2009 Vol. 30, No. 6
Members of the transforming growth factor-beta superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in fecundity of sheep. Our previous investigation confirmed that the fecundity mutations of sheep presented in highly prolific White goat individuals of Guizhou province. To illuminate other polymorphisms in Bmp15 and Gdf9 genes and the relationship of these mutations with function, we set out to clone and characterize the coding region of Bmp15 and Gdf9. Molecular models of BMP15 and GDF9 mature peptide of White goat were constructed based on the homology to experimental tertiary structure of human BMP7. Two exons encoded prepropeptide of 394 amino acids in BMP15 and 453 residues in GDF9, respectively. Apart from the FecXB mutation (S99I) in BMP15 and V79I mutation in GDF9 confirmed in White goat previously, other seven and three polymorphism sites were detected from BMP15 and GDF9 mature peptides, respectively. S32G, N66H, S99I/P99I and G107R in BMP15 could be important for the binding of dimer to receptors. Changes of P78Q and V79I in GDF9 might affect the binding of dimer to receptor type 1. Comparing the length of BMP15 and GDF9 prepropeptide in vertebrates, an increase in length of BMP15 presented along with the protein evolution from fish to mammal and the divergence of the N-terminus residues in matured BMP15 peptide might contribute to the sensitive control on the fertility of animal species with low ovulation rate. These findings gave a valuable explanation for the correlation of mutations in Bmp15 and Gdf9 genes with the control on fecundity of White goat and supported the notion that they were the pivotal factors in female fertility of White goat in Guizhou province.
2009, 30(6): 603-612. doi: 10.3724/SP.J.1141.2009.06603
The complete mitochondrial genome sequence of Aeropus licenti Chang was determined using long PCR and conserved primer walking approaches. The results showed that the entire mitochondrial genome of Aeropus licenti Chang is 15 597 bp long with A+T content 74.8%. All 37 genes are conserved in the position observed in that of Locusta migratoria. Genes are closely assembled one after the other, leaving a total of 105 bp (excluding the A+T rich region) in intergenic spacers, ranging in size from 1 bp to 21 bp. There are a total of 58 overlapping nucleotides among 10 genes with overlapping range from 1 to 17 bp. Four types of start codons were identified in the 13 protein coding genes (PCGs), in which TAA and TAG were used in 12 PCGs as stop codons, except ND5 (T). Most tRNA genes could form typical secondary structures except tRNASer (AGN), which had an absence of the DHU arm. There are six domains, 47 helices and three domains, 31 helices in the secondary structures of lrRNA and srRNA respectively. In addition, there is an A+T rich region with 712 bp in length.
Freshwater prawns (Decapoda: Caridea: Macrobrachium) play an important role in domestic fishery resources. Culturing M. rosenbergii and M. nipponense brings great economic benefits, as the two species were widely farmed in China. M. qilianensis, a native species with natural distribution limited in Gansu province, was classified into genus Macrobrachium based on external morphological characters. In order to understand the molecular genetic differences among the three species of Macrobrachium, i.e., M. rosenbergii, M. nipponense, and M. qilianensis, we analyzed sequences of mitochondrial cytochrome oxidase subunit I (COI) of them. It would provide theoretical basis of exploiting and utilizing Macrobrachium resources rationally. A total of 30 individuals (10 individuals of each species) were collected from Gansu and Zhejiang province. Samples of M. qilianensis were wild, however, that of M. rosenbergii and M. nipponense were cultured. Their mitochondrial COI gene segment sequences were obtained by using the method of PCR amplification and sequencing. After alignment, 649 bp consensus sequences of COI were obtained. One hundred and sixty-nine variable sites were detected in all 30 individuals, accounting for 26.04% of total sequence. A total of seven haplotypes were also detected. Nucleotide diversity was 0.411% within M. rosenbergii, 0.092% within M. nipponense, and 0.031% within M. qilianensis. Genetic diversity of wild M. qilianensis was much lower than that of cultured M. rosenbergii and M. nipponense. Genetic distances between different haplotypes of the three prawns ranged from 19.87% to 23.84%. It suggested that the three species were valid species, because genetic distances among them were quite great. To further determine the taxonomic status of the three prawns in family Palaemonoidae, we downloaded the corresponding COI sequences of Palaemonoidae prawns from Genbank and analyzed the phylogenetic relationships of them. Phylogenetic tree (NJ) showed that M. nipponense, M. rosenbergii and other Macrobrachium species constituted one monophyletic group. However, M. qilianensis, Exopalaemon carinicauda, and Palaemon debilis formed the other clade. Thus, results of COI sequences did not support that M. qilianensis belonged to genus Macrobrachium. The taxonomic status of M. qilianensis should be reevaluated with more comprehensive evidences.
