Species in genus Secale have provided novel rust resistance genes to protect wheat from this fungal disease. In order to transfer new stripe rust resistance gene(s) from wild Secale species, Secale africanum, we obtained a line HH41 from the advanced generation of Triticum durum-Secale africanum amphiploid (AABBRaRa) crossed with wheat(T. aestivum). Line HH41 was immune to stripe rust isolates from China, and had 42 chromosomes. Fluorescence in situ hybridization (FISH) using D-chromosome specific probe pAs1 and sequential genomic in situ hybridization (GISH) analysis using S. cereale cv. Qinglingheimai (QH) genomic DNA as probe demonstrated that a pair of wheat chromosomes 6D were absent, but a pair of S. africanum chromosomes presented in HH41. Expressed sequence tags based molecular markers also confirmed that HH41 lost 6D specific bands and amplified 6Ra specific bands, indicating that HH41 was a 6Ra(6D) substitution line. GISH analysis using genomic DNA of S. cereale cv. Qinglingheimai(QH) as a probe showed that hybridization signal covered along the chromosomes 6Ra except the telomere region of the short arm. However, the whole 6Ra chromosomes possessed hybridization signal if using genomic DNA of S. africanum for GISH analysis. FISH using Secale telomere or subtelomere tandem repetitive sequences pSc200 as a probe, no hybridization signal was detected on HH41. However, apparent signal was observed on chromosomes 6R from CS-S. cereale cv. Imperial disomic addition lines with strong signal on the telomere of the short arms were also scattered signal on the subtelomere of the long arms. It was revealed that chromosomes 6Ra of S. africanum were different from the S. cereale chromosomes 6R, especially on the telomere regions, with lost or reduced telomere or subtelomere repetitive sequences. Nine of sixteen 6R/6Ra specific markers developed by our study represented polymorphic amplification on chromosomes 6Ra of S. africanum distinct from chromosomes 6R of S. cereale, further demonstrated polymorphism exited between the two chromosome types. Based on stripe rust races evaluation, it was deduced that the stripe rust resistance in HH41 was derived from chromosomes 6Ra of S. africanum. In conclusion, the 6Ra(6D) substitution line with excellent stripe rust resistance and apparent polymorphism originated from ancient species S. africanum will serve as an important resource for translocation lines creation, valuable gene introgression and common wheat improvement

Penicillium expansum is a major postharvest pathogen and quite harmful to fruit and vegetable preservation. Therefore, it is necessary to search for an effective control method. In this study, we isolated a P. expansum from naturally infected apples(Malus domestica) and identified it by rDNA-ITS method. The relationship between exogenous nitrc oxide(NO) with different concentrations and growth or pathogenicity of P. expansum were evaluated via assessment of germination ratio, germ tube length, mycelium extension speed, and inoculation experiment of apple. In addition, by determining level of reactive oxygen species, carbonylated proteins, and evaluating the content of malondialdehyde (MDA) and ATP, the possible inhibitory mechanism of exogenous NO on P. expansum was explored preliminarily. The results indicated that exogenous NO could inhibit the growth of P. expansum and the behavior was dose-dependent. It was speculated that the inhibitory effect came from accumulation of reactive oxygen species, which led to oxidative damages. This study provides a new approach to control postharvest horticulture pathogens potentially of fruit and vegetable

This paper summarized the recent achievements of DNA barcode in animal identification studies. The DNA barcoding literature of study animals and their selected DNA barcode were analyzed. The most studies involved invertebrates and reptiles. While mammals were reported fewer, among these research mainly focused on bats and rats, but no study of Chinese local pigs. The paper also described the phylogenetic research progress in Chineselocal Pigs. The current classification system of Chinese local pigs on the molecular evidence is deficient, and the number of research involved is limited. Molecular phylogenetic studies of Chinese local pig have just started. At the same time, the paper especially emphasized the application prospects and analyzed the feasibility of DNA barcodes in genetic resources conservation for local pig studies.

