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.     

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.     

The Expression of the IGF Family During Mouse Mammary Gland Development

This study was to determine the patterns and levels of IGF family members＇ expression during postnatal mammary gland development. The authors investigated the protein expression profile of the major components of the IGF axis in murine mammary glands. All the proteins examined, IGF- Ⅰ, IGF- Ⅱ, and IGF- Ⅰ receptor （IGF-Ⅰ R） were expressed at greatly different levels and displayed unique expression profiles. IGF- Ⅱ and IGF- ⅠR were always expressed at significantly higher levels than IGF- Ⅰ. IGF- Ⅰwas localized in adipocytes as well as the epithelial and stromal compartments, but just distinctly expressed where mammary cells aggregated to form ducts, in virgins. The IGF- Ⅱ was localized only on the basal layer epithelial cell membranes of ducts and alveoli, with a peak level on the initiation of lactation. The higher level of IGF- ⅠR compared with IGF- Ⅰ was also found in adipocytes as well as in the epithelial and stromal compartments, especially during pregnancy and late lactation. The IGF- Ⅰ R pathway was obviously significant for the development of the mammary parenchyma and stroma. Overall, the comparison of the expression profiles of these different proteins would strongly suggest that they were likely to have different functions throughout the mammary gland development, and it also highlighted the potential interactions and coregulation of the members of this axis. It seems that IGF- Ⅱ was the major local modulator rather than IGF- Ⅰ by an IGF- Ⅰ R-independent pathway, especially for initiation of lactation. This study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.     

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.     

Uterine Expression of WNT7A and β-catenin After Induction of Oestrus in Sheep

WNT7A and β-catenin localisations and roles in regulating periimplantation ovine conceptus development under natural estrous conditions have been elaborated.However,their locations and expression patterns have not been reported under induction of oestrus.The localisation,expression and function of WNT7A and β-catenin in the uterine tissues of the early pregnant and non-pregnant sheep on days 10,12,14,16 and 18 following artificial induction of oestrus were investigated by means of in situ hybridisation,real-time RT-PCR,immuno-histochemistry and western blotting methods.WNT7A and β-catenin mRNA and protein were both restricted to the apical surfaces of the uterine luminal epithelium (LE) and glandular epithelium (GE).In pregnant sheep,protein localisation of WNT7A and β-catenin was observed both in the endometrial LE and GE.Their staining presented on day 10,increased between day 12 and day 16,and decreased on day 18.WNT7A and β-catenin mRNA and protein expression increased initially and then decreased from day 10 to day 18,peaking on day 16,and β-catenin reaching a peak on day 18 in the uterine tissues of pregnant sheep (p0.05).By contrast,no significant changes in WNT7A and β-catenin mRNA and protein expression levels were observed from day 10 to day 18 of the oestrus cycle in the uterine tissues of non-pregnant sheep (p0.05).Additionally,WNT7A and β-catenin mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly higher than those of non-pregnant sheep (p0.05).Treatment of endometrial epithelial cells with WNT7A increased the mRNA expressions of β-catenin,c-myc and Cyclin D1.These results provided an underlying mechanism of periimplantation ovine conceptus development under induction of oestrus.     

Molecular Cloning of Myostatin Partial cDNA of Beijing Duck and Its Expression in Breast Muscle

In this experiment, 500 bp cDNA of myostatin gene was cloned from a Beijing duck＇s breast, The duck myostatin gene was found to have 98, 96, 95, 88, and 87% sequence similarity at the cDNA level with domestic goose, chicken, domestic pigeon, human, and pig, respectively. The predicted amino acid sequence has an overall similarity with a comparable region of turkey 99%, domestic goose 98%, and chicken 99%. Conserved domains of deduced amino acids showed that it belonged to the TGF-beta family. Myostatin expression in breast muscle was higher at 28, 35, and 42 days than at 7, 14, and 21 days. The pattern of myostatin expression was closely parallel to the trend of breast muscle growth, suggesting that myostatin might play an important role in breast muscle development. It was possible to postulate that myostatin may be a major determinant of muscle mass in breast muscle, as shown in other species.     

