The rumen serves as the primary site for digestion feed and absorption of nutrition, which is the characteristic unique organ presented in ruminant animals. To investigate the changes of rumen papillae proteome affected by dietary change, in the current study, twenty-four primiparous Holstein cows(Bos taurus) with similar day in milk and body weight were randomly assigned to 2 treatments (12 cows fitted with rumenal cannulas). 1) cows were fed high concentrate diet consisted of corn(Zea mays) stalk and concentrate; 2) cows were fed low concentrate diet consisted of Chinese wildrye(Elymus chinensis), corn silage, alfalfa(Medicago sativa) hay and concentrate in a 4-wk trial. Rumen papillae was collected from ventral rumen wall of cows after a 4-wk trail. Alteration of protein were detected and identified using two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that acyl-CoA synthetase family member 2, hydroxymethylglutaryl-CoA synthase, peroxiredoxin-2 and voltage-dependent anion-selective channel protein 1 were upregulated in response to high concentrate diet, while keratin 6A and larva-specific keratin(RLK) were upregulated in response to low concentrate diet. The identified proteins were mainly associated with functions related to stress, metabolism, and signal trasduction. Based on these findings, it was concluded that the changes of rumen papillae proteins affected by dietary composition that mediate rumen epithelial adaptation to dietary change

Insulin induced gene 2 (INSIG2) plays an important role in regulating the biogenesis and metabolism of lipid, while the synthesis and metabolism of lipid in goat mammary gland is closely related to milk quality. In order to clarify the relationship between them and provide further scientific support to improve the goat dairy flavor, we used RT-PCR to clone INSIG2 from Xinong Saanen goat (Capra hircus) mammary gland total RNA and analyze its expression in ten tissues. The results showed that we got a sequence of 915 bp (GenBank accession No. JQ361767), including 678 bp of open reading frame, which encoded a protein of 225 amino acids with a predicted molecular weight of 24.8730 kD and pI 8.77. Nucleotides and amino acid (aa) sequences alignment showed that the nucleotides and aa of goat INSIG2 had the highest score similarity with bovine (Bos taurus) in GenBank. Protein structure analysis showed that INSIG2 contained six transmembrane helices; hydrophobicity analysis indicated that both N-terminal and C-terminal of the protein were hydrophilic; the genetic distance analysis showed that goat had the closest relationship with bovine, followed by pig(Sus scrofa). RT-qPCR was used to examine the mRNA expression of INSIG2 in ten tissues, and the result showed that INSIG2 was expressed in all 10 tissues, the lung had the highest expression of INSIG2, followed by muscle, there was moderate expression in mammary gland, and the minimal expression was in heart. The study of INSIG2 gene cloning and tissue expression analysis will provide a basis for function studies of the gene in goat mammary gland

Toll-like receptor 4(TLR4) is one of the most important pattern recognition receptors(PRRs) in the Toll-like receptor family capable of sensing pathogen associated molecular patterns(PAMPs). The aim of this study was to investigate the single nucleotide polymorphisms(SNPs) of TLR4 and its association with immune traits in the laying ducks. The 2 254 bp sequence of exon 3 of TLR4 gene was analyzed with PCR-SBT method in Shaoxing laying ducks and Jiyun laying ducks(Anas platyrhyncha domestica). The results showed that 10 SNPs were detected. Among them, C433G was a silent mutation that did not induce the changes of amino acid, while other mutational loci were missense mutations. The result of Chi Square test indicated that the frequencies of 7 SNPs from the exon 3 of TLR4 gene fit with Hardy-Weinberg equilibrium in this population. The association between these genotypes with some important immune traits was analyzed with generalized least squares. The results showed that T141G and T398C were significantly associated with index of bursa of Fabricius and interleukin-2(IL-2) level (P＜0.05), and individuals with AB genotype had the highest all traits. A661G、T699C and A1563G were significantly associated with thymus index (P＜0.05 or P＜0.01). The results indicated that the 5 SNPs from the exon 3 of TLR4 gene are probable important molecular markers affecting immune traits，they can be candidate molecular markers for the laying duck breeding

