Abstract At present, events about meat adulteration were common occurred, for example, mix fox flesh to cooked donkey flesh in Wal-Mart and horsemeat event spreading EU countries. Now, the method of meat adulteration has been adulterated by one ingredient to a mixture of ingredients. This kind of fraud disturbs the market order seriously. Countries have paid more and more attention to meat adulteration, the identification and traceability of meat adulteration analysis become a hot topic on the world. Traditional meat identification was authenticate the meat appearance, color, odour, falvor and hardness by vision, touch, smell and taste, these methods cannot meet the needs of meat quality and source authentication now. In recent years, with the development of modern analytical instrumentation and biotechnology, identification techniques for meat adulteration have also been well developed. Including protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) methods, chromatography analysis methods and DNA-based detection methods. Protein-based detection methods can detect quantitatively, but it is not suit for animal origin analysis. Many ELISA kits have already been commercially developed, but their application is limited due to protein activity. DNA-based detection methods have the characteristic of simple, short detection time and accurate, of them, fluorescence PCR detection method has the advantage of high sensitivity, high specificity and high throughput, it will become the future direction of the meat source identification. Multiple fluorescence qPCR was used in this experiment. In order to identify the 3 ingredients of mink source, porcine source and rat source in mutton and beef, a pair of universal primers were designed based on the mitochondrial 16S rDNA conserved region of the 3 different animals and 3 kinds of species-specific molecular beacon probes with strong specificity were designed based on the mitochondrial 16S rDNA of mustela vison, sus scrofa and mus musculus. The specific verification of the 3 probes, the sensitivity verification of the experimental system and the test of the detection limit of the samples were carried out respectively in this experiment. In the sensitivity verification experiment, the 3 probes can effectively eliminate the interference of DNA from other species and specifically amplified in both monoplex fluorescence qPCR and multiplex fluorescence qPCR systems. In the sensitivity verification of experiment, seven dilution gradients of DNA concentration were set up and 6 parallel samples for each gradient, it was determined that the sensitivity of the system was 0.01 ng/μL. In the test of the detection limit of samples, 3 kind of doping methods were set up, 7 different dopant mass ratios were set up in each doping method and 4 parallel samples for each dopant mass ratio, the sensitivity of the test was determined to be 1% (mass ratio). This method provides a scientific basis for the origin identification of multi species, puts forward a new way to control the authenticity of animal raw material sources, and provides powerful technical reference for enterprise supervision to rectify the mess of meat industry.

Abstract The selection of suitable reference genes can improve accuracy of gene expression analysis of qRT-PCR. In this study, the seedlings of Magnolia denudata were treated under salt stress in 4, 8, 12, 24, 48, 72 h. Root, steam and leaf tissues in different time were used as materials. 13 genes including ACTIN (actin), CYP (cyclophilin), EF-1α (elongationfactors-1α), GAPDH (glyceraldehyde-3-phosphat dehydrogenase), GBP (GTP binding protein), NAC (NAC domain protein), NADP (NADP-isocitrate dehydrogenase), TEF (translation elongation factors), UBC (ubiquitin-conjugating enzyme), UBQ (polyubiquitin protein), α-TUB (tubulin alpha), β-TUB (tubulin beta) and 18S (18S ribosomal RNA) were chosen as candidate reference genes. The specificity of the primers was investigated by agarose gel electrophoresis and melting curve. The softwares of geNorm, NormFinder and BestKeeper were used to analyze and screen optimum reference genes. Furthermore, two functional genes cellulose synthase-like D (CSLD) and endo-1,4-beta-d-glucanase (KOR) were selected to verify the reliability of the suitable reference genes. Electrophoresis results showed distinct PCR products with the expected size, amplification efficiency of primers were all between 95%~105%, and the single-peak melting curves further indicated the specificity of primers. GeNorm, NormFinder and BestKeeper comprehensive analysis showed that top 3 stable reference genes were GBP, UBQ and UBC, While 18S is the worst reference gene. Meanwhile, relative expression of CSLD and KOR in different tissues were analyzed by GBP, UBQ, UBC, the combination of top-stable genes (GBP, UBQ and UBC), the unstable gene 18S and GAPDH. As a result, the 2 target genes showed consistent expression profiles when normalized by 3 top-ranked reference genes and the combination, unstable genes 18S and GAPDH failed to standardize the expression data and caused deviation in the result. Therefore, GBP, UBQ and UBC could serve as stable reference genes for gene expression among different tissues in M. denudata under salt stress. The present study will provide an important reference basis for expression analysis of key genes of Magnolia in adverse situation

