The nucleotide binding site (NBS) - leucine-rich-repeat (LRR) gene family is the largest group of resistance genes cloned from plant to date. They play important roles during pathogen infection and expansion in plants. To characterize the sequences and their conserved domains of NBS-LRR type of resistance genes in tomato(Solanum lycopersicum L.), degenerate primers were designed using sequences of conserved domains and used to amplify resistance gene analogs (RGAs) from six tomato lines OH88119, Ha7981, Ha7998, PI114490, PI128216, and TR. The PCR products were sequenced and the sequences were subjected to evolutionary analysis. A total of 157 RGAs scattering on 12 chromosomes were isolated. The deduced amino acid sequences of all RGAs contained domains such as P-loop, Kinase2, Kinase3 and GLPL, which were conserved among NBS-LRR resistance genes. Phylogenetic analysis divided these RGAs into two classes, toll interleukin-1 receptor domain (TIR)-NBS-LRR and coiled-coil (CC)-NBS-LRR. These results will provide some information for isolation of disease resistance genes and marker-assisted selection in tomato.

The compact plant architecture is an important agronomic traits in cucumber(Cucumis sativus L.)breeding, it has the potential to be used in high-density, efficient cultivation and once-over mechanical harvesting of cucumber production. Compact growth habit is controlled by a simply inherited recessive compact gene (cp). Initial fine genetic mapping with 1 273 F2 plants has delimited the cp locus to a 220 kb genomic DNA region which is cosegregated with cytokinin oxidase gene(CKX). To narrow down the region of cp gene, a new set of large F2 individuals was constructed, which was derived from the same two inbred cucumber lines, WI7201(compact vining) and WI7200(regular vining). Recombinant plants were screened by the flanking markers UW084680 and UW084870, which came from cp gene fine mapping in 2011, and among the 1 348 F2 plants, there were 57 recombinants between them. Based on the cucumber genetic and physical map information in this region, sixty-eight SSRs(simple sequence repeat) and one STS (sequence-tagged sites, STS) marker between UW084686 and UW084680 were designed and screened for polymorphism between the two parents. Two SSRs and one STS marker were used to further analysis. 2 621 F2 plants were used for fine genetic mapping of cp which included 1 348 F2 plants from the new set of F2 population and 1 273 F2 plants from cp gene fine mapping in 2011. Among the 2 621 F2 plants examined, no recombination was found between the compact locus and the two markers UW057998 and cp-STS-6. Finally the cp locus was delimited to a 178 kb genomic DNA region. Mapping effort thus far resulted in two flanking markers, CKX-indel and UW058058, which was 0.04 and 0.23 cM away from the cp locus, respectively. This result excludes the possibility of CKX as a candidate gene. As a result, the candidate gene of cp is located in the 178 kb region between CKX-indel and UW058058. The results will lay the foundation not only for screening and confirmation cp gene but also for molecular marker-assisted breeding in the cucumber.

Given great biomass yield, high composition of lignocellulose, high use efficiency of feedstock, low cost, wide adaptability, Miscanthus has great potential as an excellent bioenergy crop. M. sinensis is one of the species. This study established an efficient system by using the matured seeds’ embryo and hypocotyl as explants. Significant variations were determined for the efficiency of embryogenic callus production, regeneration, tillering, cellulose and hemicellulose contents, lignin content, cellulose and hemicellulose degradation efficiency in a diverse of M. sinensis genotypes. Additionally, rooting was found during the subculture in all genotypes, which inhibited the regeneration. No significant correlation was detected between the efficiency of embryogenic callus production, regeneration and the lignocelluloses component and their degradation efficiency. For its agronomic traits, lignocelluloses feature and tissue culture result, the genotype, PMS167, was considered as model feedstock of cellulose ethanol. The study is informative for the genetic improvement and efficient micropropagation for Miscanthus.