Present broodstocks of large yellow croaker are borne from extremely small numbers of base population. Thus, it is necessary to analyze kinship of broodstocks in order to avoid inbreeding that will bring out the reduction of individual survival and growth. This paper reports kinship reconstruction and genetic diversity in 103 broodstocks of large yellow croaker by utilizing 23 microsatellite markers. Genetic diversities of 103 croakers at 23 loci pronounce that there are 134 alleles in total and an average of 5.82, and the observed average heterozygosity of 0.599 3, demonstrating that these broodstocks still maintain genetic variability to some extent. The results of sibling groups reconstructed are not identical using two methods of liklihood and 2-allele recombinatorial optimization. However, the evidence of close relationship between broodstocks is confirmed. The mating combinations are compared between these two methods, as a result of 85% identity and a final selection of 2-allele method. This study aims at finding a better way to avoid inbreeding occurred in broodstocks, and facilitating the aquaculture market of large yellow croaker, meanwhile, offering methods and statistical models to artificial propagation in other marine fish species.
Extraction of high molecular weight genomic DNA (HMW-gDNA) from spider organs and construction of an electroelution system for obtaining HMW-gDNA efficiently are described. We improved the CTAB method to isolate genomic DNA from spider pectoral muscle tissues, constructed a novel electroelution system with the use of 2-mL centrifuge tube and dialysis membrane, and utilized this self-constructed electroelution system to recover target HMW-gDNA from agarose gels. The HMW-gDNA extracted using the improved CTAB method could be over 48.5 kb in size. By trapping the HMW-gDNA with dialysis membrane, the electroelution system could recover target HMW-gDNA from agarose gels rapidly and without affecting the integrity of HMW-gDNA. The recovery rate was over 75% with the OD260/OD280 ranging from 1.8 to 2.0. The results demonstrate that the improved CTAB method can be used to extract spider HMW-gDNA, and the electroelution system is a general method for recovering target HMW-gDNA from agarose gels with low cost, simplicity, high efficiency and good practicability.
The transcription factor Sox2 is one of the earliest determinants of the neural system in vertebrate and plays crucial roles in stem cell maintenance. Through bioinformatical analysis, we found that the 3'untranslated regions (3'UTR) of vertebrate Sox2 mRNAs (especially the 300 bases at the most 3' end) are highly conserved and contain four conserved AU rich fragments. Through reporter gene analysis, we evaluated the effects of the Sox2 full length 3'UTR and the conserved fragments on gene expression in Xenopus laevis embryos and cultured cells. The results showed that the conserved fragment 2 from the 3'UTR of Xenoups laevis Sox2 was able to increase the reporter gene expression significantly, indicating the possibility that the expression level of Sox2 might be regulated post-transcriptionally through its 3'UTR.
Neural tube defects (NTDs) are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus, several tissue movements are involved in the neural tube closure process. Immediately after the neural tube fusion, the neural crest cells get monopolar protrusion toward dorsal midline and migrate to form the roof of the neural tube. At the same time, radial intercalation takes place from the ventral neural tube and forces it to be single-layered. Here, we physically block the neural tube closure to test the cell movements and the following patterning in Xenopus laevis explants. The results show that the single-layered neural tube fails to form and the neural crest cells remain at the lateral regions in the explants with NTDs. However, the patterning of the neural tube is not affected as indicated by the normal expression of the preneural genes. These results indicate a requirement of the neural tube fusion for the radial intercalation and the dorsal midline directed neural crest migration, but not for the dorsal-ventral patterning of the neural tube.