赤点石斑鱼神经坏死病毒MCP基因原核表达条件优化(英文)

[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.     

High gene flows promote close genetic relationship among finewool sheep populations(Ovis aries) in China

The aim of our present study was to construct genetic structure and relationships among Chinese fine-wool sheep breeds.46 individuals from 25 breeds or strains were genotyped based on the Illumina Ovine 50K SNP array.Meanwhile,genetic variations among 482 individuals from 9 populations were genotyped with 10 microsatellites.In this study,we found high genetic polymorphisms for the microsatellites,while 7 loci in the Chinese superfine Merino strain（Xinjiang types）（CMS）and 5loci in Gansu alpine superfine-wool sheep strain（GSS）groups were found deviated from Hardy-Weinberg equilibrium（HWE）.Genetic drift F_ST=0.019（P<0.001）and high gene flows were detected in all the 7 fine-wool sheep populations.Phylogenetic analysis showed fine-wool sheep populations were clustered in a group independent from the Chinese indigenous breeds such that the 7 fine-wool sheep clustered distinct from Liangshan semifine-wool sheep（LS）and Hu sheep（HY）reflected by different population differentiation analyses.Overall,our findings suggested that all fine-wool sheep populations have close genetic relationship,which is consistent with their breeding progress.These populations,therefore,can be regarded as open-breeding populations with high levels of gene flows.Furthermore,the two superfine-wool strains,viz.,CMS and GSS,might be formed by strong artificial selection and with frequent introduction of Australian Merino.Our results can assist in breeding of superfine-wool sheep and provide guidance for the cultivation of new fine-wool sheep breeds with different breeding objectives.     

香蕉MaPRMT1基因的分离及表达分析(英文)

[Objective] The aim of experiment was to lay molecular foundation for studying maturity mechanism of banana after harvest. [Method] The combined method of suppressing subtractive hybridization and cDNA micro-array were used to obtain cDNA segment of one PRMT gene in banana and the whole cDNA sequence of the gene was cloned.The bioinformatics analysis was operated on it,in addition, the expression profile analysis was conducted in different organs and different mature periods of banana.[Result] The whole length of cDNA in MaPRMT1 was 1 158 bp and possessed a complete open reading frame,which could encode 385 amino acids.It had high homology with PRMT in plant,containing one Methyltransf₁ domain.The MaPRMT1 gene was expressed in root,stem,leaf and fruit of banana and the expression levels in stem and leaf were relatively high.As the increase of days after harvest,the expression level declined gradually,however it reached maximum when ethylene release was biggest,then it declined.[Conclusion] MaPRMT1 belonged to the first kind of arginine methyltransferase and it was expressed differently in different organs and fruits at different mature periods.     

Effect of Glutamine Supplementation on Abilities of Antioxidative and γ-glutamyl Transpeptidase Activities in Liver and Intestine of Piglets Fed Diets Containing Raw Soybean

Eighteen male piglets weaned at 28 days age were randomly assigned to one of three treatments(1% glutamate, 1% and 2 % glutamine supplementation). The basal diet contained 5 % raw soybean. The di-ets were calculated to be isonitrogenous and isoenergetic. The level of plasma glutahione (GSH) increasedmarkedly in piglets fed glutamine, and the response was related to dose. In treatments Ⅰ and Ⅱ, the levels ofplasma GSH were significantly higher than that in the control at the 35 days age (P＜0.05). The level of plas-ma GSH in treatment Ⅱ was significantly higher than that in the control at 42 days age (P＜0.05). At 49days age, there was no significantly difference of the level of GSH in plasma, liver, spleen, intestine and mes-enteric lymph node. The level of superoxide dismutase (SOD) in liver and spleen was higher than that of thecontrol, however, the difference wasn't significant. Dietary glutamine supplementation decreased γ-glutamyltranspeptidase(γ-GT) activities in liver. The activities of γ-GT of liver protein in treatment Ⅱ were signifi-cantly lower than that in the control (P＜0. 05). The activities of γ-GT of duodenum in treatments Ⅰ and Ⅱwere also significantly lower than that in the control (P＜0.05). But there were no significant differences ofthe activities of γ-GT in jejunum and ileum. The results showed that dietary glutamine supplementation in-creased the level of plasma GSH, and decreased γ-GT activities.     

