绵羊肌肉生长激素受体基因表达的发育性变化研究(英文)

[Objective] The study aimed to explore the expression of muscular growth hormone receptor gene (GHR) in sheep at the early stage of growth and development. [Method] The GHR mRNA expression levels in longissimus dorsal muscles of male Kazak sheep and Xinjiang fine wool sheep with different ages were quantitatively analyzed by real time PCR. [Result] Sheep GHR mRNA expression level in longissimus dorsal muscle increased firstly followed by decline, and then kept steady until the end of the experiment, with the expression peak appearing on postnatal day 30. The GHR mRNA expression level of Kazak sheep was extremely lower than that of Xingjiang fine wool sheep from 2 to 90 days old (P<0.01). [Conclusion] Both age and breed had great effects on the expression of muscular GHR gene in sheep.     

Relationship Between the mRNA Expression Level of TGF-β Receptor Genes in Tissues and Ovulation Rate in Hu Sheep

Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-flR I gene in the reproductive organ was significantly higher than in the lung and muscle （P 〈 0.01）, while relative expression level of TGF-βR H in reproductive organ was significantly higher than that of other tissues （P 〈 0.01）, indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-flRH in the ovaries of the high-fecundity group were significantly higher （P〈 0.01） and obviously higher （P= 0.011） than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR H had a positive correlation with ovulation rate and the correlation coefficients were 0.562 （P〉 0.05） and 0.711 （P〈 0.05）, respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.     

Lipopolysaccharide-binding Protein Involved in Process of Mouse Embryo Implantation and Decidualization of Endometrial Stromal Cells

Lipopolysaccharide-binding protein (LBP) functions as an acute phase protein and plays a role in the innate immune response to bacterial challenge.To investigate the uterine expression of LBP during peri-implantation in mice,in situ hybridization and immunohistochemical staining were used to detect the mRNA and protein expression of LBP in mouse uteri in the early pregnancy,pseudopregnancy,artificial decidualization and hormone-treated mice.The results showed that LBP was expressed in uterine luminal epithelium (LE) and glandular epithelium (GE) during days 1-4 of pregnancy.During days 5-8,LBP was weakly expressed in the decidual cells around the embryo on the 5th day of pregnancy (implantation occurred),then gradually increased,LBP was strongly expressed in the decidual zone on the 8th day of pregnancy.The expression of LBP in pseudopregnancy was similar with pregnancy on days 1-4.In artificial decidualization mice,LBP was observed in uterine LE and GE in the control horn,whereas LBP expression was significantly higher in decidua of mouse uteri under artificial decidualization.In hormone-treated mice,the expression of LBP wasup-regulated by 17β-estradiol (E_2) and progesterone (P_4).In addition,the cultured mouse endometrial stromal cells (mESCs) were induced for in vitro decidualization with 10 nmol·L~(-1) E_2 and 1 μmol·L~(-1) P_4.Real-time PCR results showed that LBP mRNA expression was highly induced in mESCs after decidual stimulus.In vivo and in vitro experiments showed that LBP was expressed in the decidual cells,indicating that LBP involved in decidualization of mouse uteri.     

Analysis of Developmental Proteome at Egg Stage of Drone Honeybees （A. m. ligustica）

An investigation on the proteome of drone egg development of native Italian bee （Apis mellifera ligustica Spinola,1806） was carried out in order to prove up the characteristics in protein expression and regulation at egg stage and open out the molecular mechanism of the development. The experiment was carried out by two-dimensional gel electrophoresis. The results showed that there were 200, 242 and 233 proteins in a wide rang of molecular weight （12.42-169.60 kDa） and in a relatively narrow scope of pI （4.50-9.00） detected on day 1, day 2 and day 3, respectively, during the developmental process of the drone egg. Meanwhile, 164 protein spots were resolved at all the images （i.e., the protein was consistently expressed） along with the egg development, among which 7 were significantly up-expressed （P 〈 0.05） and 4 were significantly down-expressed （P 〈 0.05） while 79 had no significant differences （P 〉 0.05）. In addition, the specific proteins expressing proteins on day 1, day 2 and day 3 were 11, 18 and 18, respectively. Besides, 17 proteins expressed both on day 1 and day 2 but silenced on day 3, and 43 proteins expressed both on day 2 and day 3 but silenced on day 1, while only 8 proteins expressed both on day 1 and day 3 but silenced on day 2. The results indicate that 2-d-old eggs are at the most active expressional stage in the development of drone egg. The protein expressing at all images suggests that it should be indispensable for drone egg development, but their expression pattern is different. The proteins expressing at a specific age of egg suggest that specific proteins are needed in different developmental stages to regulate. And there are more house-keeping proteins in the developmental process of the drone egg than that of worker egg, and it will provide more targets for gene improvement.     