2009, 30(6): 645-652. doi: 10.3724/SP.J.1141.2009.06645
An experiment was conducted to compare the effects of two mouse thrombocytopenia models induced by cyclophosphamide at two different administration routes to determine a proper cyclophosphamide administration route that could cause stable thrombocytopenia. A suitable drug dosage that could induce thrombocytopenia in mouse efficiently with the definite administration route was then investigated. BALB/c mice were randomly divided into Normal, Model A and Model B groups. To Model A, 200 mg/kg of cyclophosphamide was given by vena caudalis injection as first dose and 30 mg/kg as maintenance dose by intraperitoneal injection at the following 6 days. To Model B, 150 mg/kg of cyclophosphamide was given by subcutaneous injection once a day for consecutive 3 days. All groups were under investigation for 15 days. The result suggested that a decrease in the number of blood platelets of Model B at the 7th day were significantly than that of Normal. Other platelet related indices like platelet distribution width, mean platelet volume and platelet-large cell ratio of Model B increased significantly in comparison with those of Normal group. The platelets count was reduced but fluctuated greatly, and more than half of the mice died in Model A. Therefore, subcutaneous injection of cyclophosphamide for 3 days was used for the cyclophosphamide dosage test. BALB/c mice were randomly divided into Normal, cyclophosphamide low dose (100 mg/kg), medium dose (120 mg/kg) and high dose (140 mg/kg) groups. All groups were under investigation for 11 days. Though all 3 dosages successfully initiated thrombocytopenia as the platelets number dropped at the 7th day, the low dose was considered to be a suitable one that was of high efficacy and low toxicity. Thus, BALB/c mice challenged by subcutaneous injection of cyclophosphamide 100 mg/kg per day for 3 consecutive day is one simple, feasible and stable mouse thrombocytopenia model that could be used for pharmacodynamic test of the drugs which are supposed to have platelets increasing effect.
In order to investigate whether N-methyl-D-aspartic acid (NMDA) receptor and nitric oxide synthase (NOS) made contributions to the depression induced by chronic unpredicted mild stress (CUMS)，the depression model was established by using the CUMS. Using the stereotaxic and intra-hippocampal microinjection observing rat emotion and behaviors by the change in body weight, sucrose preference test, open field test and forced swimming test, and immunohistochemistry, and the expression of NOS in hippocampus were tested. The results showed that depression-like behavior and the expression of NOS increased in hippocampus induced by rats receiving CUMS for 21 days or intra-hippocampal injections of NMDA receptor agonist. Intra-hippocampal injection of noncompetitive NMDA receptor antagonist MK-801 significantly prevented CUMS-induced depression-like behavioral changes, and decreased the expression of NOS. These results provide evidence that CUMS resulting in development of depression may induce neurotoxicity in hippocampal neurons by stimulating Glu release, inducing excessive activation of NMDA receptor, and increasing expression of NOS.
A complete survey on geographic distribution of Paa yunnanensis was conducted in 84 counties of Yunnan Province. The distribution of this species was mapped with GIS tools, based on both the data obtained from this sampling and derived from literatures. The result shows that P. yunnanensis is widespread in 71 counties of 15 prefectures, except Diqing prefecture of Yunnan Province. The altitudes and mean annual temperatures for P. yunnanensis distribution were recorded as 1 400−2 600 m and 11.54－18.50℃, respectively. Based on these features of P. yunnanensis, two possible distribution maps of the species were mapped through software DIVA-GIS 5.2. These maps may forecast the potential distribution of P. yunanensis in Yunnan, providing basic data necessary for further study and protection of P. yunnanensis.
During autumn 2007 and spring 2008, we surveyed 14 islands, with a total of 16 800 traps, for small mammals in Qiandao Lake, Zhejiang to determine their community distribution. The captured small mammals belonged to 11 species, 7 genera, 2 families and 2 orders，including 9 species of Murids（Muridae）, and 2 species of Soricds（Soricdae）. The results showed that the small mammals present a non-random nested pattern in assemblage on the islands. In terms of larger scale perspective, the species of high adaptability were able to adjust to the various environments, while ones of low adaptability only survived in particular circumstances. Plant species richness and habitat specificity of the species were the main ecological factors that influenced the formation of this pattern.
