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Background Ideal cardiovascular health (CVH) is related to a low cardiovascular disease risk profile. This study aimed to investigate CVH metrics with both the biomarkers and markers of subclinical impairments of cardiovascular diseases (CVDs) in subjects from central south China. Methods The ideal CVH score (non-smoking status; ideal body mass index; regular physical activity; healthy diet; and an optimal serum cholesterol, blood pressure, and glucose profile; 1 point for each; total score: 0–7) was collected from 3009 participants without a history of CVDs. Subclinical biomarkers were assessed using C-reactive protein, homocysteine, and microalbuminuria. The presence of subclinical disease markers was defined as having at least one of the following: increased carotid intima-media thickness, carotid plaque, left ventricular hypertrophy, left ventricular systolic dysfunction, or a reduced ankle-brachial index. The association of biomarkers and markers with the CVH score was evaluated using multivariate logistic regression and linear regression analyses. Results Only 0.2% of the study participants met all 7 ideal CVH metrics (CVH score = 7). Compared to the female participants, the male participants had poorer CVH profiles and a higher incidence of subclinical lesions (P

The design for traditional gantry planer aims at its empty stroke and low work efficiency of the two fatal flaws in this paper. We adopted the structure innovation by mechanical design and designed the gantry planer bidirectional shaping turret. And the device is simple in structure and easy in manufacture. These make improved gantry planer remove empty stroke and change one-way shaping into two-way shaping. Thus the improved gantry planer can depress energy cost consumedly and improve production efficiency. This will broaden its market greatly in the machining industry.

The design for ordinary garage aims at its small and its insufficient space of goods storage of the flaws and even only a private car can be parked. We adopted the bold structure innovation and designed the Dual Power mechanical simple garage lifting platform. And the device is simple in structure and easy in manufacture with high stability. And goods of storage is convenient which one can take it when wants to use. And two cars can been parked in the auto conditions permitting. Thus it can make the ordinary garage maximize the use of space and improve the utilization of space resources.

The differential equations of bending-torsional coupled vibration have been established with a switching crack for a Jeffcott rotor system with a transverse crack as the research object. Variable perturbation method has been used to study the stability of bending-torsional coupled vibration of a rotor system with a crack. The theoretical result shows that, when it is close to the natural frequency of 2 times, 3/2 times and 1 times, unstable vibrations exist and conditions of stable vibration. According to the above order, unstable region becomes narrow. While in 1/4 times, 1/6 times, the system is stable. The conclusions provide valuable theoretical basis for a crack rotor system in the real working conditions when corresponding measures need taking.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 GT, rs 136823901 and g.-692 CT, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P0.05) at the SNP site g.-556 GT, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P0.05). To further study the possible mechanisms by which g.-556 GT and g.-692 CT affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 GT and g.-692 CT are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

Complement component 4 (C4A) is a candidate gene that reflects complement activity. The primary role of this gene in the classical and lectin-activation pathways is to provide protection against bacterial pathogens. In the current study, the bovine complement C4A gene was screened for polymorphisms, and the associations of these polymorphisms with the hemolytic activity of the classical pathway (CH50), C4 serum levels, and milk performance traits were examined. Three novel single-nucleotide polymorphisms (rs 132741478: g.2994 AG, rs 134006517: g.3508 AG, and rs 137485678: g.3649 GC) were detected by DNA sequencing and PCR-RFLP in 1182 Chinese Holstein cows. The rs 132741478: g.2994 AG mutation in exon 10 led to methionine and valine exchange at position 362, whereas rs 134006517: g.3508 AG and rs 137485678: g.3649 GC were synonymous substitutions. The statistical analyses revealed that cows with rs 132741478: g.2994 AG-AG and rs 137485678: g.3649 GC-CC have significantly lower somatic cell scores (SCS, P0.01). Homozygote cows with GAC haplotypes have the lowest SCS, whereas AAG/AAC cows have the highest. The serum concentration of C4 by ELISA and the hemolytic and antibacterial activity of CH50 were also evaluated in the current study. The results confirmed that rs 132741478: g.2994 AG in the coding sequence of the β-chain of the bovine C4A gene is related to mastitis resistance. This polymorphism may be very important in marker-assisted selections in dairy cattle breeding programs.

