34, 38 degrees C and CT), stocking density (75, 125, 200, 275, 350 individual/L) and feeding cycle(2, 4, 6, 8, 12, 16 d). After 30 days, survival rate, weight gain rate, specific growth rate were measured. There was a significant correlation between water temperature and specific growth rate (γ = -0.066x2 + 3.543 1x -38.09, R2 = 0.837 9). Based on the regression equation, the specific growth rate of P. manillensis achieved the maximum (9.461 4) at 26.84 degrees C. And the most optimal water temperature was 26-30 degrees C. Meanwhile, the survival rates of P. manillensis was 0 at 38 degrees C in 3 d. There was significant negative correlation between density and specific growth rate (γ = -0.005 7x + 9.197 3, R2 = 0.998 3) and between feeding cycle and specific growth rate (γ = -0.468 2x + 10.574, R2 = 0.998 8).