Abstract Porcine delta coronavirus (PDCoV) is a newly emerging swine intestinal coronavirus. In this study, a reverse transcription polymerase chain reaction (RT-PCR) method based on M gene for detecting PDCoV was developed and clinically used for epidemiology survey of PDCoV in Sichuan province and detecting the tissue distribution of PDCoV in challenged piglets. Based on the results of PDCoV M gene sequence multiple alignment and comparison with M gene sequences of Porcine epidemic diarrhea virus (PEDV) and Porcine transmissible gastroenteritis virus (TGEV), a pair of specific primers was designed and the RT-PCR (reverse transcription PCR) method for detecting PDCoV was established by determining the optimum reaction conditions and the specificity, sensitivity and reproducibility was evaluated, respectively, epidemiological investigation of PDCoV in Sichuan was conducted by detecting 226 clinical diarrhea samples, and the tissue distribution of PDCoV in infected piglets after challenging 7-day-old piglets was assessed. The results showed the target M gene fragment was 654 bp by RT-PCR, the optimum annealing temperature is 60 ℃, the upstream and downstream primer volume was 1.75 μL.The method had high sensitivity and specificity with a limited detection of 8.82×109 IU/mL for PDCoV, respectively, and no cross-reaction with other reference virus such as PEDV, TGEV, Japanese encephalitis virus (JEV), Porcine rota virus (RV), Porcine respiratory and reproductive syndrome virus (PRRSV) or Classical swine fever virus (CSFV). The positive rate of PDCoV in 226 clinical samples from Sichuan was 7.1% (16/226). The M gene of PDCoV CHN-SC2015 strain was sequenced and compared with other 28 reference M genes of PDCoV strains collected from GenBank. The results showed that the nucleotide and amino similarity was 98%~100% and 99%~100%, respectively. From the challenged piglets, PDCoV could be detected from heart, liver, spleen, lung, kidney, colon, ileum and jejunum, indicating that PDCoV had a broad tissue tropism. The established RT-PCR in this study is a simple, rapid and specific assay suitable for clinically detecting PDCoV.

Abstract Detection of quantitative trait loci (QTLs) related to rice quality in different varieties and different environment can provide a basis for breeding and utilization of genes related to excellent cooking and eating quality of rice. The DH population, derived from a cross between CJ06 (Oryza sativa ssp. japonica) and TN1 (Oryza sativa ssp. indica), was used to detect QTLs of grain shape, amylose content (AC), and starch viscosity (rapid viscosity analysis, RVA). Thirteen QTLs related to grain type, amylose content and RVA parameters were obtained by QTL analysis. The QTLs were mapped to chromosome 1, 2, 3, 6, 7, 8, and 10, included qHPV-6, qCPV-6, qBDV-6, qCSV-6, qSBV-1, qSBV-6, qSBV-7, qRL-2, qRLW-2, qRLW-3, qRLW-8-1, qRLW-10, and qAC-6. Seven QTLs on chromosome 1, 6, and 7 that accounted for 7.03% to 49.05% of the total phenotypic variance and their LOD values ranged from 2.55 to 13.18. In these QTLs, contribution rate of different single QTL was more than 25% except qSBV-1 and qSBV-7. Furthermore, qHPV-6, qCPV-6, qBDV-6, qCSV-6, and qSBV-6 were mapped to the same region of chromosome 6, a main effect QTL qAC-6, whose contribution rate was 45.21%, was also detected in the region. Bioinformatics analysis indicated that the region contained a reported Waxy (Wx) gene which regulates amylose synthesis. Five QTLs with additive effects were detected in grain shape. The LOD values ranged from 3.16 to 8.47 and single QTL contribution rate was 8.95% to 27.23%. Three regions of qRLW-2, qRLW-3, and qRLW-8 contained 3 major reported QTLs GW2, GS3 and GW8. The QTL analysis in present study provides basic data for molecular breeding of rice quality.