The implication of the revelation of the inverse leaf aging sequence (ILAS) of wheat needs to be probed in theory and practice. Since 2005, the comparison experiment of the ILAS and the conventional leaf aging sequence (CLAS) has been carried out to measure the canopy temperature and some important biological parameters of wheat. In nature, there existed the phenomenon that the wheat leaves aged in an sequence opposite to the conventional sequence and some of the leaves of ILAS wheat aged sequentially differed from those of CLAS wheat, i.e., the penultimate leaves rather than the flag leaves aged last among the leaves at different leaf positions; in correspondence with the inverse leaf aging sequence, there appeared an unconventional leaf color structure at the late fruiting stage, which had yellow upper leaves and green lower leaves, opposite to the conventional leaf color structure that had green upper leaves and yellow lower leaves; the chlorophyll concentrations, soluble protein concentrations, transpiration rates, and net photosynthetic rates of the penultimate leaves of ILAS wheat unconventionally surpassed those of their flag leaves as their growth moved forward from one stage to another stage, and the ILAS wheat characteristically presented cold canopy temperature, i.e., a cold tail canopy temperature at the late fruiting stage, or a cold canopy temperature at the whole fruiting stage; because ILAS wheat was unique in physiological process, its kernel weights were obviously higher than those of CLAS wheat, which closely related to the “relay” kernel-filling mode, which was different from the kernel-filling mode of CLAS wheat under which the flag leaves act as the main nutrient supply source at the whole fruiting stage. This study provided a new idea and approach for the theoretical exploration on wheat fruiting and aging, wheat yield further improvement, and cold type wheat and cold tail wheat breeding.

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P >0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P

The polymorphisms of HSP70-1 gene in 253 Chinese Holstein dairy cows were studied, and the association between the polymorphisms and somatic cell score (SCS) were analyzed. PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the coding region of HSP70-1 gene. The G-->A-->C mutation at 1 623 bp and G-->A mutation at 2 409 bp were found and both of them were silence mutations that caused no alteration in amino acid sequence. Chi-square test showed both loci were'nt at Hardy-Weinberg disequilibrium in Chinese Holstein. In the meanwhile, the association of 2 409 locus and SCS was not significant. However, the polymorphism at 1623 locus affected SCS significantly (P

In this research, PCR-SSCP technique was used to analyze the polymorphisms of the exon 11 of Nramp1 gene in Chinese Holstein cattle (n=344), and correlation between polymorphisms of Nramp1 with somatic cell score (SCS) and milk production traits was analyzed. The results show that three genotypes namely AA, BB, and AB were detected. Allele A was predominant and the frequencies of alleles A and B were estimated to be 0.767 and 0.233, respectively. Chi-square test indicated that the polymorphic locus in Chinese Holstein fitted Hardy-Weinberg equilibrium (P>0.05). Sequencing analysis showed two polymorphic sites at positions 200 bp (C/G) and 254 bp (T/G), which resulted in amino acid alteration Ala356Pro and Leu374Met. The least squares means of SCS in Holstein cattle was lower for genotype AA than that for genotypes AB and BB (P

Complex vertebral malformation (CVM), a lethal autosomal recessive inherited defect in Holstein calves, was newly reported worldwide. The molecular cause of CVM was a substitution of guanine by thymine (G-->T) in a solute carrier family 35 member 3 gene (SLC35A3), encoding UDP-N-acetylglucosamine transporter. It was characterized by stillborn, abortion, and premature birth. The objective of this study was to study the actual carrier frequency of the CVM mutation in a population of Chinese Holstein (=Chinese Black-and-White) normal cattle. The normal 436 Holstein cows and 93 Holstein bulls were genotyping by using the Created Restriction Site PCR (CRS-PCR) and Allele-specific PCR (AS-PCR) methods. There were two bulls and one cow in three observed CVM-carriers. In the Holstein dairy cattle and Holstein bull population, the percentages of CVM carriers were estimated as 0.60% and 2.20% respectively. This study provided a more reliable and useful method for extensive screening of CVM and also offers a theoretical basis for molecular diagnosis in Holstein calves.