Promoter is a key cis-acting element to regulate gene expression, which is also an important element of the expression vector in genetic engineering. Understanding the structure and function of promoters contributes to constructing vector and controlling the gene expression, further we can achieve the goal that induces protein expression selectively. Therefore introducing a proper method to clone the promoter presents significance and wide application prospects. Since the invention of polymerase chain reaction (PCR) technique, several PCR-based chromosome walking methods have been developed to amplify unknown promoter sequences flanked by a known segment. In this review, we introduce the PCR-based methodologies for the rapid acquisition of unknown promoter, including Inverse PCR, Ligation-mediated PCR, Semi-random primer PCR. These principles and research progresses of each approach are discussed.

Piriformospora indica is a root endophytic fungus, which can promote the growth of many plants, enhance crop yield, and also confer biotic and abiotic stress tolerance to its host plants. In this study, in order to confirm the influence of P. indica on drought resistance of Brassica napus, the differences of malondialdehyde (MDA) content, the relative conductance, proline(Pro) content, antioxidant enzymes(superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT)) activity and expression level of drought-related genes between B. napus colonized and un-colonized by P. indica were analysed after treating by 20% polyethylene glycol-6000(PEG) solution. The results showed that the concentration of MDA and the relative conductance in the leaves of B. napus colonized by P. indica was significantly lower than that in un-colonized ones. The Pro content in the leaves of B. napus colonized by P. indica were significantly higher than that in un-colonized ones, and it was 1.3 folds of that after PEG treatment for 72 h. The activity of SOD, POD and CAT in the leaves of B. napus colonized by P. indica were significantly higher than that in un-colonized plants, they were 1.17, 1.38 and 1.27 folds of the control ones after PEG treatment for 24 h, respectively. RT-PCR analysis showed that the expression of drought-related gene 575 which codes lipid-transfer protein was upregulated in leaves of B. napus colonized by P. indica, and the expression level was 3.2 folds of control plants after PEG treatment for 9 h. This study indicated that the drought resistance improvement of B. napus conferred by P. indica was related to MDA and Pro contents, plasmamembrane permeability, antioxidant enzymes activity and the expression level of drought-related genes. P. indica may enhance drought resistance of B. napus through improving total antioxidant ability, maintaining cell biomembrane integrity and cell osmotic pressure, and reducing the level of membrane lipid peroxidation of plants. This study preliminary confirms the role and partly mechanism of drought resistance of B. napus conferred by P. indica, and lays the foundation for further research on the role and related mechanisms of stresses resistance in B. napus conferred by P. indica.

p53 is a kind of tumor suppressor genes, it plays a role in the process of controlling cell cycle arrest, senescence, autophagy and apoptosis, and it was espressed widely in various tissues of organism. The objectives of this study were to construct a eukaryotic expression vector pVenus-p53 and research the expression and localization of p53 gene in bovine oocyte. Firstly, we cloned the total CDs sequence of p53 gene from bovine cumulus cells and linked the CDs sequence of bovine p53 gene into pMD19-T, then we identified pMD19-T-p53 by double restriction enzyme digestion and cloned the p53 into expression vector pVenus, and then we constructed eukaryotic expression vector of pVenus-p53. After pVenus-p53 recombinant plasmid transfected into Hela cells mediated by Lipofectamine 2000, we confirmed the expression and position of the recombinant plasmid pVenus-p53 in Hela cells. We found that pVenus-p53 recombinant plasmid mainly located in the nucleus of the Hela cells. Then p53-Venus was transcribed into cRNA using in vitro transcription kit. After that, we observed the expression and localization of p53-Venus cRNA in bovine oocyte after microinjection. We found that it expressed not only in cell nucleus, but also in cytoplasm, but the expression amount was much smaller than that in nucleus. Finally, we collected bovine oocytes in different developmental stages in vitro culture, and detected the changes of p53 mRNA expression in different developmental stages in vitro culture via the Real-time PCR. We used 2-ΔΔCt analysis to analyze the expression level of p53/β-actin and the result showed that the expression of p53 mRNA lifted quickly from GⅤ stage to MⅠperiod and the expression quantity was the highest in MⅠperiod, and then it gradually decreased. In this study we constructed the p53 eukaryotic expression vector pVenus-p53 and the p53-Venus cRNA which could be expressed and localized correctly in bovine oocyte. We also detected the changes of p53 mRNA expression in different developmental stages of bovine oocyte in vitro culture. Our study provides reference material for further research in the role of p53 gene in bovine oocyte in vitro maturation.