A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z

This study was to discuss the relationship among the change in the activity of Bombyx mori carboxylesterase （BmCarE） in the midguts, the differential expression of BmCarE gene （bmcare） in the midguts, and the ability of Bombyx mori resistant to densonucleosis virus （BmDNV）, and to elucidate the molecular mechanism of resistance to BmDNV-Z. With two silkworm strains, HUABA, which is susceptible to BmDNV-Z, and BC8 （a near isogenic line of HUABA）, which is completely resistant to the same virus, as materials, the activity of BmCarE in the midgut was determined by Bio-Tek Synergy, and the differential expression of bmcare between the two strains was investigated by real-time fluorescence quantitative PCR, both at 12, 36, and 72 h post oral inoculation of the two strains with virus （hereafter referred as inoculation）. While the activity of BmCarE in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculated group, and the HUABA control group by 3.28, 2.26, and 3.02 times, respectively, with the difference being highly significant （P 〈 0.01）, there was no statistical difference among the other groups. The relative expression level of bmcare in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculation group, and the HUABA control group by 17.714, 21.76, and 15.09 times, respectively, with the difference being highly significant （P 〈 0.01）, and there was no statistical difference among other groups. The elevation of BmCarE activity and expression level of bmcare in the resistant strain at 12 h post inoculation may relate to the resistant gene （nsd/nsd） and the stimulation of BmDNV-Z. The molecular basis for the elevation of BmCarE activity in the resistant strain BC8 may be the change in the expression level of bmcare.     

低温胁迫对甘薯S-腺苷甲硫氨酸合成酶mRNA表达水平的影响（英文）

[Objective] The mRNA expression level changes of S-adenosylmethionine synthetase(SAMS)under low temperature stress was studied.[Method]Total RNA were extracted from leaves,stem and earthnut of sweet potato 0,12,24,48 and 72 h after low temperature treatement.mRNA expression level was analyzed by reverse expression and Real-time PCR technique.[Result]The expression quality of the gene extracted from leaves,stem and earthnut increased and the expression quality reached the peak point 24,72 and 72 h after low temperature treatment respectively.The expression change of earthnut was the biggest.[Conclusion]Low temperature was good for increasing mRNA expression of relevart genes.     

Study on FSHR and LHR mRNA Levels of Different BMPRIB Genotypes of Small Tail Han Sheep During the Oestrum

The relationship between different BMPRIB genotypes of Small Tail Han sheep and FSHR and LHR mRNA levels during the oestrum was studied using semiquantitative PCR. The results indicated that FSHR mRNA extracted from the right ovary of BB (1.14 ± 0.11) ewes showed higher levels compared with AA (0.44 ± 0.11) and AB (0.36±0.08) ewes (P ＜ 0.01), and LHR mRNA extracted from the right ovary of BB (0.42±0.02) ewes showed significantly higher levels compared with AA (0.23 ±0.02) and AB (0.25 ±0.04) ewes (P＜0.01); however, the mRNA extracted from the left ovary showed no significant difference in levels among the genotypes during the oestrum. It indicated that the fecundity induced by a mutation of BMPRIB in Small Tail Han sheep may be related to the changes of the mRNA expression of LHR and FSHR in ovary.     