Low temperature wheat genotypes are a group of wheat with a slightly low canopy (plant) temperature, and the research on their biological characters and utilization in wheat breeding has been done at home and abroad for more than 20 years, and has made great progress. The research contents and advances include the following respects: Wheat genotypes with slightly low canopy temperature have been verified to exist in nature; these wheat genotypes, which present cold temperature, are superior to conventional wheat materials in some important biological characters and particularly prominently in metabolic function and cellular structure; when they suffer stresses such as drought, high temperature and overcast and rainy weather, they still retain their superiority in some of their important biological characters and therefore have a wide range of ecological adaptability; slightly low canopy temperatures of these genotypes are closely correlated with low temperatures of their second heat sources and their vigorous plants; since their low canopy temperatures can be inherited, they can exert favorable influence on the temperatures of their offspring while crossing with other wheat materials, and in particular, the discovery of cold-source wheat as a contributor to low temperature, has further formed good conditions for breeding high and stable quality low temperature wheat varieties with a high and stable yield. Thus, low temperature wheat genotypes are of great research importance and have great prospects.

K-casein gene was regarded as a candidate gene for milk production traits of cows. In this study, a 779 bp fragment of k-casein gene of Chinese Holstein was amplified by polymerase chain reaction (PCR), the polymorphisms of three loci of k-casein gene were detected by PCR-RFLP with restriction endonuclease Taq, Hind, Pst. After sequencing, T/C single nucleotide polymorphism (SNP) was identified at nucleotide 10 891C/A SNP was identified at nucleotide 10 927 and G/A SNP was identified at nucleotide 10 988 in exon4 of k-casein gene. Both alleles (A and B) of three loci were found in the population that showed low polymorphism. The gene frequencies of A and B were 86.03% and 13.97%, respectively. The genotype frequencies of AA, AB, and BB were 73.71%, 24.63%, and 1.66%, respectively. Statistical results of c2 test indicated that three polymorphism sites in the population fitted with Hardy-Weinberg equilibrium (P > 0.05). Meanwhile, the effect of polymorphism of k-casein gene on milk production traits was analyzed. The results indicated that in the three loci, the different genotype of k-casein gene had no significant influence on milk yield and milk protein percent (P > 0.05). The cows with genotypes BB and AB showed higher milk fat percent than those with genotype AA ( P < 0.05 ) ; with genotype AB showed higher fat protein ratio than those with genotype AA ( P < 0.05 ). The polymorphism of the three loci in the experimental population is closely linked. The conclusion is that k-casein B allele can be used as the molecular genetic markers of modifying milk fat percent in Chinese Holstein cows.

The nucleotide sequence of the melanocortin-1-receptor (MC1R) gene was studied with the help of the polymerase chain reaction (PCR), in which the protein structure in Chinese Holstein was predicted, and the molecular mechanism of the red coat color was investigated. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to genotype the individuals. The bioinformatics and biotechnology softwares were used to predict the secondary structure of MC1R. The results showed that the EE genotype was the dominant genotype in Chinese Holstein Black and White herd, whereas, it was ee in Chinese Holstein Red and White herd. The secondary structure of the mutational MC1R protein was changed and the deletion mutation caused an earlier termination in translation, which led to the formation of the red coat color. The allele E was mainly associated with the black coat color, whereas, e was associated with red.

An HPLC method has been developed for the separation of new complexes of tetrakis(4-methoxylphenyl)porphyrin (TMOPP) with four heavy rare earth elements (RE = Y, Er, Tm, and Yb). The function of amine and acid in the mobile phase has been investigated and a reasonable explanation is presented. Successful separation of the RE-TMOPP-Cl complexes is accomplished in 10 min with a mobile phase consisting of methanol-water-acetic acid-triethanolamine. The detection limits (S/N= 3) for the four complexes are 0.01 microg/mL. This method is rapid, sensitive, and simple.

Six new thulium porphyrin complexes have been separated by high performance liquid chromatography with triethanolamine (TEOLA) and acetic acid (HAc) as additives in the methanol–water mixture. The function of TEOLA and HAc has been described. The whole analysis was completed on a C18 column in 16 min at a flow rate of 0.5 mL min−1, with the mobile phase of methanol–water (93:7,v:v) containing 75 mmol L−1 TEOLA and 200 mmol L−1 HAc. The detection limits (S/N = 3) of thulium porphyrins at 420 nm were 1.0, 1.0, 1.0, 1.0, 5.0, and 5.0 ng mL−1, respectively. Compared with the methanol system, it can attain the equivalent separation with the mobile phase of ethanol–water (90:10,v:v) containing 25 mmol L−1 TEOLA and 200 mmol L−1 HAc pumped at 0.25 mL min−1.