Mannose binding protein (MBP) is a subgroup of plant lectin and thought to play a role in various biological processes such as cell-to-cell and host-pathogen interactions. In our previous experiments, an apple MBP gene was found among the highly expressed genes induced by inoculation with Botryosphaeria dothidea, a causal agent of a disastrous disease, apple ring rot. To investigate the role of the MBP gene in the defense responses of apple(Malus domestica) against B. dothidea, we cloned the full-length cDNA sequence of the MBP gene and designated as MdMBP2(GenBank accession No. JX126857). The full-length cDNA was of 1 553 bp with a 1 368 bp of open read frame (ORF) encoding a protein of 455 amino acid residues. The calculated molecular weight and isoelectric point of the MdMBP2 protein were 50.4 kD and 8.68, respectively. Sequence and structure analysis indicated that MdMBP2 possessed a B-lectin domain and PAN-apple domain. There was a predicted signal peptide of 27 amino acids in the N terminus. BLAST analysis revealed that MdMBP2 had high identity to epidermis-specific secreted glycoprotein from Vitis vinifera (66%) and Glycine max (42%), and mannose binding protein from Arabidopsis thaliana (57%). Phylogenetic analysis revealed that all B-lectin domain containing proteins could be grouped into two classes and MdMBP2 belonged to the ClassⅠwhich lacked SLP domain and PKc (protein kinase) domain compared with the proteins in ClassⅡ. The expression of MdMBP2 gene was observed in all tissues examined here, and the highest expression of MdMBP2 gene was found in bark and leaf, and the lowest in root. This expression pattern suggested that MdMBP2 might be involved in many physiological pathways. The responses of MdMBP2 expression to B. dothidea infection were also examined using quantitative Real-time PCR. The results indicated that MdMBP2 expression was significantly enhanced by B. dothidea infection, and the temporal expression was related with the course of disease, which suggested that MdMBP2 might be involved in the defense responses against B. dothidea infection. The recombinant protein of MdMBP2 was prepared with prokaryotic expression system and used to test carbohydrate binding activity. The results indicated that MdMBP2 exhibited strong affinity toward oligomannosides and high-Man N-glycans, but interacted weakly with D-Mannose. The results indicated that MdMBP2 gene might be involved in the recognition of fungal pathogen. All together, in the present study, we identified a new apple MBP gene and analyzed its mRNA expression and biological activity of recombinant protein. The results revealed that the gene is involved in the defense responses against B. dothidea and may play a role in recognition of the pathogen.

In order to study the molecular mechanism that determines the formation of the coat color in different coat color of Tibetan sheep(Ovis aries) skin tissues, agouti signaling protein gene(Agouti) as the main candidate gene about coat color, Agouti gene's polymorphisms were detected firstly, then associated coat color with the exon 2, exon 4 function mutations and the gene copy number variation(CNV) of Agouti, as well as the Real-time fluorescent quantitative PCR was adopted to detect mRNA's relative expression quantity of Agouti gene and microphthalmia-associated transcription factor gene(MITF), so as to analyze the discrepancy of relative expression quantity of the same gene in different skin tissue with different coat color and then had a correlation analysis of mRNA expression quantity of the two genes in the same parts of the same individual. The results showed that there was no correlation between Agouti gene exon's SNP polymorphism and coat color, so did the CNV of Agouti gene and coat color. Brown was significantly higher than that of other color skin tissue in mRNA expression of Agouti gene (P<0.05), mRNA expression quantity of MITF gene in black skin tissue had a great difference to other color skin tissue (P<0.05). Through correlation analysis, there was no significant correlation for mRNA's relative expression value of both genes in the same color of skin tissue of the same individual. The results presumed that the polymorphism of Agouti gene may have no evident correlation to the coat color of Tibetan sheep. the mRNA expression of MITF gene of the skin tissue around the eye socket plays a certain role in the formation of the black coat color of Tibetan sheep. mRNA expression of Agouti and MITF genes may have to do with the black brown hair; mRNA expression of Agouti and MITF genes has no evident correlation to the white hair, respectively.