Based on “Presence data”, we assessed the habitat quality according to the physical factor utilized by Tibetan fox in Gouli Township, Dulan County, Qinghai Province, China. The spatial carrying capacity of Tibetan fox was also estimated according to their minimum space requirements. A total of 4 topographical factors, elevation, slope, terrain ruggedness and slope position, were used to conduct habitat assessment for Tibetan fox. The suitability of habitat factors were determined by comparing the utilization by Tibetan fox and its availability based on 95% Bonferroni confidence interval. A comprehensive assessment criterion was set up for all topographic factors. For Tibetan fox, the elevations of 4 050 - 4 300 m, the slopes of 5 -20°, the upper and lower slope positions were suitable habitat. Tibetan fox use terrain ruggedness non-selectively. According to the utilization of Tibetan fox on different topographic factors and the consistence of habitat utilization by different fox individuals, we set the comprehensive criterion as: Xi = （X2elevation×X2slope position×Xslope×Xterrainrug gadness）1/6.The area of suitable habitat is about 17.4 km2, which take a percentage of 38.8% of research area. The area of most suitable habitat is only 0.4 km2. The home range of Tibetan fox ranges from 2.53 km2 to 4.99 km2, and the overlap index (OI) of different individuals were 0.16 -0.66. The minimum space requirements of Tibetan fox are 2.09 -3.55 km2, and the spatial carrying capacity of Tibetan fox in suitable habitat is 7-12. According to the habitat resource and monitoring results, we infer that the Tibetan fox population is keeping steady.
The diurnal behavioral rhythm and time budgets of dwarf blue sheep were studied by method of scan sampling. The behaviors recorded were classified into five groups: feeding, moving, standing, bedding, and other activities. During summer, there are three feeding peaks in diurnal time (6:00 -9:00, 10:00 -11:00 and 16:00 -20:00), and one bedding peak (11:00 -15:00). The dwarf blue sheep was found to use 30.14% (±3.32) of their time feeding, followed by bedding (26.44%±3.59), standing (24.90%±4.27), moving (15.73%±1.26), and others (2.82%±1.00).We also found that adult males spend more time on feeding and standing than other individuals, juveniles spend more time moving and other activities, while females spend more time on bedding.
In November, December 2007 and March 2008, we studied the diurnal activity budgets of the Mongolian Gazelle (Procapra gutturosa) using group-scan-sampling for the time before, during and after the rut around DaLai Lake, Inner Mongolia. The activities of Mongolian Gazelle were divided into 6 categories: feeding, standing, moving, lying, rutting and others. The results showed: 1) Before the rut, the major behavior of the female was feeding, the time budget of feeding occupied 44.9±3.8% of all diurnal time, the lying time was 32.3±4.8%; During the rut the females spent 43.5±4.0% on feeding and 29.2±2.9% on lying; After the rut the time budget of feeding occupied 46.2±3.1% of all diurnal time, the lying time was 28.0±4.8%. Activity budgets of the female on standing, rutting, others changed significantly among the three periods (P<0.05)，but activity budgets on feeding, moving, and lying did not change significantly among the three periods (P>0.05). 2). Before the rut, the major behavior of the males was feeding, the time budget of feeding occupied 52.6±3.8% of all diurnal time, the lying time was 13.4±6.4%; During the rut the males spent significantly less time on feeding (17.5±2.8%) and more time on lying (24.2±4.1%) compared with before the rut. After the rut, the time budget of feeding occupied 29.8±4.8% of all diurnal time, while the lying time was 44.2±4.7%. Activity budgets of the male on feeding, standing, moving, lying, rutting, others changed significantly among the three periods (P<0.05). The time budget on feeding and lying reflects on energy balance strategies. We conclude from the results that; females do not change their strategy significantly among the three periods - they try to increase their energy intake; before the rut, the male is the same as the female, but during and after the rut, the males adopt an energy saving strategy to optimize their energy balance; males aim to save energy by spending more time lying.
By binocular microscope examination and hand-drawing, we observed the pharyngeal bones and teeth of 39 Gobininae species to explore their morphological variations and functional adaptation. The results showed that, in the Gobioninae, pharyngeal bones could be divided into three morphological types: wide, intermediate, and narrow; pharyngeal teeth into five morphological types: conical, molar, coarsely compressed, compressed and extremely compressed. Different types of pharyngeal bones and teeth cooperate together to deal with different types of food. Combination of the pharyngeal bones and teeth in the Gobioninae exhibit various types, and this variation occurring in trophic apparatus provides the diverse feeding organ adapting for the ecological resource utilization and the different types of dietary habits.
One hundred and fourty-nine specimens of Sinocyclocheilus grahami from six different localities of Lake Dianchi area were examined. Twenty frame and nineteen general characteristics, thirty-nine characteristics in sum were measured for multivariate morphometrics analysis. Based on the results of principal components analysis, specimens clustered together by scatter plots of scores could not be divided. It indicated that there is no morphological diversification under species level.