Objective To study the effects of methyl jasmonate on multidrug resistance in a mouse model of hepatocellular carcinoma. Methods Multidrug resistant H22 (H22/FAP) hepatocellular carcinoma cells were produced in vitro by continuous exposure to increasing doses of doxorubicin, cisplatin and 5-fluorouracil (FAP regimen). Cell toxicity was measured using the 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. Survival time was calculated for BALB/c mice that received intraperitoneal injections of H22/FAP cells followed by treatment with methyl jasmonate or verapamil in combination with FAP for 7 days. Adenosine triphosphate (ATP) hydrolysis was used to measure the activity of permeability-glycoprotein (P-gp) ATPase activity in plasma membranes. Results The MTT assay showed that methyl jasmonate significantly enhanced the cytotoxicity of the FAP regimen in multidrug resistant H22/FAP cells. Methyl jasmonate (10 mg/kg and 5 mg/kg) combined with FAP significantly increased survival time in BALB/c mice by 44.25% and 48.01%, respectively, compared with FAP. Methyl jasmonate increased P-gp ATPase activity. Conclusion The combined use of methyl jasmonate and the FAP regimen might be a novel strategy for overcoming the multidrug resistance often observed in hepatocellular carcinoma.

[This corrects the article DOI: 10.1371/journal.pone.0159719.].

The design and function of an amperometric dual-microelectrode is communicated which can operate as a collection sensor. The device consists of two individually addressable microelectrodes of parallel cylindrical geometry. Dual-cylinder microelectrodes have been prepared from micrometre platinum wire and carbon fibre to extremely good precision using a microscope fabrication technique. The shielding and feedback properties of the new microelectrode assembly have been evaluated with respect to electrode separation for the case of the hexacyanoferrate (iii)/(ii) couple. Carbon fibre (CF) devices with cylinder length of 585 μm are suitable for the cycling of organic redox couples between narrow gap electrodes. A discussion follows reporting the selective sensing of 1,4-dihydroxybenzene at a narrow gap CF device where the chemical and electrochemical effects of ascorbate interferences are largely filtered out to concentrations of 200μM.

To evaluate the use of Delorme procedure for full-thickness rectal prolapse.A series of 25 patients with full-thickness rectal prolapse were treated by Delorme procedure in four institutions between March 2005 and June 2010. The clinicopathological data were analyzed retrospectively.There were 9 males and 16 females. The mean age was 52(46-72) years old. All the procedures were successfully performed. There were no perioperative deaths. The mean operative time was 65(45-150) min. The intraoperative bleeding was 58(20-200) ml. The mean length of hospital stay was 8.5(5-14) days. Anastomosis dehiscence occurred in 1 patient at post-operative day 7 who was managed under anesthesia. Minor complications occurred in 8(32%) patients, including urinary retention(n=3), intractable pain(n=1), and bowel obstruction(n=4). The follow up time ranged from 2 to 6 years with a median of 3.5 years. Prolapse recurrence was observed in 1(4%) patient during the follow up. The remission rates of fecal incontinence, constipation, bleeding were 37.5%(6/16), 45.5%(5/11), and 15.4%(2/11), respectively. The Wexner incontinence score significantly decreased (median, 5.0 vs. 9.0, P0.01). The resting pressure and maximum squeeze pressure increased significantly after surgery, while the initial volume and maximal tolerance volume decreased significantly(All P0.01).Delorme procedure is safe and easy to perform. The anorectal function is improved after surgery. Therefore it should be considered the procedure of choice for rectal prolapsed.

The feedback and collection diffusion current properties of platinum dual-cylinder microelectrodes have been investigated using bipotentiostatic chronoamperometry. The study focuses on the influence of the interelectrode spacing on the steady-state response at independently controlled generator and collector microcylinders. A series of such devices were fabricated onto polyethylene terephthalate (PET) strips using a microscope manipulation method. Micro-manipulation techniques allowed dual-cylinder devices to be constructed using 25 µm platinum wire with gap spacings from 3.5 to 188 µm and cylinder lengths 250–500 µm. Recycling efficiencies, expressed as an ic/ig ratio, observed with these feedback dual-microelectrodes could be as high as 83 % for closely spaced microcylinders. Electrochemical measurements further indicate that a convergence of the generator and collector currents would follow as the interelectrode distance diminishes below the µm scale where the strength of the recycling flux field at the device is high enough to minimise bulk solution exchange. The response of the new amperometric device is discussed with reference to the relevant theoretical stationary flux model proposed for dual-cylinder microelectrodes.