Echinochloa crus-galli is one of the most noxious grass weeds in Chinese rice paddies, which has evolved resistance to quinclorac after continuous application since 1990. But little is known about its resistance mechanism. In order to explore the mechanism involved, we described isolation and mRNA expression of a quinclorac-responsive gene MYB in E. crus-galli. Transcriptal factor MYBs played important roles in regulating plant growth and development and withstanding environmental stresses. But, little is known about expression profile of MYBs mRNA in herbicide-resistant barnyardgrass (E. crus-galli) under quinclorac stress. A homologue sequence of MYB was obtained from quinclora-resistant-identified E.crus-galli using RACE(rapid amplification of cDNA ends). The gene, designated EcMYB1 (GenBank accession number: JX518599) with whole length 1 844 bp, had a 1 527 bp open reading frame predicted to encode a protein of 508 amino acids with molecular weight 55.4 kD and isoelectric point 8.6. The predicted EcMYB1 contained three conserved MYB domains that make the EcMYB1 belong to the R1R2R3 gene family which was not common in plants. Based on phylogenic analysis of protein sequence between EcMYB1 and others MYB, EcMYB1 had the closest relationship with the MYBs of gramineous plants, such as Sorghum bicolor, Zea mays. The result of whole-plant dose-reponse experiments demonstrated that the near iso-genicline material used in this study was susceptible or resistant to quinclorac. The GR50 (for 50% growth reduction) was 158.3 g·AI/hm2 for quinclorac-susceptible (S) biotypes or 858.0 g·AI/hm2 for quinclorac-resistant (R) biotypes. Ratios (R/S) of GR50 value was 5.4. EcMYB1 transcriptional expression was monitored in seedlings (leaves and roots) and adult plants (leaves, roots, stems and seeds) of R and S biotypes of E.crus-galli through Real-time PCR experiment with EcActin (GenBank accession number: HQ395760) as the reference sequence. The relative expression of EcMYB1 in transcriptional level was 26.22~52.50 in R biotypes and 6.85~34.62 in S biotypes, such as 52.50 in leaves of seedling of R biotypes and 6.85 in leaves of S adult biotypes, and 1.22~4.78 times greater in the R plants than that in the S plants. After exposure to quinclorac, its increased relative expression was 189.66~395.45 in R biotypes and 61.91~144.29 in S biotypes，such as 395.45 in roots of seedling of R biotypes and 61.91 in stems of S adult biotypes, and 2.46~4.06 times greater in the R plants than that in the S plants. Its expression was higher in all tissues tested of R biotypes than in that of S plants before or after quinclorac treatment. The difference expression of EcMYB1 mRNA in R and S biotypes suggested that EcMYB1 maybe involve in resistance to quinclorac in E. crus-galli. The results of this study provide basic information for the further research of function of the EcMYB1 in resistance to quinclorac in barnyardgrass.

Molecular marker technique has become the important tool for genetic diversity analysis. Miniature inverted-repeat transposable element(MITE) transposable marker is a type of PCR marker with high polymorphism and easy detection by agarose gel electrophoresis in peanut(Arachis hypogaea L.). 33 pairs of AhTE primers were used to amplify 115 peanut accessions (including 26 varieties and 89 advanced generation lines), 31 pairs got better results and each pair produced 1~3 bands with average bands of 1.84. Out of 57 bands amplified among 115 peanut accessions, 54 bands were polymorphic with polymorphic ratio of 96.49%. Average coefficient of genetic similarity(GS) was 0.6902 ranged from 0.4035 to 0.9825. The maximal GS was from peanut variety Mi-2 and Mi-3 and minimum was from lines F0316-1-1 and H0502-5-2-1. Cluster analysis was carried out using NTSYS-pc V2.10 software based on unweighted pair-group method with arithmetic means(UPGMA). At the GS of 0.65, all accessions were classified into 3 groups A, B and C. Group A consisted of 9 varieties and 11 lines, Group B was made up of 94 accessions including 16 varieties and 78 lines, and Group C had only one line NP13-2-1-3. Lines derived from same hybridization combination or varieties from same province were clustered into one group, which showed genetic relationship evaluated by MITE transposable markers consisted with their pedigree or origin. Results above suggested that AhMITE transposable marker is a useful marker for genetic diversity of peanut varieties and lines.The study also revealed that it is worth considering the use of AhMITE as DNA markers, AhMITE has many useful characteristics that other DNA markers do not have, it will contribute to the construction of linkage maps, evolutionary studies on the Arachis genome, and as a convenient tool for molecular breeding, it can assist in the progression of genetics, genomics and the breeding of peanut and its relatives.

Transmissible gastroenteritis virus (TGEV) can make the pig infectious gastroenteritis disease, and lead to death of large numbers of the pigs(Sus scrofa). Our study investigated that the inhibitory activities of anthocyanins from Sambucus williamsii Hance to TGEV in vitro. Anthocyanins structure of S. williamsii Hance was identified by LC-MS. Prevention and treatment roles of anthocyanins in the TGEV-infected different phase were studied, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and inverted microscope assay were used to observe the swine testicular (ST) cell survival rate and cell morphological changes. The results showed that S. williamsii Hance contained six major anthocyanins, and one of the main component was cyanidin-3-glucoside. ST cell was well-grown without the cytotoxic response when treated with anthocyanins lower than 50 μg/mL. Anthocyanins could restrain the proliferation of TGEV under 10 μg/mL, and the inhibition effect showed as dose-dependent manner in the safe concentration. In cell culture condition, the prevention was better than treatment effects about anthocyanins anti-TGEV. In short, this paper identified that there were at least six kinds of anthocyanins component of Sambucus williamsii Hance, and the best activity of anthocyanins against TGEV was about 50 μg/mL, and this will provide theoretical basis for the development of the new plant source of antivirals medicine.