In order to investigate the relationship between PsPⅡ gene and dormancy release by low temperature, using tree peony(Paeonia suffruticosa) 'Lu He Hong' as plant materials, the full length of PsPⅡ, bioinformatics analysis and expression pattern were conducted under different chilling treatments. The main results were as follows: The full-length cDNA was 899 bp, containing an open reading frame of 603 bp, which encoded a 200 amino acid polypeptide. Its 5' untranslated regions was 70 bp, 3' UTR was 226 bp, including a 24 bp poly(A) tail. It was predicted that PⅡ protein of tree peony was non-secretory hydrophilic protein, modulating the activity of glutamine synthetase. Homology and phylogenetic relationships analysis revealed that PⅡ protein in rice was on the same branch with PⅡ protein in tree peony and there was an identity of 83.6% at amino acid homology. The following species were Nicotiana tabacum, Arabidopsis Thaliana and Pinus pinaster, respectively. By Real-time PCR, we demonstrated that the expression of PsPⅡ was enhanced and continuously increased in the buds of tree peony during the release of dormancy by chilling treatment, reaching the highest value during 15 days chilling treatment. Significant positive correlation between the expression level of PsPⅡ and α-starch hydrolytic enzymes, the activity of glutamine synthase and soluble carbohydrate was observed. This result indicted that the role of PsPⅡ was to increase the activity of enzymes associated with nutrient metabolism during the process of dormancy release.

Secretory proteins in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematodes parasitism of plant. A new venom allergen-like protein gene (Dd-vap-1) was cloned from Ditylenchus destructor by RT-PCR and RACE. The full length cDNA of Dd-vap-1 was 1 346 bp. The cDNA sequence consisted of a 1 209 bp open reading frame encoding 402 amino acid residues that were franked by a 69 bp 5'-untranslated region and a 68 bp 3'-untranslated region(GenBank accession No. JQ341057). The theoretical molecular weight and isoelectric point of the putative protein were 45.3 kD and 6.68, respectively. The sequencing analysis showed that this gene was classified as a member of venom allergen-like protein. In situ hybridization analysis showed that the transcript of Dd-vap-1 accumulated exclusively within the subventral oesophageal cell of D. destructor. The result suggested that Dd-vap-1 may play an important role in the early stage when infecting host plants.

In order to investigate the differences of hepatic lipid synthesis between fat and lean lines of broiler chickens, the gene expression levels of acetyl CoA-carboxylase (ACC), fatty acid synthase (FAS), sterol regulatory element binding protein-1 (SREBP1), liver X receptor (LXR), glycerophosphate acyltransferase (GPAT), apoprotein A-1 (ApoA-1), glucose transporter (GLUT2) and apoprotein B (ApoB) genes were analyzed in livers of the 14th generation of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) population using Real-time RT-PCR. The results revealed that, the expression levels of ACC, FAS, SREBP1, LXR, GPAT and ApoA-1 genes, on the whole, were higher in fat line than that in lean line. In particular, the expression levels of ACC, FAS and SREBP1 genes were dramatically higher in fat line than that in lean line at the age of 10 weeks (P<0.01). In addition, the expression of LXR gene was significantly higher in fat line than that in lean line at the age of 9 and 10 weeks (P<0.01), and the expression of ApoA-1 gene was significantly higher in fat line than that in lean line at the age of 6, 8, 10, and 11 wk (P<0.01 or P<0.05). However, the expression of GLUT2 gene was significantly higher in lean line than in fat line at the age of 5 and 8 weeks (P<0.05), and the expression of ApoB gene was also significantly higher in lean line than that in fat line at the age of 7 weeks (P<0.05). Taken together, these results suggested that fat broilers have higher a capacity for hepatic fat synthesis than that of lean broilers, which may lead to divergent phenotype for fatness traits. Our findings provide basic research material for further study on the molecular mechanisms of broiler fatness traits.

