Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens.  This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase.  Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme.  Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B.  To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As.  The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene).  To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued.  Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV.  Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Secondary metabolites are closely related to the nutritional quality and health functions of plants.  We investigated the secondary metabolites of both wild (n=23) and cultivated (n=27) pomegranate plants (Punica granatum L.) growing in China.  The total flavonoid (TF) and tannin (TT) contents from the peel and juice were determined and the secondary metabolites in the peel (ZLP) and juice (ZLZ) of ‘Zela 4’ were identified using liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS).  Analysis of variance (P<0.05) showed that there were significant differences in the TF content of peel (TF (P)) and juice (TF (J)), and the TT content of peel (TT (P)) and juice (TT (J)) among different pomegranate accessions.  Pearson correlation analysis showed that latitude and altitude might be the main environmental factors affecting TF and TT contents in pomegranates.  In this study, 279 secondary metabolites were identified in the ZLP and ZLZ.  In addition, we report for the first time 227 secondary metabolites in pomegranates.  Using orthogonal partial least squares discriminant analysis, 90 differential metabolites were identified in ZLP and ZLZ.  In addition, we screened eight specific germplasms (high-TF (P), ‘Junyong 3’; low-TF (P), ‘Yanzhihong’; high-TF(J), ‘Zela 4’; low-TF (J), ‘Yudazi’, high-TT (P), ‘Junyong 4’; low-TT (P), ‘Anba 1’; high-TT(J), ‘Yeba 1’; and low-TT (J), ‘Baihuayushizi’).  The results of our study provide a reference for the development and utilization of wild pomegranate resources and pomegranate breeding in China.

CCCH (C3H) Zinc finger (Znf) transcription factors (TFs), as a novel type of Znf gene, regulate the expression of genes by binding to their mRNAs and play important roles in plant growth and development and abiotic stress resistance.  Longan (Dimocarpous longan) is a tropical/subtropical fruit tree of great economic importance in Southeast Asia.  However, genomic information on C3H and their functions in longan are still unknown.  In this study, a comprehensive analysis of the longan C3H (DlC3H) gene family was carried out.  A total of 49 DlC3H genes in three clades were identified from the longan genome database.  Characteristics of the genes were analyzed with respect to gene structure, motif composition, phylogenetic tree and potential functions.  The analysis of alternative splicing (AS) events suggested that AS events in DlC3H genes were related to the transformation from longan non-embryonic to embryonic cultures.  Promoter analysis indicated that most of the DlC3H genes included cis-acting elements associated with hormones and stresses responses.  Quantitative real-time PCR (qRT-PCR) analysis indicated that 26 of the 49 DlC3Hs, which possess methyl jasmonate (MeJA) and abscisic acid (ABA) responsive cis-acting elements, showed differential expression patterns under treatment with ABA, MeJA and their endogenous inhibitors, suggesting that DlC3Hs might be involved in the ABA and MeJA signaling pathways.  The expression profiles of 17 of the 49 DlC3Hs in non-embryonic callus and three tissues of embryonic cultures showed that only five of the 17 DlC3Hs had the same expression trends as the FPKM trends in transcriptome data; the expression levels of DlC3H07/14/16/36/49 in embryogenic callus and DlC3H04/38 in globular embryos were high, suggesting that they have different functions in embryonic development.  Further, we verified that DlC3H01/03/05/11/19/39 were regulated by sRNAs by a modified 5´ RLM-RACE method.  This study provides the first systematic analysis of C3H genes in longan, and found that C3H genes may be involved in hormone and stress responses, and somatic embryogenesis.  Our preliminary investigation may provide clues to further studies on the characteristics and functions of this family in longan.