Enterotoxigenic Escherichia coli (ETEC) is the main cause of Piglet early weaning diarrhea(PWED) and immunoprophylaxis is one of the most efficient measure to solve this problem. This paper aims to use E. coli F18 fimbrial adhesion F(FedF) recombinantly expressed as antigen to explore in mice(Mus musculus) the possibility of using FedF as the target to prevent PWED. Primers added with EcoRⅠand SalⅠsites were devised and synthesized according to the sequence F18 fimbriae antigen gene fedF on GenBank. Full length fedF gene fragment of 908 bp with 20 signal peptide sequence was amplified by using E. coli F18 genome as template. Then the target fragment was subcloned into the prokaryotic expression vector pET-30a (+) and the recombinant plasmid pET-fedF was transformed into E.coli BL21. After induction with IPTG, recombinant FedF about 32.9 kD was detectable. Then BALB/c mice were orally immunized by the recombinant strain E. coli [pET- fedF]. The results showed that the recombinant strain could induce high level of sera FedF specific IgG (P/N >2.0) and significant increase in intestinal sIgA by 2.07 fold (P<0.05); finally oral immunization of the recombinant strain could greatly protect mice against lethal dose of enterotoxin E. coli administration, the immune protective rate reached up to 62.5%. All the above results implied that the recombinant strain E. coli [pET- fedF] can protect mice from ETEC attach by induction of sera FedF specific IgG and intestinal sIgA, and it may be a promising weapon against PWED

R2R3-MYB is a principal member of MYB transcription factor superfamily, which has been showed to play an important role in secondary metabolism and abiotic stress responses. In order to obtain a full-length sequence of R2R3-MYB transcription factor gene and its expression profile under different abiotic stresses, a full-length cDNA sequence of sugarcane(Saccharum complex) R2R3-MYB-like gene named as Sc2RMyb1 (GenBank Accession No. JQ823165) was cloned by combined sugarcane EST databases, electronic cloing technology and PCR amplification. Genomic DNA sequence of Sc2RMyb1 was 1 807 bp in length, including 3 exons and 2 introns, and the complete coding sequence was 1 248 bp encoding 427 amino acids. The recombinant protein with an estimated molecular mass of 52 kD was produced in positive prokaryotic strain induced by IPTG. The recombinant Escherichia coli cells exhibited better growth than that of the negative strain in liquid LB medium with addition of NaCl. When 25rRNA gene was used as the internal control, the expression profiles of Sc2RMyb1 gene responsive to different stresses were detected by Real-time qPCR in sugarcane tissue culture seedling. The transcript levels of Sc2RMyb1 gene in sugarcane seedling reduced under H2O2 and NaCl stress, respectively. It was suggested that the Sc2RMyb1 gene, as a negative adjustment factor, might involve in the response to salt stress. The results of this study provide basic information for the further research of the Sc2RMyb1 gene in sugarcane stress tolerance mechanisms and resistance breeding

In order to study the iron absorption and utilization molecular mechanism of different Fe efficiency genotypes apple rootstocks, we used the Malus baccata Borkh as material, which is an iron-inefficient genotype apple rootstock. Through the full-length of citrate synthase gene MxCS1 was obtained from M. xiaojinensis, which is an iron-efficient genotype apple rootstock and previous studies have shown that it is related to iron absorption and transport, and their specific primers were designed to clone the citrate synthase gene(CS) from cDNA of M. baccata Borkh by using RT-PCR, which was composed of 1 422 bp and had high homology with the gene encoding citrate synthase in Golden Delicious(M. domestica Borkh ) and M. xiaojinensis, thus we designated it as MbCS1(GenBank accession No. JQ898346). The bioinformatics analysis showed that citrate synthase gene from M. baccata Borkh encoded 473 amino acids, whose relative molecular weight was 54.26 kD and theoretical isoelectric point was 8.95. Subcellular localization assay of the MbCS1 protein, which used transient expression of MbCS1::GFP fusion protein in onion(Allium cepa) cells by particle bombardment, displayed that MbCS1 encoding protein localized in cell membrane. RT-PCR and Real-time PCR analysis suggested that MbCS1 gene was expressed in roots, shoots and leaves in M. Baccata Borkh under normal supply of iron; and the expression of MbCS1 gene was enhanced obviously in roots, shoots and leaves under a low iron concentration(EDTA-NaFe, 4 μmol/L) and reached to the highest value at the ninth day, then decreased; the expression of MbCS1 gene was different in roots, shoots and leaves, enriched in shoots than that in leaves and roots under ion deficiency induced. The expression pattern of MbCS1 gene in M. baccata Borkh was very different from MxCS1 gene in M. xiaojinensis. The result provides solid foundation for further study of the resistance mechanisms in higher plants and provides basis for improvement of iron-inefficient apple rootstocks