Electronic Scanning of Uterine Endometrium in Postpartun Cow

Two postpartum cows were used to study the ultrastructural changes of uterine endometrium by using scanning electron microscope,The results showed that the process of repair of uterine endometrium after calv-ing was demonstrated by scanning electron microscope through a series of endometrium biopsy.Some part of the endometrium was damaged after calving and its adjacent endometrium cells became necrosis and exfoliated during the first 7days post-partum;the cilium and microvillus of the epithelial cell in the umdamaged area of the endomketrium disappeared,By26days postpartum the damaged area reduced and the cilium and microvillus increased in their numbers.The damaged tissues were all repaired by day60postpartum.     

Gene Cloning and Tissue-Specific Microplitis mediator （Hymenoptera： Expression of G Protein β Subunit in Braconidae）

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

CDC25B Involved in Proliferation of Sertoli Cells of New Born Calves Through FSH and Possibly Being Key Regulating Factor

The effects of FSH on the proliferation of sertoli cells of new born calves were studied in order to provide some data for theoretical research and practical use of spermatogenesis in vitro. Different concentrations of FSH(0, 0.01, 0.02, 0.04, and 0.08 IU · mL~(-1)) were taken to treat bovine sertoli cells in vitro culture, the number of sertoli cells and the expression of seven genes were determined at 6, 12 and 24 h after FSH treatments. FSH could significantly promote the proliferation of in vitro cultured sertoli cells. FSH had no significant effects on the expression of CDC25A and could significantly improve the expression of CDC25B. 0.04 IU · mL~(-1) and 0.08 IU · mL~(-1) FSH treatments decreased the expression of CDC25C at 12 h. 0.08 IU · mL~(-1) FSH treatment decreased the expression of CDC25C at 24 h. 0.04 IU · mL~(-1) FSH could significantly decrease the expression of GSK-3β and improve the expression of β-catenin at 6, 12 and 24 h. 0.02, 0.04 and 0.08 IU · mL~(-1) FSH treatments enhanced the expressions of CYCLIND1 and C-MYC. In conclusion, FSH promoted the proliferation of sertoli cells and 0.04 IU · mL~(-1) FSH concentration could significantly promote the proliferation of in vitro cultured sertoli cells. FSH promoted the proliferation of sertoli cells by CDC25B and WNT/β-catenin and CDC25B might be the key regulator to the proliferating rate of sertoli cells of bovine calf.     

Effect of Atipamzole on Fos Protein Expression Induced by Telazol/Xylazine in Rat Cerebral Cortex and Thalamencephal

The aim of the study was to assess effect of the atipamezole on talezol/xylazine induced expression of c-fos in rat brain. Rats were injected with the mixture of 13.81 mg · kg~(-1) telazol and 5.21 mg · kg~(-1) xylazine, following 10 min later0.522 mg · kg~(-1) atipamezole injected, and then the cerebral cortex and thalamencephal were removed at 1 h after injected. Level of Fos protein was measured in the brain tissue by western-blot. The results revealed that telazol/xylazine induction Fos protein expression in the thalamencephal and cerebral cortex during the period of anesthesia, atipamezole attenuated telazol/xylazine induction Fos protein expression in the thalamencephal and cerebral cortex. The results indicated that atipamezole could inhibite telazol/xylazine-induced c-fos expression in the rat brain, and played a protective role of neuronal injury.     