Three novel SNPs were found by DNA sequencing, PCR-RFLP and CRS-PCR methods were used for genotyping in 979 Chinese Holstein cattle. One SNP, G1178C, was identified in exon 2 of POU1F1 gene. Two novel SNPs, A906G and A1134G, were identified in 5'-flanking regulatory region (5'-UTR) of PRL gene. The association between polymorphisms of the two genes and milk performance traits were analyzed with PROC GLM of SAS. The results showed that GC genotype at 1178 locus of POU1F1 gene was advantageous for milk yield, milk protein yield, and milk fat yield. AG genotype at 906 locus was advantageous for milk yield. There was no significant difference between 1134 locus and milk performance traits of 5'-UTR of PRL gene. Analysis of genotype combination effect on milk production traits showed that the effect of combined genotype was not simple sum of single genotypes and the effects of gene pyramiding seemed to be more important in molecular breeding.

Based on the NSP4 sequence of bovine rotavirus (BRV), the shRNA was designed and synthesized, and a shRNA recombinant lenti-virus vector RNAi-H1-89 was constructed. The recombinant RNAi-H1-89 Lenti-virus was packaged by transfecting the 293T cell with the recombinant vector RNAi-H1-89 and two helper plasmids using lipofectamine, and then used to infect MA104 cells. The MA104 cells were further infected with BRV strain G6 24h post-infection, with the LacZ shRNA recombinant lenti-virus as control. Thirty-six hours later, the CPE of the infected cells was observed under microscope, shRNA of NSP4 gene inhibited CPE in MA104 cell; the shRNA against NSP4 gene also inhibited NSP4 gene expression by RT-PCR, The virus titer in the cell culture supernatant was significant lower compared with the control group. The above results showed that RNAi-H1-89 against NSP4 gene could specifically silence NSP4 gene expression, and inhibit the proliferation of BRV.

Mannan-binding lectin (MBL), a pattern recognizing serum protein, participates in the innate immune system of mammals as an opsonin. In humans, single-nucleotide polymorphisms (SNPs) in MBL2 gene were found to cause various innate immune dysfunctions. In the present study, we discovered three single-nucleotide polymorphisms of the MBL1 gene in Chinese native cattle and analyzed their associations with milk traits. By screening the genetic variation of MBL1 in 1053 individuals of three Chinese native cattle breeds including China Holstein, Luxi Yellow and Bohai Black using created restriction site-polymerase chain reaction (CRS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing techniques, three new SNPs, g.855G>A, g.2651G>A and g.2686T>C, were found to have allele frequencies of 0-12.65%, 24.07-42.39% and 56.95-73.68%, respectively. While SNP g.855G>A is located within intron І, the other two SNPs reside in the exon II region with one mutation being non-synonymous (GTT (Val)>ATT (Ile)) and the other synonymous (GCT (Ala)>GCC (Ala)). Among the 596 Chinese Holstein cattle with at least 3 lactation Dairy Herd Improvement (DHI) records, eight different haplotypes and 19 genotype combinations were detected. Statistical analyses revealed no correlation between either g.855G>A or g.2686T>C and somatic cell score (SCS), however significant association was found between g.2651G>A and SCS, suggesting a possible role of this SNP in the host response against mastitis. Our data also suggested that the combined genotypes of GGC/AAC with the lowest SCS, AAT/AAT with the highest protein content and AGC/AGC with the highest 305-d milk yield were favorable combinations for mastitis resistance and milk production traits. Therefore, GGC/AAC, AAT/AAT and AGC/AGC can be used as possible candidates for marker-assisted selection in the dairy cattle breeding program.

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P0.01) or AB (P0.05). In position -478(/G), the cow with genotype CC showed significantly lower SCS in contrast to cow with genotype CD and DD (P0.01). In conclusion, genotype AB in position +72(T/C) and genotype CC in position -478(/G) of LF gene were advantageous genotype, which can be used as candidate markers for mastitis resistance selection in dairy cattle.