In order to study the fungistasis and antifungal mechanisms of biocontrol strain against Setosphaeria turcica, one endophytic bacterium strain YY1 was isolated from leaves of healthy maize(Zea mays) plants and was identified as Bacillus subtilis based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis in this study. Meanwhile, antifungal substances from the fermentation liquor of strain YY1 were deposited by the method of ammonium sulfate and exhibited a high antifungal activity as precipitated by 50% ammonium sulphate. These results implied that antifungal substances produced by strain YY1 may be proteinous material. It was shown on PDA plates that many plant pathogens such as Fusarium graminearum, Botryosphaeria dothidea, Botrytis cinerea and Curvularia lunata were inhibited effectively by both the fermentation liquor and the crude protein extracts. After treated by crude protein extracts, the substrate hyphal morphology had an aberration from filiform to bead-like, however the inhibited aerial hyphae were the same with that of the control. The conidial germination would start to be inhibited at the concentration of 0.35 μg/μL and could be inhibited completely at the concentration of 0.78 μg/μL, the IC50 was 0.46 μg/μL, but the crude protein extracts could not split the conidia, even the concentration was up to 17.36 μg/μL. Through the crude protein extracts incubation, the protoplast membrane was broken and the intracellular materials were dying out gradually at the concentration of 0.78 μg/μL. It was suggested that the crude protein extracts may change the structure or permeability of the plasma membrane. The semiquantitative RT-PCR results indicated that in the inhibited conidia of S. turcica, the expression of G protein γ subunit-encoding gene (Stgg-1) was inhibited completely, while other genes encoding three different subtypes of G protein α subunit (Stga-1, Stga-2 and Stga-3) and G protein β subunit (Stgb-1) were not expressed in both CK group and treatment group. The expression of protein kinase A regulated subunit gene(StPKA-r) and protein kinase A catalytic subunit gene(StPKA-c), which encoded the catalytic subunit and regulatory subunit of cAMP-dependent protein kinase A, decreased significantly, but adenylate cyclase encoding-gene (StAC) had slightly increased transcription level. The transcription level of mitogen-activated protein kinase kinase kinase gene(Stk1k) involved in mitogen-activated protein kinase (MAPK) signaling pathway and protein kinase C gene(StPKC) in Ca2+ signaling pathway displayed no difference during the process of treated or untreated conidial germination. The inhibitory ratios of crude protein extracts on RNA interference transformants of Stgg-1, StPKA-c and StPKA-r knockout mutant were decreased markedly, while the hyphal growth of Stk1k-silenced transformant had no distinction compared with that of the wild-type strain during face-to-face-culturing with those extracts on PDA media. So it was inferred that the molecular mechanisms of the crude protein extracts against S. turcica may mainly be mediated by cyclic adenosine monophosphate (cAMP) signaling pathway. This work will lay a foundation for finding new control techniques against Setosphaeria turcica

The aim of the study was to investigate that characterization and antibiotic resistance of Escherichia coli isolated from bovine mastitis in some areas of China for raising therapeutic effect and decreasing drug residues. Milk samples of clinical and subclinical bovine mastitis were collected from seven provinces in China and a total of 95 E. coli isolates were further identified by microbiological tests. Following, serotype, pathogenicity and antibiotic resistance of 95 strains were also studied. The results showed that for the 95 E. coli strains, 54 isolates were identified as 37 serogroups, 2 isolates were self-clumpinged and 39 isolates could not be serotyped, 093 and 09 serotypes were the most common serogroups. Obviously pathological changes were observed in internal organs of dissected experiment mouse(Mus musculus). Among the 95 strains, the rate of drug fast to 8 out of 16 antibacterial drugs exceeded 50%, and a same strain could resisted 14 kinds of antibacterial drugs at most and 2 kinds of antibacterial drugs at least. E. coli strains which were resistant above 6 antibacterial drugs occupied 51.58%. So we concluded that serotypes of E. coli isolates from bovine mastitis were complicate in China, and E. coli isolates had produced different level resistance to many kinds of antibacterial drugs and presented serious multiple antibiotic resistance. This research provides a theoretical basis to further prepare bovine mastitis vaccine and treat bovine mastitis clinically