Expression of Inflammatory Factors in Bovine Reproductive Tract During Follicular and Luteal Phases

In order to improve reproduvtive efficieny and understand reproduvtive defense mechanism, the oviduct, uterine horn and uterine body of bovine were used to detect the changes of inflammatory factors and the relationship between estrogen and progesterone receptor protein during estrous cycle by real-time PCR and Elisa method. The results showed that interleukin-4(IL-4), interleukin-6(IL-6), interleukin-10(IL-10), interleukin-1α(IL-1α) and interleukin-1β(IL-1β) were expressed in cow oviduct, uterine horn and uterine body. In the follicular phase and the luteal phase, m RNA expression of five inflammatory factors in the uterine horn and uterine body was higher than that in the oviduct. In the follicular phase, IL-10 was highly expressed in the uterine horn and uterine body, IL-4 was highly expressed in the uterine horn, uterine body and oviduct. Additionally, in the luteal phase, IL-6 and IL-1β were highly expressed in the uterine horn, uterine body and oviduct, and the highest expression of IL-1β was observed in the uterine horn. The levels of Estrogen Receptor(ERα) protein in the oviduct, uterine horn and uterine body significantly increased in the follicular phase. The levels of Progesterone Receptor(PR) protein in the same portions of the reproductive tract in the luteal phase were significantly higher than those in the follicular phase. IL-4 and IL-10 in the cow reproductive tract might play a major role in the follicular phase, while IL-6 and IL-1β might play a major role in the luteal phase. The expression of five inflammatory factors was not directly regulated by ERα and PR.     

Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator (Hymenoptera: Braconidae). The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head (without antennae), thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Developmental Changes of the LPL mRNA Expression and Its Effect on IMF Content in Sheep Muscle

This study investigates the developmental changes of the lipoprotein lipase （LPL） mRNA level in sheep muscle and its effect on intramuscular fat （IMF） accumulation. Male Kazak and Xinjiang Merino sheep at 2-120 days old were selected. Six animals of each breed were slaughtered at 2, 30, 60, 90, and 120 days （only the Xinjiang Merino sheep at 120-day old were available） to collect samples from longissimus dorsi muscle for the purpose of determining the IMF content and extracting total RNA that was used to investigate the developmental changes of the LPL mRNA expression by real-time PCR. The results showed that in male Kazak sheep, the IMF content increased with the progress of development and there were significant differences （P〈0.05） between the age groups. However, there was no difference （P〉0.05） between age groups in Xinjiang Merino sheep. Furthermore, the IMF content of the male Kazak sheep was significantly higher （P 〈 0.01） than that of the Xinjiang Merino sheep aged from 30 to 90 days. The highest LPL mRNA expression appeared at day 2 and it was significantly higher than that of all other age groups （P 〈 0.01）, while animals at 60-day old had the lowest LPL mRNA expression in the male Kazak sheep. In Xinjiang Merino sheep, the highest one occurred at 30-day old （P〈0.01）, followed by a continuous decrease to the lowest level at 90-day old, and then it started to increase slightly. At 2 to 60-day old, the LPL mRNA expression was negatively correlated to the IMF content （r=-0.625, P 〈 0.05） in male Kazak sheep, but no such relationship was detected in the male Xinjiang Merino sheep.     

Characterization of dual enzyme resulted from bicistronic expression of two β-glucanases in porcine cells

Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four artificial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only p Bg A and p Egx showed high activity in transfected pig kidney cells. To improve the p H range and p H stability of β-glucanase, the two β-glucanases, p Bg A and p Egx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of p Bg A3 p Eg and p Bg2 Ap Eg showed significantly enlarged p H range and significantly increased p H stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.     

Cloning and Expression of Actinobacillus pleuropneumoniae Gene Coding for TbpA and Development of an Indirect TbpA-ELISA