The polymorphisms of HSP70-1 gene in 253 Chinese Holstein dairy cows were studied, and the association between the polymorphisms and somatic cell score (SCS) were analyzed. PCR-SSCP, PCR-RFLP and DNA sequencing were used to investigate mutations in the coding region of HSP70-1 gene. The G--A--C mutation at 1 623 bp and G--A mutation at 2 409 bp were found and both of them were silence mutations that caused no alteration in amino acid sequence. Chi-square test showed both loci were'nt at Hardy-Weinberg disequilibrium in Chinese Holstein. In the meanwhile, the association of 2 409 locus and SCS was not significant. However, the polymorphism at 1623 locus affected SCS significantly (P0.05). The SCS of genotype CC was significantly lower than that of genotype AG and GG (P0.05), so genotype CC was mastitis resistant. These results suggest that genotype CC of HSP70-1 gene may be used as a molecular and genetic marker to improve the phenotype of anti-mastitis in Chinese Holstein dairy cows.

K-casein gene was regarded as a candidate gene for milk production traits of cows. In this study, a 779 bp fragment of k-casein gene of Chinese Holstein was amplified by polymerase chain reaction (PCR), the polymorphisms of three loci of k-casein gene were detected by PCR-RFLP with restriction endonuclease Taq, Hind, Pst. After sequencing, T/C single nucleotide polymorphism (SNP) was identified at nucleotide 10 891C/A SNP was identified at nucleotide 10 927 and G/A SNP was identified at nucleotide 10 988 in exon4 of k-casein gene. Both alleles (A and B) of three loci were found in the population that showed low polymorphism. The gene frequencies of A and B were 86.03% and 13.97%, respectively. The genotype frequencies of AA, AB, and BB were 73.71%, 24.63%, and 1.66%, respectively. Statistical results of c2 test indicated that three polymorphism sites in the population fitted with Hardy-Weinberg equilibrium (P0.05). Meanwhile, the effect of polymorphism of k-casein gene on milk production traits was analyzed. The results indicated that in the three loci, the different genotype of k-casein gene had no significant influence on milk yield and milk protein percent (P0.05). The cows with genotypes BB and AB showed higher milk fat percent than those with genotype AA ( P0.05 ) ; with genotype AB showed higher fat protein ratio than those with genotype AA ( P0.05 ). The polymorphism of the three loci in the experimental population is closely linked. The conclusion is that k-casein B allele can be used as the molecular genetic markers of modifying milk fat percent in Chinese Holstein cows.

Complex vertebral malformation (CVM), a lethal autosomal recessive inherited defect in Holstein calves, was newly reported worldwide. The molecular cause of CVM was a substitution of guanine by thymine (G--T) in a solute carrier family 35 member 3 gene (SLC35A3), encoding UDP-N-acetylglucosamine transporter. It was characterized by stillborn, abortion, and premature birth. The objective of this study was to study the actual carrier frequency of the CVM mutation in a population of Chinese Holstein (=Chinese Black-and-White) normal cattle. The normal 436 Holstein cows and 93 Holstein bulls were genotyping by using the Created Restriction Site PCR (CRS-PCR) and Allele-specific PCR (AS-PCR) methods. There were two bulls and one cow in three observed CVM-carriers. In the Holstein dairy cattle and Holstein bull population, the percentages of CVM carriers were estimated as 0.60% and 2.20% respectively. This study provided a more reliable and useful method for extensive screening of CVM and also offers a theoretical basis for molecular diagnosis in Holstein calves.

Interferon a gene was cloned from genomic DNA of Chinese Luxi yellow cattle by PCR, and the PCR product was inserted into vector pET32a( + ) to make a recombinant plasmid pET32a( + )/BoIFN-alpha. The expression of BoIFN-alpha in Escherichia coli was induced by addition of IPTG. Sequence analysis showed that the Chinese Luxi yellow cattle IFN-alpha gene is composed of 498 nucleotides, encoding a mature polypeptide of 166 amino acids. Compared with other BoIFN-alpha subtypes, it shares the highest identity of 97.6% to the C-subtype. SDS-PAGE results showed that recombinant proteins were expressed in inclusion bodies in Escherichia coli with molecular weight of 40 kD and the recombinant proteins accounted for 26.7% of the whole proteins.The expressed product was purified by affinity chromatography with immobilized nickel chelating NTA (Ni-NTA) and its antiviral activities were tested on MDBK/VSV cell system. Its antiviral activities were 5 x 10(5) u/mg on MDBK/VSV cell system. The results showed that the expression plasmid was successfully constructed and BoIFN-alpha C2 protein was expressed in Escherichia coli. Moreover the purification had good effects on antiviral activities.