Abstract Nicotinamide could regulate lipid metabolism. However, the regulatory effects of maternal nicotinamide supplementation on lipid metabolism of offspring are still unknown. The aim of this study was to explore the effects and mechanism of nicotinamide supplementation in perinatal dairy goat (Capra hircus) on lipid metabolism of the offspring. Fifteen perinatal dairy goats (prenatal 21 d) were paired and assigned randomly within block to 3 groups. In group C (control group), ewes were fed with basal diets without nicotinamide supplementation. In group P (postpartum group), ewes were fed with basal diets with nicotinamide supplementation from postpartum 1 to 28 days. In group EP (prenatal group), ewes were fed with nicotinamide supplementation from prenatal 21 d to postpartum 28 d. The lambs from each treatment were named group LC, group LP and group LEP respectively. Fasting blood from lambs was obtained from jugular vein 3 h after morning feeding on 14th and 28th d, to measure the concentration of triglyceride (TG), free fatty acid (FFA), total cholesterol (TC) and the activity of lipase. Lambs were slaughtered at the age of 28 d, and abdominal adipose tissues were collected to measure the concentration of TG, FFA, TC and the gene expression of key enzymes involved in lipid metabolism. Results at 14-day age showed that the concentration of TG, FFA and TC in plasma were not significantly (P＞0.05) different among groups. Results at 28-day age showed that the concentration of TG in plasma in group LEP was extremely significantly (P＜0.01) increased compared with group LC and group LP, and the activity of lipase in group LP and group LEP were extremely significantly (P＜0.01) higher than that of group LC. A significant (P＜0.05) increase of FFA in abdominal adipose tissue occurred in group LEP compared with group LC. The concentration of TG in abdominal adipose tissue in group LEP and group LP were extremely significantly (P＜0.01) increased compared with group LC, and the concentration of TG in abdominal adipose tissue in group LP was significantly (P＜0.01) higher than that of group LEP. The mRNA expression of peroxisome proliferator-activated receptor-γ coactivator α (PGC1α), sterol regulating element binding protein-1 (SREBP1), fatty acid synthetase (FAS) in abdominal adipose tissue increased (P＜0.05) in group LEP compared with group LC and group LP. The mRNA expression of glycerol-3-phosphate acyltransferase mitochondrial (GPAM), 1-acylglycerol-3-phosphate acyltransferases 6 (AGPAT6) were extremely significantly (P＜0.01) increased in group LEP. Besides that, the mRNA expression of AGPAT6 gene increased (P＜0.01) in group LP compared with group LC. It is concluded that nicotinamide supplementation in perinatal dairy goat facilitated fat deposition in abdominal adipose tissue by improving the metabolism of lipogenesis of lambs. The results of this study provided a clue to explore the effects of maternal nicotinamide supplementation on the growth and development of lambs.

Abstract MicroRNAs (miRNAs) are a class of recently identified regulatory RNAs which are short non-coding RNA. They participate in diverse biological functions including embryogenesis, development, metabolism, and oncogenesis. Let-7 family members (Let-7s) are a fundamental tumor suppressor miRNA, which was first identified as a heterochronic gene in Caenorhabditis elegans. In recent years, studies have shown that Let-7s plays an important regulatory role in the growth and development of animals. It is not only related to tumor development, but also participates in the reproduction of females. In order to analyze the sequence of let-7s and to study its expression pattern during the goose reproductive cycle, the current literature and miRBase were used to search the let-7s. The NCBI and Ensembl databases were used to identify the location of let-7s in the genome of each species. At the same time, qRT-PCR was used to detect the expression of let-7s in geese's ovary tissues at the reproductive cycle. A total of 300 vertebrate let-7s sequences were found, containing 11 members (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-5p, let-7g, let-7h, let-7i, let-7j, let-7k) widely distributed and highly conserved in eukaryotes. Most of the members were located in the intergenic region (IGR), only let-7e, let-7f-5p and let-7g were located in the gene region in mammals. The multiple sequence alignment of the mature sequences of let-7s showed that let-7s have high homology of mature sequences with similar length of sequences. And there is nearly half of the conserved bases in the let-7s which indicating that let-7s is highly conserved across species. In addition, the let-7s family may have mutated during evolution. The results of qRT-PCR showed that there was no significant difference in the expression levels of let-7a, let-7b, let-7c and let-7f-5p between ovulation and broody phase (P＞0.05). However, the expression levels of let-7g and let-7h in broody phase were significantly less than those at pre-laying stage, ovulation and oviposition (P＜0.01). Therefore, let-7s were highly conserved in evolution, and let-7g and let-7h played a regulatory role in reproductive cycle of geese. This study will provide a reference for investigating the regulation of let-7 family in geese.