 

Blumeria graminis f. sp. tritici, the pathogen that causes wheat powdery mildew, is one of the most important diseases affecting wheat production in China, and the oversummering is the key stage of wheat powdery mildew epidemic.  The more oversummering regionalization of wheat powdery mildew has played an important role in disease prediction, prevention and control.  In this study, we analyzed the correlation between oversummering data of wheat powdery mildew and the meteorological factors over the past years, and determined that temperature was the key meteorological factor influencing oversummering of wheat powdery mildew.  The average temperature at which wheat powdery mildew growth was terminated (26.2°C) was used as the threshold temperature to regionalize the oversummering range of wheat powdery mildew.  This regionalization was done using the GIS ordinary kriging method combined with the Digital Elevation model (DEM) of China.  The results showed that annual probability of oversummering region based on Model 26.2 were consistent with the actual survey of the more summer wheat powdery mildew.  Wheat powdery mildew oversummering regions in China mainly cover mountainous or high-altitude areas, and these regions form a narrow north-south oversummering zone.  Oversummering regions of wheat powdery mildew is mainly concentrated in the high-altitude wheat growing areas, including northern and southern Yunnan, northwestern Guizhou, northern and southern Sichuan, northern and southern Chongqing, eastern and southern Gansu, southeastern Ningxia, northern and southern Shaanxi, central Shanxi, western Hubei, western Henan, northern and western Hebei, western Liaoning, eastern Tibet, eastern Qinghai, western Xinjiang and other regions of China.

Genomic selection has been demonstrated as a powerful technology to revolutionize animal breeding.  However, marker density and minor allele frequency can affect the predictive ability of genomic estimated breeding values (GEBVs).  To investigate the impact of marker density and minor allele frequency on predictive ability, we estimated GEBVs by constructing the different subsets of single nucleotide polymorphisms (SNPs) based on varying markers densities and minor allele frequency (MAF) for average daily gain (ADG), live weight (LW) and carcass weight (CW) in 1 059 Chinese Simmental beef cattle.  Two strategies were proposed for SNP selection to construct different marker densities: 1) select evenly-spaced SNPs (Strategy 1), and 2) select SNPs with large effects estimated from BayesB (Strategy 2).  Furthermore, predictive ability was assessed in terms of the correlation between predicted genomic values and corrected phenotypes from 10-fold cross-validation.  Predictive ability for ADG, LW and CW using autosomal SNPs were 0.13±0.002, 0.21±0.003 and 0.25±0.003, respectively.  In our study, the predictive ability increased dramatically as more SNPs were included in analysis until 200K for Strategy 1.  Under Strategy 2, we found the predictive ability slightly increased when marker densities increased from 5K to 20K, which indicated the predictive ability of 20K (3% of 770K) SNPs with large effects was equal to the predictive ability of using all SNPs.  For different MAF bins, we obtained the highest predictive ability for three traits with MAF bin 0.01–0.1.  Our result suggested that designing a low-density chip by selecting low frequency markers with large SNP effects sizes should be helpful for commercial application in Chinese Simmental cattle.

The aim of this study was to evaluate the growth of rape (Brassica napus L.) seedlings under different light intensities to select appropriate conditions for cultivation in an indoor system.  Seedlings were grown under different light intensities of red and blue light provided by light-emitting diodes (LEDs) and their self-adjustment ability and changes in leaf microstructure were evaluated.  Light was supplied by red LEDs with peak wavelengths of 630 (R₁) and 660 nm (R₂) and by blue LEDs (B) with a peak wavelength of 445 nm (the light intensity ratio of R₁:R₂:B was 3:3:2), at intensities of 400 (R₁R₂B400), 300 (R₁R₂B300), and 200 μmol m^–2 s^–1 (R₁R₂B200).  Natural solar light served as the control (C).  Plant height, stem diameter, root length, leaf area, and dry weight of rape seedlings gradually increased with increasing light intensity.  The seedlings in the R₁R₂B400 treatment grew more vigorously, while those in the R₁R₂B200 treatment were weaker.  The photosynthetic pigment contents did not differ significantly between the R₁R₂B400 treatment and C, but were significantly lower in the R₁R₂B300 and R₁R₂B200 treatments.  The highest intercellular CO₂ concentration, stomatal conductance, and transpiration rate were in the R₁R₂B300 treatment.  The highest photosynthetic rate was in the R₁R₂B400 treatment, and was related to more compact leaves, thicker and tidier palisade and spongy tissues, and well-developed chloroplasts.  In contrast, the seedlings in the R₁R₂B200 treatment had disordered mesophyll cells, round chloroplasts, and fractured and fuzzy grana lamellae, all of which inhibited plant growth.  In conclusion, the seedlings in the R₁R₂B400 treatment had well-developed leaves, which favored photosynthesis.  Compared with the light intensities below 300 μmol m^–2 s^–1, the light intensity of 400 μmol m^–2 s^–1 provided by a combination of red and blue LEDs was beneficial for cultivating strong and healthy rape seedlings in an artificial system.  