This study presents the cloning and expression of gene encoding transferrin-binding protein A from Actinobacillus pleuropneurnoniae in Escherichia coli expression system and the development of an indirect TbpA-ELISA. The gene coding TbpA was amplified from the A. pleuropneumoniae serotype 2 genome using polymerase chain reaction and cloned to pET-28b expression vector under the control of strong, inducible T7 promoter. The recombinant plasmid was expressed in E. coli BL21 （DE3）. The expressed fusion protein was analyzed using SDS-PAGE and Western blotting. The diagnostic potential of recombinant TbpA （rTbpA） was evaluated through an antibody-detection indirect ELISA based on the purified rTbpA. The TbpA antibodies were detectable in mice on day 7 after vaccination with purified rTbpA protein or infection with A. pleuropneumoniae serotype 10 with the TbpA-based ELISA. In addition, the TbpA-ELISA was able to detect 12 serotyping rabbit antisera postinoculation （PI） with A. pleuropneumoniae 12 serotypes experimentally. The comparable result was obtained by detecting the 117 clinical serum samples using, respectively, the TbpA-ELISA and indirect hemagglutination test （IHA） based on multiplex antigen. The result indicates that the TbpA-ELISA was the more sensitive method compared with the Mix-IHA method because of its consistent presence in A. pleuropneumoniae serotypes. In conclusion, the conserved TbpA of A. pleuropneumoniae can be used for the development of a cross-serotype diagnostic method for the detection of antibodies against A. pleuropneurnoniae.     

Influence of Gossypol on Ultrastructure and mRNA Expression of Bax and Bcl-2 in Mice Testis

The objective was to evaluate the toxicity effect of gossypol on ultrastructure of mouse testis and the expression of Bax mRNA and Bcl-2 mRNA of sperm cells in mice.Forty-eight male mice were randomly divided into four groups:control group,L-group(30 mg·kg~(-1)·d),M-group(60 mg·kg~(-1)·d)and H-group(120 mg·kg~(-1)·d)and were orally administrated with gossypol diluted by sodium carboxymethyl cellulose(SCC)or SCC(control group)for 20 days.On the 21st day,all the mice were killed and ultrastructure changes of testis were observed by TEM.mRNA expression of Bax and Bcl-2 in testis was measured by semiquantitative RT-PCR.The results showed that the testicular ultrastructure in three treated groups was gradually damaged,according to the dosage of gossypol and cellular structure disordered and organelle degenerated,manifesting vacuolation of mitochondria,expansion of endoplasmic reticulum.mRNA expression of Bcl-2 in testis significantly increased(p0.05)in L-group and then significantly decreased(p0.05,p0.01)in M-group and H-group compared with that in the control group;mRNA expression of Bcl-2 in M-group and H-group significantly decreased(p0.05,p0.01)than that in L-group and Bcl-2 mRNA expression in H-group showed a significant decrease(p0.05)compared with that in M-group.On the other hand,mRNA expression of Bax significant increased(p0.05,p0.01)in M-group and H-group than that in the control group.The ratio of Bcl-2/Bax significantly reduced(p0.05,p0.01)in the treated group than that in the control group and was found to be an obvious dose-dependent.It demonstrated that the gossypol could induce the changes on ultrastructure of mice testis,down-regulate mRNA expression of Bcl-2 and up-regulate mRNA expression of Bax,which indicated that sperm cells were induced apoptosis.     

The Levels of Genetic Differentiation of Small-Tailed Han Sheep and Tan Sheep Populations Using Structural Loci

Using the method of ＂random sampling in typical colonies of the central area of the habitat＂ and several electrophoresis techniques, the variations of 17 structural loci encoding blood proteins in 60 Small-Tailed Han sheep and 73 Tan sheep were examined and compared with those of 14 other sheep populations in China and other countries to investigate their levels of genetic differentiation. The average heterozygosities of Small-Tailed Han sheep and Tan sheep were 0.2360 and 0.2587, respectively. The average polymorphic information content values were 0.1974 and 0.2102, respectively. The average effective numbers of alleles were 1.5723 and 1.5751, respectively. The coefficients of gene differentiation in the four groups （including 4, 6, 13, and 16 sheep populations, respectively） were 0.049323, 0.059987, 0.1728, and 0.201256, respectively, indicating that the degree of gene differentiation at the structural loci was the least in Hu sheep, Tong sheep, Small-Tailed Hart sheep, and Tan sheep; followed by the above-mentioned four sheep populations and two Mongolian sheep populations; and was the highest in sheep populations belonging to the Mongolian sheep group, South Asian sheep, and European sheep. The earlier researchers＇ conclusions that both Small-Tailed Han sheep and Tan sheep evolved from Mongolian sheep were further verified by the results of this study. Hu sheep, Tong sheep, Small-Tailed Han sheep, and Tan sheep were decreasingly affected by the bloodline of Mongolian sheep to different degrees. The relationships among sheep populations were not closely related to the geographical distances among sheep populations.