Abstract The ATP synthase subunit beta (ATP5B) is one of key factors in ATP synthesis, participating in various metabolic activities. Nanyang cattle (Bos taurus) is the one of the five major Chinese native cattle varieties, famous for its adaptability. This study investigated the genetic variation in ATP5B promoter regions and the association of Nanyang cattle growth traits; estimated the conservation of ATP5B among different species, and examined the mRNA expression. It was sequenced the ATP5B promoter region to determine the genetic variants and their association with Nanyang cattle growth traits. To find the reason of this association, It was detected the promoter activity of each genotype promoter at these SNPs site using dual luciferase assay. In the results, there were 14 similar motifs and 5 conservative functional domains in the ATP5B of 7 species, such as, Bos taurus, Sus scrofa, Ruttus and so on. The ATP5B was widely expressed in many tissues in Nanyang cattle, and the muscle tissue had the significant high mRNA level than other tissues (P＜0.05), the ATP5B mRNA in fat tissue was following, the next was in heart, kidney, lung, and liver; the ATP5B mRNA in spleen was the lowest compared with other tisuses (P＜0.05). 3 SNPs in ATP5B promoter regions were identified, named as g.-428T>A, g.-390T>C and g.-322C>G, respectively. After association analysis, there were 3 mutations fell into Hardy-Weinberg equilibrium significantly (Chi-Squared test, P＞0.05). The polymorphism information content (PIC) classification indicated that the 3 SNPs were moderately polymorphic (0.25＜PIC＜0.50). At g.-428T>A locus, individuals with TT genotype exhibited significantly higher body weight, body height and chest circumference than those with AA genotype (P＜0.01). Such as, TA individuals had greater body weight than AA individuals (P＜0.01), but not than TT individuals (P＞0.05). In body height, the individuals with TT or TA had greater level than AA individuals (P＜0.05). In chest circumference, the TT individuals had greater level than the individuals with TA or AA (P＜0.01). So, the TT was the preferable genotype. At g.-390T>C locus, the body height and chest circumference of animals with TT genotype were significantly higher (P＜0.01) than these traits of animals with CC genotype. TC individuals had greater body height than CC individuals (P＜0.05), but less than TT individuals (P＜0.01). TC individuals had less chest circumference than TT individuals (P＜0.01). So, the TT was the preferable genotype. Moreover, the g.-322C>G locus were significantly affected body weight and body length (P＜0.01 or P＜0.05), including that, the CC individuals had greater body weight than CG individuals (P＜0.05) and GG individuals (P＜0.01), meanwhile, CG individuals had greater body weight than GG individuals (P＜0.05). In body length, the individuals with CC or CG had greater level than GG individuals (P＜0.05). So that, CC was the preferable genotype. Last, according to the report of dual luciferase assay, although there were many difference between each promoter style at each SNP site, but all of them not reached at significant level (P＞0.05). The molecular mechanism of regulation between these SNPs and growth traits need to further search. To sum up, these results suggested that the SNPs of ATP5B promoter region, the TT at -428T>A locus, TT at -390T>C locus, and CC at -322C>G locus, affected the growth traits of Nanyang cattle, and they could be used as candidate genotype for breeding programs.

Abstract Cadmium (Cd) is a highly toxic and persistent environmental poison, its increasing level in environment exerts a wild range of adverse effects on wheat (Triticum aestivum) and even the health of human beings by food chains. In order to clarify the mechanism of Cd poisoning on wheat, we studied the effects of different concentrations (0, 10, 20, and 40 mg/L) of Cd solution (CdCl2·2.5H2O) on Zhongyu 10, Zhoumai 18 and Luomai 23 in seed development and physiological mechanism. We studied the effects of different Cd concentrations on some physiological indexes including peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), soluble sugar, soluble protein, malondialdehyde (MDA), proline content, root vigor, and photosynthetic indexes. Then the ultrastructure of root cells was observed. Research results showed that the germination rates of Zhongyu 10 (strong Cd tolerance), Zhoumai 18 (medium Cd tolerance) and Luomai 23 (low Cd tolerance) decreased by 12%, 20% and 42% under the treat with 20 mg/L Cd solution, respectively. The growth of wheat was inhibited as the Cd solution concentration increased. Under 40 mg/L Cd solution, the root length of Zhongyu 10 decreased by 69%, and the root length of Luomai 23 decreased by 80%. Besides, POD activity, SOD activity, CAT activity, soluble protein content, soluble sugar content, MDA content, proline content and intercellular CO2 concentration all increased in flag leaves at seeding stage. Zhongyu 10 increased more than Luomai 23, and exhibited some adaptability. While root activity, net photosynthetic rate, transpiration rate, and stomatal conductance all decreased, and Luomai 23 decreased more than Zhongyu 10. In addition, root cells were observed by transmission electron microscopy, and showed that the damages of mitochondria, cell wall and cell membrane of root cells in Luomai 23 were more serious than that of Zhongyu 10. The present study explored the responses of 3 strains of wheat with different Cd tolerance including growth morphology, physiological metabolism and ultrastructure, which might provide certain reference for wheat genetic breeding.