    The antioxidant of reduced glutathione (GSH) is the most abundant thiol in cells for the maintenance of the intracellular redox balance. The study aimed to assay the effect of sperm treatment with GSH before incubation with oocytes on the development potential of embryos obtained by in vitro fertilization (IVF). Also the mitochondrial membrane potential (ΔΨm), plasma membrane integrity (viability), DNA fragmentation, reactive oxygen species (ROS) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, methane dicarboxylic aldehyde (MDA) level as indices of lipid peroxidation in sex-sorted and unsorted sperm from three bulls were investigated using flow cytometry and enzyme-labeled instrument individually. The results showed that 2 mmol L^–1 GSH increased significantly the cleavage rate (86.68% vs. 82.78%), 4- to 8-cell rate (82.30% vs. 73.43%) and blastocyst rate (43.15% vs. 35.24%) of IVF embryos compared with untreated group. Furthermore, addition of GSH increased significantly the ΔΨm and viability, decreased the ratio of DNA fragmentation in sex-sorted or unsorted semen (P<0.05), except the sex-sorted semen from bull 019. Similarly, activities of SOD, CAT and GPx were increased significantly. However, the contents of MDA were decreased significantly both in sex-sorted and unsorted semen treated with GSH (P<0.05). These results suggest that sperm pretreatment with GSH during IVF can maintain better the viability and fertility of sperm through reducing apoptosis and increasing the antioxidant capacity, which improves the IVF embryos development.

  Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are essential components required for normal cellular function and have been shown to have important therapeutic and nutritional benefits in humans. But humans or mammals cannot naturally produce ω-3 PUFAs, due to the lack of the ω-3 fatty acid desaturase gene (fat-1 gene). Previously, fat-1 gene has been cloned from Caenorhabditis elegans and transferred into mice, pigs and sheep, but not yet into beef cattle. We attempt to transfer it into beef cattle. The object of this paper is to edit the fat-1 gene from C. elegans to express more efficiently in beef cattle and verify its biological function in mice model. As a result, the fat-1 gene from C. elegans was modified by synonymous codon usage and named it Bfat-1. We have demonstrated that degree of codon bias of Bfat-1 gene was increased in beef cattle. Moreover, Bfat-1 gene could be transiently expressed in mouse liver and muscle, the ω-6/ω-3 PUFAs ratio of 18 and 20 carbon was decreased significantly in liver (P<0.05), and the ratio of 20 carbon decreased significantly in muscle 24 and 72 h after injection (P<0.05). This confirms that the Bfat-1 gene modification was successful, and the protein encoded was able to catalyze the conversion of ω-6 PUFAs to ω-3 PUFAs.

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

In order to clarify the impact posed by wheat powdery mildew (Blumeria graminis f. sp. tritici) on the yield and yield components in different epidemic seasons, field trials were conducted in three growing seasons, 2009-2010, 2010-2011 and 2011-2012, in Langfang City, Hebei Province, China. The relationships between 1000-kernel weight, crude protein content of grain and yield and disease index (DI), as well as area under disease progress curve (AUDPC) were studied. The models of the percentage of loss of 1000-kernel weight, crude protein content and yield were constructed using DI at critical point (CP) of growth stages (GS) and AUDPC in the three growing seasons, respectively. The CPs for estimating 1 000-kernel weight, crude protein content of grain and yield of wheat caused by powdery mildew were GS 11.1, GS 10.5.3 and GS 10.5.3, respectively. Models based on DI at CP to estimate the percentage of loss of 1 000-kernel weight, crude protein content of grain and yield were better than models based on AUDPC. And models of the percentage of loss of 1 000-kernel weight, crude protein content and yield for 2011-2012 season were significant different from these for 2009-2010 and 2010-2011 seasons. These results indicated that besides powdery mildew, weather conditions also had influence on 1 000-kernel weight, crude protein content of grain and yield loss of wheat when powdery mildew occurred.