Dynamic Changes in the Calcium Content of Several Apple Cultivars During the Growing Season

Dynamic changes in calcium content were investigated in eight apple cultivars. The results showed that the calcium concentration in leaves and shoots increased with fruit development. The cultivars displayed only a small difference in the calcium concentration during the early stage of development, the difference became very significant at the late stage of development, especially in shoots. In shoots, for example, calcium content was highest in Starkrimson （19 638.6 mg kg^-1） and lowest in Fuji （8 751.3 mg kg^-1）. Calcium concentration was highest in young fruits and was found to decrease with the growth of fruit, and was characterized by a dramatic drop at the rapid expansion stage. There was a significant difference among cultivars. Young Starkrimson fruits contained the highest calcium concentration of 506.52 mg kg^-1 among cultivars tested, followed by Pink Lady and Fuji. The calcium concentration in mature fruits from high to low is as follows： Starkrimson, Sansa, Pink Lady, Senshu, Gala, Fuji, Red General and New Century. In this study, it was found that eight cultivars continuously assimilated calcium during the whole growing season, especially at the young stage when fruit took up 35-46% of total calcium. The calcium content in fruitlets was low in all cultivars; in the expansion stage, there was rapid absorption of about 30% of total calcium, whereas in the ripening fruit, content of calcium was reduced. The calcium accumulation increased with fruit growth in stalk, similar to that in fruit.     

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.     

Mutagenesis of Arachidonic Acid-producing Mortierella Isabellina and Analyses of △6-desaturase Role by qPCR

Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.     

AMPK Subunit Expression Regulates Intramuscular Fat Content and Muscle Fiber Type in Chickens

The objective of this study was to assess the role of AMPK in intramuscular fat(IMF) and fiber type in chicken muscle. The chickens were slaughtered and their muscles were collected at the ages of 4, 8, and 16 weeks so as to determine the IMF contents, as well as the expression levels of AMPK subunits, regulators of adipogenesis. In addition, the myosin heavy chains(My HCs) in thigh muscle tissues were also measured. The results showed that the IMF contents in 16-week old chickens were higher than those in 4 and 8-week-old chickens(P0.05).The expression levels of fatty acid synthase(FAS) and fatty aicd translocase CD36(FAT/CD36) m RNA were increased significantly in samples collected at the ages of4 and 16 weeks(P0.05). The expression levels of My HC IIa and IIb differed significantly among all the developmental stages(P 0.05). The AMPKα2, AMPKγ1,and AMPKγ3 m RNA levels were dramatically decreased with the increase of age(P 0.05). To examine the role of AMPK in adipogenesis regulation, the SV cells were cultured in an adipogenesis medium and treated with AICAR and Compound C respectively, the specific activator and inhibit of AMPK. The Compound C induced dramatically a greater expression of C/EBPβ, SREBP1 and PPARγ(P 0.05). In conclusion, the expression of AMPKα2, AMPKγ1, and AMPKγ3 m RNA is significantly correlated with the adipogenesis in skeletal muscle of chickens.     

SFRP2 affects prenatal muscle development and is regulated by micro RNA-1/206 in pigs

Secreted frizzled-related protein 2(SFRP2), a member of the SFRPs family, is associated with cell growth and differentiation in myogenesis. Our previous study suggested that SFRP2 was a potential target of micro RNA(mi RNA)-1/206, which was considered as myomiR s. To further explore the biological function and regulation mechanisms of the SFRP2 gene in porcine skeletal muscle development, we first analyzed the sequence structure of the porcine SFRP2 gene. Subsequently, we detected its tissue distribution in adult Tongcheng pigs(a Chinese indigenous breed) and investigated its dynamic expression in developmental skeletal muscle(13 prenatal and 7 postnatal time points) in Tongcheng pigs. An interaction analysis between SFRP2 and myomi Rs was also performed. The results showed that the expression pattern of the SFRP2 varied greatly across diverse tissues. It exhibited abundant expression in prenatal skeletal muscle and peaked at 55 days post coitus(E55), and had a lower expression in postnatal skeletal muscle, indicating that the SFRP2 gene might affect porcine embryonic skeletal muscle development. Co-expression analysis revealed that the expression levels of SFRP2 correlated negatively with mi RNA-1(r=–0.570, P-value=0.009) and mi RNA-206(r=–0.546, P-value=0.013), but positively with SFRP1(r=0.613, P-value=0.004). The bioinformatics analysis and dual luciferase assay verified that the SFRP2 was a putative target of mi RNA-1/206 in pigs. Therefore, this study is helpful for understanding the biological function and molecular regulation of the SFRP2 gene during porcine skeletal muscle development.     

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.