Abstract As early precursors for sperm cells, primordial germ cells (PGCs) are widely used to study spermatogenesis. Fibroblast growth factors (FGFs) are essential in regulating the formation of PGCs. In order to investigate the regulation of FGF8 on the formation of PGCs, we established a chicken embryonic stem cell line with FGF8 low expression, through constructing a small hairpin RNA (shRNA) lentivirus vector for chicken (Gallus domesticus) FGF8, and further investigate the role that FGF8 plays in the differentiation of male germ cells. Three shRNA interfering vectors targeting chicken FGF8 were constructed and transfected into chicken DF1 cells transiently. qRT-PCR was used to detect the interference efficiency of different interference targets on FGF8, and the shRNA vector with the best interference efficiency was packaged in lentivirus. The induction of retinoic acid (RA) was performed in an ES cell lines knocked down by FGF8, and the morphological changes of the cells in each group were observed, also, the cells of each group were collected on day 0, 2, 4 and 6, respectively. The expression of related reproductive genes, like Nanog, Oct4, Cvh, C-kit, Blimp1 and so on, and the efficiency of PGCs formation were detected by qRT-PCR, flow cytometry, and immunofluorescence. The results showed that 3 lentiviral interference vectors and 1 negative vector for FGF8 were successfully constructed and named as shFGF8-1, shFGF8-2, shFGF8-3, shNC. By transfecting DF1 cells, shFGF8-2 had been found the highest interference efficiency relative to shFGF8-1 and shFGF8-3. Hence, shFGF8-2 was packaged with lentivirus and the titer was 5×108 TU/mL, the interference efficiency was (70±4.31)% in ESCs. For ESCs with low expression of FGF8, we found that under normal RA-induced for 4 d, PGC-like cells were significantly increased, and the pluripotency associated gene Nanog, was significantly down regulated (P＜0.01). Meanwhile, the germ cell marker gene Cvh, and C-kit, were significantly up-regulated (P＜0.01). Moreover, the results of immunofluorescence and flow cytometry further confirmed that FGF8 knockdown could induce significant excess of CVH+PGCs (P＜0.01), compared to the control group after 4 d of induction. Collectively, a FGF8-specific lentivirus vector stably transfected with chicken embryo stem cells was successfully constructed, and a novel function of FGF8 in the formation of PGCs in vitro was covered.

Abstract Potato late bright is the most serious disease of Solanum tuberosum, and its pathogen is Phytophthora infestans. In order to screen culturable myxobacteria resistant to the pathogen of potato late blight from soil samples, myxobacteria were isolated from the soil sample by Escherichia coli inducing method in this study. The strains resistant to the pathogen of potato late blight were screened by plate confrontation assay and identified by morphological observation, physiological and biochemical characteristics, and the 16S rRNA sequence analysis. Then, the antimicrobial activities of the strain were determined by plate confrontation method. Then, the growth curve of the target strain was determined by the weighing method and the distribution of antibiotic substances was measured by filter paper disc method. The fermentation parameters were studied by the combination of univariate analysis and orthogonal optimization. Five strains were isolated in this experiment and 3 of them presented antagonistic activity against Phytophthora infestans. The activity of strain X6-II-1 was the strongest among them and the shortest distance from the edge of mycelia of Phytophthora infestans to the edge of colony of strain X6-II-1 was 5 mm. The identification results showed that this strain belonged to Myxococcus stipitatus. The strain could kill and dissolve E. coli and inhibit the growth of Bacillus subtilis. The antibiotic substances against Phytophthora infestans were mainly present in the extracellular matrix. The optimal fermentation conditions of strain X6-II-1 were as follows: Inoculum size 10%, shaking speed 180 r/min, incubation temperature 30 ℃, incubation time 7 d. The strain X6-II-1 can produce antibiotic substances against the pathogen of potato late blight, which has the potential value for developing biological pesticides resistant to potato late blight. These findings may lay a foundation for isolation and identification of the antibiotic substances and the development of new pesticides resistant to potato late blight.