Wheat (Triticum aestivum L.) often experiences photoinhibition due to strong light during the grain filling stage. As such, increasing the tolerance of wheat to photoinhibition is very desirable in breeding efforts focused on increasing grain yields. Previous reports have suggested that PROTON GRADIENT REGULATION 5 (PGR5) plays a central role in the generation of a proton gradient across the thylakoid membrane (DpH) and in acclimation to high light intensity conditions. Three PGR5 homoeologues were isolated from wheat, and mapped onto chromosomes 7A, 7B and 7D, respectively. The TaPGR5s shared highly similar genomic sequences and gene structures. The transcripts of TaPGR5s were found to be abundantly expressed in the flag leaves, and were transiently up-regulated by treatment with high light. High light treatment inhibited the net photosynthetic rate (Pn) and the maximal quantum yield of photosystem II (Fv/Fm). Further, these inhibitions were more evident in the leaves with reduced expression of TaPGR5s achieved using virus-induced gene silencing methods. Moreover, reducing TaPGR5 expression impaired the induction of non-photochemical quenching (NPQ), which caused more severe cell membrane damage and lipid peroxidation in high light. Additionally, we observed that TaPGR5s transcripts were more abundantly expressed in the wheat genotypes with higher ms-delayed light emission (ms-DLE), a value reflecting transthylakoid DpH. These results suggested that TaPGR5s play important roles in the tolerance of wheat to photoinhibition.

Phosphorus (P) is an essential element for plant growth and yield. Improving phosphorus use efficiency of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. Soybean (Glycine max (L.) Merr.) is sensitive to phosphorus (P) in the whole life history. Soybean cultivars with different P efficiencies were used to study P uptake and dry matter accumulation under different P levels. Under low P conditions, the P contents of leaf in high P efficiency cultivars were greater than those in low P efficiency cultivars at the branching stage. The P accumulation in stems of high P efficiency cultivars and in leaves of low P efficiency cultivars increased with increasing P concentration at the branching stage. At the late podding stage, the P accumulation of seeds in high and low P efficiency cultivars were 22.5 and 26.0%, respectively; and at the mature stage were 69.8 and 74.2%, respectively. In average, the P accumulation in whole plants and each organ was improved by 24.4% in high P efficiency cultivars compared to low P efficiency cultivars. The biomass between high and low P efficiency cultivars were the same under extended P condition, while a significant difference was observed at late pod filling stage. At the pod setting stage, the biomass of high P efficiency cultivars were significant greater (17.4%) than those of low P efficiency cultivars under high P condition. Meanwhile, under optimum growth conditions, there was little difference of biomass between the two types of cultivars, however, the P agronomic efficiency and P harvest index were significant higher in high P efficiency cultivars than those in low P efficiency cultivars.