Previous Solexa sequencing analysis from miRNA in the hypothalamus of 1-day-old and 36-weeks Gushi chickens demonstrated that let-7a is one of the most abundant and differential expression miRNA during development of chicken(Gallus domesticus) hypothalamus. For a thorough knowledge of let-7a function, the let-7a expression profiles in 13 tissues in two stages of Gushi chicken were characterized by Real-time quantitative PCR. The results indicated that the let-7a was widely expressed in the detection tissue and the expression in hypothalamus from the Solexa sequencing was consistent with those from Real-time RT-PCR measures. Let-7a was varied among different tissues，with most abundant in the hypothalamus and kidney, low abundance in the lung and moderate expression in other tissues at 1-day-old chickens. Except for the lung, the let-7a expression was lower at the age of 36 weeks than that at 1 day. In addition, The total 107 target genes were predicted by two kinds of algorithm overlap with gene ontology(GO) and kyoto encyclopedia of genes and genomes(KEGG) analysis, the results showed that target genes were rich in the regulation biological processes, especially in the minerals and phosphorus metabolism regulation process and significantly existed in the regulatory pathway of focal adhesions, extracellular matrix receptor interaction, the mitogen-activated protein kinases(MAPK) signaling pathway (P<0.01). These results provide useful clues for further study of let-7a function.

Burkholderia gladioli pv. alliicola is an important plant pathogen. This research was to establish the specific, sensitive and fast Taqman Real-time PCR for detection of B. gladioli pv. alliicola. The specific genome region of B. gladioli pv. alliicola was used for the design of Taqman probe and specific primers. The reaction conditions was optimized. Fourteen reference strains were used for the specificity test, the sensitivity of the Real-time PCR was tested as well. The sensitivity of bacterial suspension of Real-time PCR reached 1.02×102 CFU/mL and the sensitivity of DNA reached 1.73×10-4 ng/μL. Total reaction time took about 1 h or so. Among 14 reference strains belong to the genus of Burkholderia, Real-time PCR showed an extremely high specificity. The simulation test was performed on the onion (Allium cepa) seeds mixed with bacterial suspension of B. gladioli pv. alliicola, the results revealed that the Real-time PCR was practical in the test of B. gladioli pv. alliicola. The Real-time PCR established in this research is useful for the diagnostics of B. gladioli pv. alliicola. It provides an easy, fast and accurate method for molecular identification of B. gladioli pv. alliicola.

Kimchi is a kind of traditional fermented vegetable, and it is also one well-know beneficial food. The number of viable lactic acid bacteria (LAB) is an important indicator to assess the nutritional value of kimchi. Ethidium monoazide in combination of quantitative PCR (EMA-qPCR) has been considered as a rapid and effective method to enumerate viable cell. In this study, EMA-qPCR method was established to detect viable LAB(Lactobacillus plantarum) rapidly and precisely in kimchi. For non-viable LAB, the maximum ΔCt (with EMA - without EMA) was achieved at an EMA concentration of 10 μg/mL, and there were no significant differences (P>0.05) in the ΔCt values for LAB treated with different EMA concentrations of 10, 25, 50 and 100 μg/mL. Moreover, the ΔCt increased significantly (P<0.05) as the photoactivation time of EMA increased from 0 to 20 min at 10 μg/mL EMA. But this effect was not observed among viable LAB, so EMA treatment could not affect the enumeration of viable cells. Therefore, optimum EMA treatment (10 μg/mL and 20 min light activation) lead to effective discrimination between viable and dead LAB. Under these conditions, results from EMA-qPCR (viable LAB of 109, 108, 107, 106, 105 and 104 CFU/mL) correlated well with that of plate counting (R2=0.999), and PCR efficiency reached to 104%. Due to sublethally injury of LAB with fermentation proceeding, EMA could penetrate into sublethal injured cell, and EMA-qPCR method consequently underestimated cell counts. Incubating cells in MRS medium for 30 min before detection could offset this error. That because incubation for suitable time could recover the sublethally injured cells but could not increase cell number. That indicated that EMA-qPCR could not only discriminate viable and non-viable cells, but also detect the sublethally injured cells. The viable LAB counts detected by EMA-qPCR increased with fermentation time increasing in the early stage of kimchi fermentation. As fermentation proceeded, lactic acid produced by LAB accumulated, leading to high environmental pressure to the survival of microorganisms. This gave rise to the sublethal injure of cells. Eventually LAB died and the cell number decreased. Viable LAB could be successfully enumerated at different fermentation times in kimchi through the establishment of EMA-qPCR method. Through exploration of suitable conditions, this method differentiating viable and non-viable cells can also be applied in other foods and environments in the future investigation.