The soil microbial diversity has been characterized as an important soil quality indicator. In this study, the soil bacterial community constitution of Jasminum sambac Ait was analyzed by the high throughput sequencing (Illumina Miseq) of the 16S rRNA V3-V4 hypervariable region, to investigate differences of the soil bacterial community structure among different months during the jasmine flower season and the relatedness between community structure of bacteria and soil physic-chemical properties. The results indicated that the soil bacterial diversity of J. sambac was very rich, including 41 phyla, 98 classes, 225 orders, 427 famlies and 799 genera. The 6 phyla with high abundance were Proteobacteria, Acidobacteria, Chloroflexi, Actinobacteria, Bacteroidetes and Firmicutes. The most abundant phylum in each month was Proteobacteria (22.0%~28.4%). The abundance of the phylum Acidobacteria was the lowest (11.7%) in June, suggesting the best soil quality in this month. The Shannon index and Simpson index were the maximum (6.9264) and the minimum (0.002901) in October, respectively, which reflecting the highest bacterial diversity. These results demonstrated that the low temperature was beneficial to increase the soil bacterial diversity. The results of clustering and principal component analysis showed that the bacterial community structure of the soil samples in July, August and September possessed the highest similarity, while those of the soil sample in June and October exhibited obviously difference. Heatmap and redundancy analysis indicated that soil bacterial diversity were significantly influenced by soil properties including the soil pH, organic matter, total and alkali hydrolyzable nitrogen, total and available phosphorus, total and available potassium. Among them, pH, total phosphorus and available potassium were the three most important environmental factors. Moreover, there were positive correlations between the three main bacterial phyla (Firmicutes, Actinobacteria and Chloroflexi) and the six soil environmental factors, including pH, organic matter, total nitrogen, available nitrogen, total phosphorus and total potassium. This work provides useful informations for field management of J. sambac during flower season and soil conservancy ecological balance.

Abstract Late embryogenesis abundant protein (LEA) is a kind of cytoprotective protein accumulated under abiotic stress in plant. LEA gene family is the hot spot of stress research. Salvia miltiorrhiza is an important medicinal plant and its dry roots or rhizomes have been widely used as traditional Chinese medicine. Its yield and quality are easily affected by adversity. The objective of this study is to identify LEA proteins in S. miltiorrhiza and analyze the gene sequence, physical and chemical properties, tissue expression specificity and expression profile under different stress treatments. Based on S. miltiorrhiza genome and transcriptome database, SmLEA genes were identified and the physical and chemical properties were analyzed by bioinformatics methods. The phylogenetic tree, conservative motifs, gene structure and promoter region were analyzed by MEGA5.0, MEME, GSDS2.0 and BDGP bioinformatics tools, and the expression profile of SmLEA genes was analyzed by using the qRT-PCR and public RNA-seq databases. A total of 23 SmLEA genes were systematically identified and divided into 7 groups, SmLEA-1, SmLEA-2, SmLEA-3, SmLEA-4, SmLEA-5, SMP and Dehydrin, respectively. The theoretical of isoelectric points of SmLEAs ranged from 4.51 to 10.3 and most SmLEAs belonged to the highly hydrophilic protein. Prediction results showed that SmLEAs were distributed in different subcellular compartments, such as nucleus, mitochondrion, chloroplast, cytoplasm, and extracellular matrix. Obvious differences were observed in motif composition in genes among different groups. The gene structure was conserved and introns were relatively few. The promoter region contained a large number of MBS (MYB binding site), TGACG-motif, TCA-element, low temperature response (LTR), high temperature response element (HSE), GARE-motif, CGTCA-motif and abscisic acid responsive element (ABRE) abiotic stress response cis-element. The results of qRT-PCR showed that SmLEA genes expressed in root, stem, leaf and apical bud, with a higher expression level in root and stem. The expression of SmLEA genes in the seedlings of S. miltiorrhiza was tested after treatment by salt, dehydration, injury, high temperature and low temperature. It was found that the expression of most genes were up-regulated, of which SmLEA1-3, SmLEA4-2, SmLEA4-6, SmLEA7-2, SmLEA7-4 were significantly up-regulated in the 5 stress treatments. The results of digital gene expression profiles showed that after treatment with methyl Jasmonate (MeJA) and salicylic acid (SA), SmLEA genes were more than doubled in the expression of 9 genes and 2 genes respectively, indicating that SmLEA genes had a higher response to methyl jasmonate (MeJA). SmLEA proteins were highly conserved and the conserved motifs of different subgroups were specific. The promoter regions of SmLEA genes contained a large number of abiotic stress response cis-element. Most of the genes responded to salt, dehydration, injury, high temperature, low temperature stress, MeJA and SA hormone treatment, suggesting that the family genes played an important role in abiotic stress. This study analyzed the gene sequences, physicochemical properties, tissue expression specificity and expression levels of SmLEA family genes under different stress treatments, which provides the basic data for for further study of SmLEA family genes and the resistance mechanism and breeding new resistant varieties of S. miltiorrhiza.