Super high-yielding soybean cultivar Liaodou 14, soybean cultivars from Ohio in the United States, and the common soybean cultivars from Liaoning Province, China, with similar geographic latitudes and identical pod-bearing habits were used as the study materials for a comparison of morphological traits and production characteristics to provide a theoretical basis for the breeding of improved super high-yielding soybean cultivars. Using a randomized block design, different soybean cultivars from the same latitude were compared under conventional and unconventional treatments for their production characteristics, including morphological traits, leaf area index (LAI), net photosynthesis rate, and dry matter accumulation. The specific characteristics of the super high-yielding soybean cultivar Liaodou 14 were analyzed. The results showed that the plant height of Liaodou 14 was significantly lower than that of the common cultivars from Liaoning, whereas the number of its main-stem nodes was higher than that of the cultivars from Ohio or Liaoning. A high pod density was observed in Liaodou 14 under conventional treatments. Under both conventional and unconventional treatments, the branch number of Liaodou 14 was markedly higher than that of the common cultivars from Liaoning, and its branch length and leaf inclination angle were significantly higher than those of common cultivars from Liaoning or Ohio. Only small changes in the leaf inclination angle were observed in Liaodou 14 treated with conventional or unconventional methods. Under each treatment, Liaodou 14 exhibited the lowest amplitude of reduction in SPAD values and net photosynthesis rates from the grain-filling to ripening stages; the cultivars from Ohio and the common cultivars from Liaoning exhibited more significant reductions. Liaodou 14 reached its peak LAI later than the other cultivars but maintained its LAI at a higher level for a longer duration. Under both conventional and unconventional treatments, Liaodou 14 produced a higher yield than the other two cultivars, with significant differences from the Ohio cultivars. In summary, super high-yielding soybean cultivars have several main features: suitable plant height, high pod density, good leaf structure with strong functionality, and slow leaf senescence at the late reproductive stage, which is conducive to the accumulation of dry matter and improved yield.

Wheat dwarf bunt, caused by Tilletia controversa Kühn (TCK), is an important quarantine wheat disease throughout the world. Based on published research results of the biology and the epidemiology of the disease, the main factors including temperature, humidity, snow cover, and their parameters relating to teliospore germination, infection and epidemics of TCK were determined. The geophytopathological models for the risk analysis of wheat dwarf bunt establishment were modified. Fifty-year meteorologic data from about 500 weather stations in China were used to calculate the probabilities of TCK establishment in different geographic sites with the models. The map that displays the establishment risk of TCK in winter wheat growing regions in China was generated by using geographical information system (GIS). The zones showing high, moderate, low, and very low, including no risk, of TCK establishment accounted for 27.33, 27.69, 38.12, and 6.86% of total winter wheat growing areas in China, respectively. These results will provide useful information to formulate quarantine regulations and wheat importation policy in China.

The ultra-structure of mother and outer daughter scales of Lilium Oriental hybrid Sorbonne were studied using transmission electron microscope to examine the sub-cellular localization of starch and lipid droplets during growth and development from shoot emergence to senescence. The contents of starch granules and lipid droplets in the cell of the mother scales decreased significantly from shoot emergence to anthesis, indicating that these scales served as a source for growth and development. After flowering, the number of starch granules and lipid droplets increased dramatically, and finally the cells were filled with the above molecules indicating that the bulb becomes a major sink during bulb enlargement. Ultrastructure observation also showed that symplastic pathway is the main pathway in cells in the exchange and transportation of material during bulb development. The activity of β-amylase, one of the key enzymes catalyzing starch breakdown, showed a similar trend. The enzyme sub-cellular localization via immune-gold electron-microscopy showed that β- amylase was predominantly located together with starch granules, while the gold particles were scarcely found in other sub-cellular compartments. The result suggested that this enzyme is compartmented together with its functional substrate supporting its function in catalyzing starch breakdown in living plant cells.

EST-derived SSR marker has been developed in many species, but few methods of high efficiency have been reported for the exploitation of EST-SSR markers. Thus, a high efficiency method for mining millions of redundant EST data is needed. A modified method for the EST-SSR development with high efficiency was established based on the redundant EST data of soybean in this study. The method achieved its function through classifying ESTs according to the same SSR motif and detected candidate loci with redundant sequences. In this study, a total of 80 polymorphic EST-SSR markers of soybean were developed, 50 of them were exploited by this modified method which proved the higher speed and efficiency of this method. All the 80 polymorphic EST-SSRs were mapped on soybean physical map through in silico mapping and 15 markers were integrated on a genetic map constructed in previous study. A software named hpSSR (high polymorphic SSR) was programmed based on the concept of the up-built method for EST-SSR development. This method is not only pragmatic for EST-SSR exploitation in soybean, but also effective for the development of the marker in other species if the redundancy EST data is available.

