Gao Xue-jun

Contents of Trace Metal Elements in Cow Milk Impacted by Different Feedstuffs

Abstract Feedstuff intake plays key role in impacting the yield and quality of milk. In this study, we discussed the contents of trace metal elements in cow milk impacted by different feedstuffs. We detected the contents of Pb, Cd, As, Cu, Mg, Ca, Fe and Zn in different feedstuffs and milk by atomic absorption spectrophotometry. The determinations of Pb, Cd and As contents were by graphite furnace AAS and Cu, Mg, Ca, Fe and Zn was by flame atomic absorption spectrometry. The results showed that Pb, Cd, As and Cu presented in feedstuffs, but Pb, Cd and As were lowly detected in milk samples, and Cu was not detected in milk samples. The content of Mg in concentrates was lower than that in forages. However, the content of Mg in milk from concentrate feed was higher than that in forage feed. This suggested that the utilization of Mg in concentrate feed was higher than that in forage feed. The contents of Ca and Zn were opposite to Mg, and the utilization of Ca and Zn in forage feed was higher than that in concentrate feed. There was no obviously change law of Fe in different feedstuffs and milk samples. The relationship between the contents of trace metal elements in milk to feedstuffs would provide a theoretical basis for dairy farming. It would be useful for improving the milk yield and milk quality of dairy production.

Application of Protein Feed Processed by Microbial Fermentation to Dairy Cow

Methionine (Met) and lysine (Lys) have been reported as the first two limiting amino acids (AA) for maximum milk yield and milk protein production. Supplying these AA may improve microbial protein synthesis and therefore improve milk production without adding excess N to the environment. This observation utilized fermented soybean meal (SBM), cottonseed meal (CSM), rapeseed meal (RSM) and corn by Bacillus subtilis 168 and Leuconostoc mesenteroides as core feedstuffs to produce special biological protein feed for dairy cow. The results showed that the milk production, milk protein percentage, milk fat percentage and milk DM percentage of test groups in trial period were significantly more than those of the control group (P<0.01), the results showed that adding fermenting protein feed in dairy cow diets could significantly improve milk yield, milk protein and milk fat content. The economic benefits of actual application were analyzed, the group of 0.5% was the best compared with the other groups.

Proteomic Identification of Differentially Expressed Proteins in Vaccaria segetalis-treated Dairy Cow Mammary Epithelial Cells

To investigate the potential effects of Vaccaria segetalis, we employed the proteomic approach on the dairy cow mammary gland epithelial cells (DCMECs). A total of 35 differentially expressed nuclear proteins were visualized by 2-DE and silver nitrate staining. Of these, five proteins also displayed significant expression changes upon treatment of the water decoction from Vaccaria segetalis, and such alterations were further confirmed by RT-PCR. Together, at both the mRNA and protein levels, the water decoction from Vaccaria segetalis increased the expression of proteins ethylmalonic encephalopathy 1 (ETHE1), vesicle amine transport protein 1 homolog (VAT1), parkinson disease protein 7 homolog (Protein DJ-1), proteasome subunit alpha type-2 (PSMA2) and SUMO-activating enzyme subunit 1 (SAE1). This study would enable a better understanding of the molecular mechanisms underlying this water decoction effects at the protein level.

Expression of a Lysine-rich Gene in Bacillus subtilis 168

Lysine-rich protein gene (lys) was cloned from winged bean (Psophocarpus tetragonolobus (L.) DC), and cloned into prokaryotic expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

Abstract TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate C t value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained C t into the standard curve equation. The standard curve equation of endogenous reference gene was y =−3.422x+35.201, R 2 =0.998; the standard curve equation of exogenous gene was y =−3.495x+35.303, R 2 =0.999. The sample copies was got by putting C t value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

Study on Lysine and Methionine Content Promotion of Soybean Meal by Probiotic Fermentation Process

Abstract Soybean meal (SBM) is commonly used for livestock feeds, but its application in diets for livestock is limited due to some antinutritional factors. The contents of methionine and lysine of soybean meal were promoted by Bacillus natto and Leuconostoc mesenteroides fermentation, benefial for the livestock feeds. It was crude protein (CP) 56.8%, methionine 43.56 mg • g −1 , and lysine 74.87 mg • g −1 , cows fed a diet with FSBM milk yield raised 14.2%, the change in the milk protein, the lactose and the dry matter content had also obvious increase. This convenient technique offers helpful exploration for industrialization of soybean meal fermentation.

Expression and localisation of glucose transporter 1 (GLUT1) in dairy goat mammary gland at different physiological stages

Glucose is the major energy source for mammary epithelial cells, as well as an important substrate for lactose synthesis. Mammary epithelial cells take up glucose from extracellular fluid into the cell through glucose transporter (GLUT). This study was aimed at investigating the expression of GLUT1 glucose transporter in dairy goat mammary gland during puberty, pregnancy, lactation, and involution. Using real-time reverse transcription PCR (qRT-PCR) and Western blotting, we analyzed the expression of GLUT1 mRNA and protein in dairy goat mammary gland. GLUT1 mRNA and protein expression increased during pregnancy and lactation, especially at peak lactation, and decreased strongly after weaning. Furthermore, the location of GLUT1 protein was determined by immunofluorescence laser confocal microscopy. GLUT1 protein localised to the basal and apical plasma membrane of epithelial cells, and also in the cytoplasm. The results from this study showed that GLUT1 is expressed in the dairy goat mammary gland with the...

SYBR® Green qPCR Screening Methods for Detection of Anti-herbicide Genes in Genetically Modified Processed Products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of GMOs are necessary in order to verify compliance with labelling requirements. There are few effective screening methods for processed GM (genetically modified) products. Three anti-herbicide genes (CP4-EPSPS, BAR and PAT) are common exogenous genes used in commercialized transgenic soybean, maize and rice. In the present study, a new SYBR® Green qPCR screening method was developed to simultaneously detect the three exogenous anti-herbicide genes and one endogenous gene in a run. We tested seven samples of representative processed products (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, maize protein powder, maize starch, and maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products, and the sensitivity was 0.1%. These results indicated that SYBR® Green qPCR screening method was appropriate for qualitative detection of transgenic soybean, maize and rice in processed products.

Potential Genes for Regulation of Milk Protein Synthesis in Dairy Goat Mammary Gland

Abstract The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages: days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution (three animals per group). Mammary tissue RNA was isolated for quantitative real-time RT-PCR of four casein genes alpha-s1 casein (CSN1S1), alpha-s2 casein (CSN1S2), beta-casein (CSN2) and casein kappa (CSN3), four whey protein genes lactoglobulin (LGB), lactalbumin (LALBA), lactofarrin (LTF), and Whey acidic protein (WAP) and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor (PRLR), AKT1, signal transducers and activators of transcription 5 (STAT5), E74-Like Factor 5 (ELF5), eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1), S6kinase (S6K) and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.

Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene in Bacillus natto

Abstract Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene ( zein ) from maize endosperm protein with lysine-rich gene ( Cflr ) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43 /zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto , and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.