Abstract Escherichia coli served as an important zoonotic pathogen that cause diarrhea in human and neonatal livestock, and the E. coli with fimbriae of the F4 family are one of the major causes of diarrhea. Previous study showed that the Mucin 4 gene (MUC4) was likely to be the receptor of E. coli F4ab/F4ac. Analysis of codon usage patterns contribute to a better understanding of the molecular mechanism and the evolution of a particular gene. To reveal the codon usage patterns of MUC4 gene, this study performed codon usage bias analysis of the complete coding sequences of the MUC4 gene from 13 different mammals. The indexes of codon usage bias of the MUC4 gene including nucleotide composition, effective number of codon and relative synonymous codon usage were calculated. Meanwhile, the analysis of codon usage bias parameter were performed to investigate the main factors that influence the codon usage bias of the MUC4 gene. The results showed that the codons ending with G or C were preferentially used in the MUC4 gene of all the analyzed species. RSCU analysis showed that all of these species had 6 optimal codons, whose biased strongly codons were CUG, AGG, GCC, AUC, GUG, ACA. Moreover, the codon usage patterns of the MUC4 gene were found to be mainly influenced by GC content at the third position. Clustering analysis by RSCU values demonstrated that Sus scrofa and Felis catus, Homo sapiens and Canis lupus familiaris were clustered respectively, which disagreed with taxonomic relationship. In conclusion, this study systematically provides insights into the codon usage patterns of the MUC4 gene. These results can provide the scientific basis for selecting a suitable recipient animal in animal genetic improvement, enhancing exogenous expression level of the MUC4 gene to improve the resistance to E. coli F4ab/F4ac infection by the methods of gene engineering and codon optimization.

Abstract Primordial germ cells (PGCs) are the progenitor cells of gametes involving in the development of embryonic germ cells (ESCs). 3β-hydroxysteroid dehydrogenase 2 (Hsd3β2) can promote cell differentiation in the steroid hormone pathway, but its specific regulation mechanism on the formation of PGCs is still unknown. The study aims to construct a short hairpin RNA (shRNA) interfering vector of Hsd3β2, a key gene in the process of steroid hormone synthesis and to observe its effect on the differentiation of chicken (Gallus gallus) ESCs into PGCs. Hsd3β2 was sythesized, amplified, and ligated into the lentiviral plasmid pGMLV-SC5. Gene sequencing was done to identify the successful construction of the vector. DF1 was transfected by 3 vectors and a control vector to choose the best one for the next lentiviral package. On the basis of retinoic acid (RA) induction, ESCs were transfected by sh-Hsd3β2 and the cell morphology was observed at 2, 4 and 6 d, respectively. Cell samples were collected for further experiments. qRT-PCR was used to detect the expression of Hsd3β2, marker genes chicken KIT proto-oncogene receptor tyrosine kinase (Cvh) and chicken KIT proto-oncogene receptor tyrosine kinase (C-kit), downstream genes cytochrome P450 subenzyme (Cyp1a1), UDP glucuronosyltransferase family 1 member A1 (Ugt1a1) and 17beta-hydroxysteroid dehydrogenase7 (Hsd17b7). Flow cytometry in vitro showed the number of the positive cells. After injecting the vector in vivo, genital ridges were collected in 4.5 d. qRT-PCR was used to detect the expression of Hsd3β2, marker genes and downstream genes. Flow cytometry was used to analyze the number of PGCs. PGCs were observed by optical microscopy and periodic acid-schiff stain (PAS). The target gene was successfully ligated into the lentiviral vector. The vector had a high efficiency and shRNA-2 has a better interference efficiency in DF1. qRT-PCR showed that after transfecting sh-Hsd3β2 and pc-Hsd3β2, the expression of Hsd3β2 increased. After packaging with lentiviral, the viral titer was 5×106 TU/ mL. qRT-PCR showed that after interfering Hsd3β2 in vitro/in vivo, the expression of downstream genes Cyp1a1, Ugt1a1 and Hsd17b7 in the steroid hormone signaling pathway was significantly decreased (P＜0.01). After inhibiting Hsd3β2 during the induction experiment, the number of embryoid body decreased and embryoid body appeared later. qRT-PCR showed that marker genes Cvh, C-kit significantly decreased (P＜0.01). Flow cytometry indicated that after inhibiting Hsd3β2, the number of positive cells (3.27±0.19)% compared with control group (7.39±0.09)% significantly decreased in vitro. In vivo, after inhibiting Hsd3β2, PAS staining showed that the number of PGCs had been descreased. qRT-PCR indicated that marker genes decreased (P＜0.01). Flow cytometry showed that after inhibiting the expression of Hsd3β2, DDX4+ positive cells decreased.Hsd3β2 could regulate the steroid hormone synthesis pathway to positively affect the differentiation of ESCs to PGCs.