To our knowledge, no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) by controlled freezing and vitrification. This experiment, therefore, was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification. Adenosine-5´-triphosphate (ATP) content and reactive oxygen species (ROS) level in blastocysts were also analyzed. Firstly, for each type of blastocyst (IVF, ICSI or SCNT), significant differences were observed between the survival rates of the controlled freezing ((81.56±2.33), (68.18±4.72) or (47.89±5.83)%) and OPS vitrification groups ((92.24±4.54), (82.40±3.76) or (78.71±5.91)%; P<0.05). Secondly, for each type of blastocyst (IVF, ICSI or SCNT), ATP content was significantly decreased after controlled freezing or vitrification, and the ATP content in the controlled freezing group (0.43±0.06), (0.35±0.05) or (0.21±0.02) pmol) was significantly lower than that found in the OPS vitrification group (0.62±0.04), (0.46±0.03) or (0.30±0.01) pmol; P<0.05). Thirdly, ROS level in fresh IVF ((47.33±3.56) c.p.s (counted photons per second), ICSI ((36.51±2.58) c.p.s) or SCNT blastocysts ((26.44±1.49) c.p.s) was significantly lower than that found in the OPS vitrification group ((72.14±4.31), (58.89±3.89) or (40.11±5.73) c.p.s; P<0.05), but higher than that of the controlled freezing group (34.41±3.32), (23.13±1.26) or (15.46±2.45) c.p.s; P<0.05). The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF, ICSI or SCNT than controlled freezing. Furthermore, both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.

The aim of this study is to reveal the regulation mechanism of the effect of Semen vaccariae and Taraxacu mogono on the cell-cell adhersion molecule, E-cadherin and b-catenin on the proliferation role and secretion function of bovine mammary epithelial cells cultured in vitro. Firstly, the epithelial character of bovine mammary epithelial cells was authenticated using immunofluorescence, then the cell grow curve was observed and investigated after S. vaccariae and T. mogono treatment. On the effect of S. vaccariae and T. mogono, cell adhesion molecules E-cadherin, b-catenin and CycinD1 mRNA and protein were detected by qRT-PCR and Western blotting, respectively. The results showed that the cellular keratin 18 expressed positively and proliferated vigorously after S. vaccariae and T. mogono treament. The mRNA and protein levels of E-cadherin and CycinD1 were remarkably higher (P<0.05) in 36 h after S. vaccariae and T. mogono treatment. The cell proliferation at 36 h was increased significantly (P<0.05). In conclusion, S. vaccariae and T. mogono have a positive impact on the cell proliferation and an effect on the adhesion molecules E-cadherin, b-catenin and CycinD1 in the Wnt signaling pathway.

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression (SAGE) tags for gene identification (GLGI) to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5´-end of RNA transcript (SMART) technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

In previous experiment, we isolated a compound dibutyl phthalate (DBP) from Vaccaria segetalis which had galactopoieticfunction on mammary gland epithelial cells of dairy cow (DCMECs). In this experiment, we ascertained the metabolicregulation function of DBP on DCMECs. Many genes related to lactation including Stat5, AMPK, â-casein, Glut1, SREBP-1,PEPCK, and ACC were detected by real-time PCR. Furthermore, Stat5 and AMPK were detected by Western blot andimmunofluorescence co-localization, respectively. The results showed that DBP stimulates the expression of Stat5 andp-Stat5, thus activates Stat5 cell signal transduction pathway and stimulates â-casein synthesis. DBP also raises theactivities of Glut1 and AMPK to stimulate glucose uptake and glycometabolism and activates the expression of AMPKdownstream target genes PEPCK and ACC and expression of SREBP-1 to stimulate milk fat synthesis. In addition, theactivities of HK, G-6-PDH, ICDH, ATPase, and energy charges were stimulated by DBP to increase the energy metabolismlevel of DCMECs. The results showed DBP stimulates energy metabolism related to galactopoietic function in DCMECs.