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis. It is one of the three major infectious diseases which threaten public health, the other two are AIDS(acquired immune deficiency syndrome) and malaria. Exported repeated protein (Erp) is an important virulence factors of M. tuberculosis. It has potential value in the clinical application. In order to study the function of Erp, short fragments of DomC and Dom8 synthetized by solid-phase phosphoramidite method were assembled by SOE-PCR. M. tuberculosis erp gene PGLTS length of 8 motif, C , N and CN terminal end mutantion of the 8 motif were cloned by overlap extension PCR. The clones in Escherichia coli BL21 (DE3) pLysS strain were expressed. The mutated genes were subcloned into the prokaryotic expression vector pET-28a (+). Positive plasmids were selected by enzyme digestion, PCR, and DNA sequencing. The recombinant protein expression of M. tuberculosis Erp was detected by SDS-PAGE and Western blot. The results showed that the sizes of the amplified fragments areas were as expected. The erp gene PGLTS motif length of 8 motif, C, N and CN terminal end mutant were amplified successfully by overlap extension PCR and built into the prokaryotic expression vector. Recombinant BL21 (DE3)pLysS strains was induced by IPTG. Detected by tricine/SDS-PAGE, the relative molecular weights of the expression products were 13.0, 17.1 and 6.7 kD, respectively. Prepare the Freund's adjuvant vaccine and vaccinate rabbits. The antibody titers were determined with ELISA. When the titer got 1∶512, the heart blood could be drawn and separated. After preparing the antiserum, we used Western-blot to detect the extracted proteins. The primary antibody was l∶200 dilution of rabbit positive sera. The secondary antibody was 1∶800 dilution of HRP-labeled sheep anti- rabbit IgG. The results of Western blot showed that the proteins of MutC8 and MutN8 possessed good antigenicity, which were identified by the rabbit antisera against Erp of M. tuberculosis. The experiment results showed that the target proteins of BL21 (DE3) pLysS (pET28a-C8) and BL21 (DE3) pLysS (pET28a-N8) were expressed specifically, which were identified by the rabbit antisera against Erp in Mycobacterium tuberculosis. Meanwhile, the proteins mentioned above had excellent antigenicity. The experiments provides basic data for the diagnosis of bovine tuberculosis and the recombinant vaccine antigen of the gene in the future

Plants have been used for human medicine for many centuries and account for about one fourth of present prescription drugs. Advances in modern biotechnology have further extended the potentials and applications of plants for human medicine development. Many important recombinant pharmaceutical proteins have been successfully expressed in plants and some of them are even in the commercial pipeline. Among several production strategies, chloroplast engineering offers a number of unique advantages including high-efficiency and stable expression, multi-gene expression in single transformation event, environment-friendliness, etc.. More than 40 functionally active recombinant pharmaceutical proteins such as vaccines and various other therapeutic proteins have been successfully produced through chloroplasts engineering. In this paper, we gave a review on recent advances in recombinant pharmaceuticals and vaccines production in chloroplasts. Some considerations for the chloroplast-derived pharmaceuticals production were also discussed

To explore broad-spectrum and durable rice resistance in breeding, as well as effective prevention and treatment of rice diseases, 43 SSR primers of functional genes related to blast and bacterial leaf blight(BLB) resistance were used to detect genetic diversity among 76 Chinese three-line hybrid rice(Oryza sativa ssp. indica) parents. The result indicated that 36 primers showed polymorphism and 87 alleles loci were detected; While the number of effective alleles (Ne) were 61.96, account for 71.22%, and Nei's genetic diversity index (He) ranged from 0.358 to 0.974, on an average of 0.629. The genetic similarity(Gs) of 76 varieties ranged from 0.341 to 0.925, on an average of 0.550. UPGMA cluster analysis showed that the 76 accessions could be classified into two distinct classes, maintainer lines and restoring lines, at similarities coefficient 0.624. The coefficient of genetic differentiation(Fst) was 0.158, showed high variation level, and the Nei's genetic distance(D) was 0.201. The study suggested that the functional gene markers had high DNA polymorphisms detection efficiency, and could be used as useful tool for measuring genetic diversity accurately and reliably; overall, the backbone parents studied in research showed nearer genetic relationship and higher homology sort of genetic basis. However, there still showed higher genetic differentiation between maintainer lines and restoring lines, which suggesting that, with more and more functional locus going to be expiscated, there can be more value in use to rice resistance by heterosis breeding.