Abstract Soybean cyst nematode (SCN, Heterodera glycines) seriously endangers the yield and quality of crops. SCN as one plant-specific endoparasitic nematode, is the most destructive pathogen of soybean (Glycine max) and other legumes, while tobacco (Nicotiana tabacum) is its non-host. However, in recent years, our research team found a special SCN population (SCNT) in Shandong province, which could infect tobacco, but whose pathogenicity to soybean was very poor. In order to reveal the molecular mechanism of significant difference in pathogenicity of the same host infected by SCNT and the normal SCN population, the Illumina platform HiSeqTM 2500 high-throughput sequencing technique was conducted to detect transcriptional profile of the 2nd Stage Juvenile (second stage juvenile, J2), which were derived from SCN and SCNT, respectively. qRT-PCR was performed to validate the result of transcriptome sequencing. The result demonstrated that there were a total of 1 628 differentially expressed genes (DEGs) between SCN and SCNT, among which, 1 347 DEGs were up-regulated in SCNT with 281 DEGs being down-regulated. GO (Gene Ontology) analysis was carried out to classify functions of DEGs. In the category of biological processes, the relative up-regulated and down-regulated genes were significantly enriched in the translation process, including 139 and 72 DEGs respectively. The two GO terms under cell component representing cytosolic small ribosomal subunit and the cytosolic large ribosomal subunit were notably enriched in the relative up-regulated genes, while other apparent GO terms in the relative down-regulated genes associated with DEGs were ribosome and cytosolic small ribosomal subunit. A notable number of genes (204 up-regulated and 80 down-regulated) were categorized under the GO term of structural constituent of ribosome belonging to molecular function. Furthermore, gene expression patterns were integrated with biochemical pathway from KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. 639 DEGs might involve in 277 different metabolic pathway branches, among which, a significant number of genes participating ribosome pathway were enriched, including 210 relative up-regulated and 73 relative down-regulated genes. Other pathway of oxidative phosphorylation was notably enriched. 76 DEGs (70 up-regulated and 6 down-regulated) were categorized under phosphorylation. 6 down-regulated genes were localized to cytochrome b gene, cytochrome c oxidase CO I, CO Ⅱ, CO Ⅲ subunit, NADH oxidoreductase ND5 subunit and ATP enzyme subunit, while ND1, ND2, ND3, and subunits involved in NADH oxidoreductase were up regulated. In addition, pathways associated with nematode growth and development and parasitism (such as Nicotinate and nicotinamide metabolism, Glutathione metabolism and Metabolism of xenobiotics by cytochrome P450) were enriched, followed by 13 DEGs encoding esophageal gland cell secretory proteins. There were only two genes encoding secretory proteins (pel2 and Hgg-20) suppressed, while the other 11 genes performed different degrees of down-regulation in SCNT population. The qRT-PCR data were correlated with transcriptome sequencing result, since the correlation coefficient R2=0.98, which indicated transcriptome sequencing data was reliable. This is the first report describing differences on transcriptional levels of different pathogenic SCN and SCNT populations, which will provide a scientific basis for further analysis on molecular mechanisms of pathogenicity differences of SCN.

Abstract The cultivated strawberry, Fragaria×ananassa (2n=8x=56), is an octaploid perennial herb plant with complicated genetic background. Most agronomic and quality traits of strawberry are controlled by polygenes, resulting in difficulty in breeding. DNA molecular markers showed the genome variances and mutants directly, so it had been widely used in marker associated breeding, gene mapping, genetic map construction, QTL mapping, cultivar identification and genome research since 1990s. And it greatly improved the efficiency of breeding, especially for the perennial fruit trees which were highly heterozygous and had long growth period. In this paper, we reviewed the current status of the applications of molecular marker techniques in strawberry breeding and production, aiming to provide foundations for further studies and push forward strawberry breeding and the whole industry.

Abstract ANT (AINTEGUMENTA) gene family is a plant-specific transcription factor, which contain two highly conserved AP2 domain. However, the other regions of amino acid sequences were changeable. It is found that ANT genes, belonging to AP2/EREBP(APETALA2/ethylene-responsive element binding proteins)family transcription factors, are involved in the control of organ growth and development and responses to various external environment stressesin plant. This review focused on ANT protein, genetic characteristics, biological function and regulation mechanisms which to provide some references and inspiration for further research of plant ANT like transcription factor.