In order to evaluate theirs genetic diversity and temporal trends, 200 accessions, including the core wheat(Triticum aestivum L.) parent Funo and its derivative varieties (lines), were analyzed using 29 informative markers selected from 304 SSR markers. A total of 197 alleles were detected and the alleles with frequency lower than 5% accounted for 14.21%. The scale of the number of alleles was from 3 to 15 with an average of 7.41 per SSR marker. The number of alleles declined trend among Funo and its derivative varieties (lines). The polymorphism information content (PIC) value varied from 0.5499 to 0.9082 with an average of 0.7763 and was high in the tested materials. In the three genomes of wheat, the average genetic richness were 7.14, 7.73, and 7.00 (Genome B＞A＞D), respectively, and the genetic diversity indices were 0.7687, 0.7763 and 0.7517 (Genome B＞A＞D), respectively. The genetic diversity of B genome was the most abundant. Among the seven homoeologous groups, the average genetic richness were group 6＞2=4＞1=3＞7＞5, and the genetic diversity indexes were group 2＞7＞6＞4＞1＞3＞5, and therefore group 5 possessed the lowest genetic diversity. Funo specific bands of SSR markers Xgwm268, Xgwm400, Xwmc398, Xwmc125, Xwmc817, Xgwm272 and Xgwm383 were with the higher inheritable rates among the tested materials, but their contribution ratio differed among the different genomes and chromosomes. The similarity coefficient of the 200 materials varied from 0.4162 to 0.9442 with an average of 0.6619. The ranges of similarity coefficient were becoming narrower and narrower from the first generation to the forth generation, and the genetic similarity coefficient of Funo's pedigree had the tendency of increasing with time slightly. UPGMA (unweighted pair group method with arithmetic mean) cluster analysis revealed that all the materials could be divided into five categories in the genetic similarity coefficient of 0.624, of which 180 (90.0%) species were clustered in the same class. The genetic basis of Funo derivative varieties(lines) was very narrow, and the genetic diversity was poor. The above results demonstrated that the genetic diversity and temporal trends of Funo and its derivative varieties (lines) implicated in the molecular level, and 7 SSR markers with the higher inheritable rates were detected, and thus this may useful to study the genetic basis of Funo

Plant height is one of important agronomy traits affecting wheat yield with very high sensitivity of water environment. Known better the quantitative genetic and its interactions with water environments of plant height (PH) development in common wheat, conditional quantitative trait loci (QTLs) were studied for PH using a recombinant inbred lines (RIL) with 120 progeny lines, derived from a cross between Longjian 19 (drought tolerant) and Q9086(water sensitive)(Triticum aestivum L.). The PH phenotypes at different stages were evaluated under rainfed (drought stress, DS) and well-watered (WW) conditions in Zhenyuan and Lanzhou, Gansu Province, respectively. Conditional QTLs analyses were performed by a mixed linear model approach. The total of 26 additive QTLs (A-QTLs) and 56 pairs of epistatic QTLs (AA-QTLs) were detected for conditional PH of wheat at different growth stages in two locations. In all A-QTLs, six major loci, Qph.acs-1A-1, Qph.acs-4B-2, Qph.acs-5A-1, Qph.acs-5D-1, Qph.acs-6B-2 and Qph.acs-7D-1, were identified in repetition at pre-flowering stages, showed higher genetic contribution percentage (H2(A)) ranged from 7.39% to 31.04%. All of AA-QTLs for PH were mainly composed of interactions between different QTLs with non-significantly additive effects, explaining H2(AA) ranged from 1.38% to 24.27%. Those AA-QTLs significantly influenced the PH at later growth stages. 61.54% of A-QTLs and 58.93% of AA-QTLs were greatly interacted with water regimes. Under rainfed, genetic effects of water environmental interaction could decrease the PH. At the jointing stage, additive effects of conditional A-QTLs for PH showed higher than that of other stages. And then, additive effects were gradually decreased but more emphasized of epistatic effects. The result indicated that quantitative genes controlled the PH development could be easily interacted with water environments, proceeding to some temporal-spatial expressions at different growth stages in wheat. The information in this study can be useful for the molecular genetic improvement of drought tolerance in wheat