The metabolism of adipose tissue is known to regulated by gonadal steroid hormones such as testosterone. Castrated and intact male pigs(Sus scrofa) demonstrate striking difference in body fatness, but up to now, the molecular mechanisms underlying such difference are still not clear. To determine whether the increased body fat mass in castrated male pigs is associated with the changes of gene expression in adipose tissues, we compared the mRNA expression levels of genes related to fat deposition in both subcutaneous and abdominal fat adipose tissue depots between intact and castrated male pigs using Real-time PCR method. Moreover, effects of castration on body fatness trait and serum hormones concentrations were also investigated in the present study. The results showed that castration could significantly reduce serum testosterone concentrations (P<0.01), while serum leptin concentrations were significantly increased by castration in male pigs (P<0.01). The castrated male pigs had lower body weight compared to the intact male pigs (120.6±4.77) kg vs (133.29±7.25) kg(P<0.05), but the levels of adiposity were higher in castrated male pigs than in intact male pigs. For example, the carcass fat weight, leaf fat weight, back fat thickness at shoulder and back fat thicknesses at buttock were all significantly higher in castrated male pigs than those in intact male pigs (P<0.05). Additionally, castration of male pigs caused a significant increase in peroxisome proliferator-activated receptor γ(PPARγ), fatty acid synthase(FAS), acety-CoA carboxylase(ACC), steraroyl-CoA desaturase(SCD) and malic enzyme 1(ME1) mRNA expression in subcutaneous fat adipose tissue (SAT) (P<0.05). The mRNA expression levels of sterol regulatory binding protein-1C (SREBP-1C) gene was not significant different in SAT adipose tissue between intact and castrated male pigs (P>0.05). The expression changes of lipogenic genes in castrated male pigs were different between adipose tissue depots. In abdominal fat adipose tissue (ATT), only the mRNA expression levels of FAS and ACC genes were significantly induced by castration in male pigs (P<0.05). However, no significant changes of PPARγ, SREBP-1C, SCD and ME1 mRNA expression level in AAT adipose tissue were induced by castration in male pigs (P>0.05). In conclusion, our results demonstrated that castration could increased body fat accumulation in male pigs, and castrated male pigs had higher expression levels of PPARγ, FAS, ACC, SCD and ME1 in both SAT and ATT adipose tissue. These results suggest that the increased fat accumulation in castrated male pigs is partly caused by the increased of expression levels of lipogenic genes in adipose tissues.

To investigate the effects of different diets on nutritive components in the muscle of genetic improvement of farmed Tilapia(GIFT) in high-density fishery mode, the nutritional components in the muscle of the inflated floating feeds group and the common sinking feeds group of GIFT were tested and analyzed with routine methods. The results showed that the contents of crude protein、 crude fat and crude ash in the muscle of the inflated floating feeds group were significantly higher compared with the common sinking feeds group(P＜0.05), while the content of moisture appeared in contradiction(P＜0.05). Eighteen amino acids were found in the muscle of two groups, and significant differences(P＜0.05) in the content of many amino acids were also found between two groups. Contents of delicious amino acid(DAA)、essential amino acid(EAA) and total amino acid(TAA) of the inflated floating feeds group were significantly higher compared with the common sinking feeds group(P＜0.05) . The essential amino acid indexs (EAAI) of two groups were 65.64 and 62.62, respectively, but the inflated floating feeds group was conformed to FAO/WHO criterion only. According to amino acid score(AAS) and chemical score(CS), the first limited amino acid was Trp of two groups, but the second limited amino acids were different. Among the 11 fatty acids, the contents of most saturated fatty acid(SFA) in the inflated floating feeds group were significantly higher compared with the common sinking feeds group(P＜0.05) , while the content of most unsaturated fatty acid(UFA) appeared in contradiction. According to the composition of trace elements in the muscle of GIFT, Zn was the highest content in the inflated floating feeds group, while Fe was the highest content in the common sinking feeds group. In a word, the inflated floating feeds were better than the common sinking feeds. The results of this study provide the basis for the nutritional quality of GIFT of high-density fishery mode.

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from the mesoderm, which have ability to differentiate into osteoblasts, chondrocytes and adipocytes. In this experiment, the effects of cyclopamine (CPA), a specific inhibitor of hedgehog signaling pathway, on cell division cycle and adipogenic induction and osteogenic induction of bovine(Bos taurus) adipose mesenchymal stem cells (AMSCs) were studied. After isolation and identification of AMSCs, CPA was used to treat AMSCs, then effects of CPA on cell cycle and adipocyte differentiation and osteoblast differentiation associated gene expressions were investigated using flow cytometry and Real-time PCR. The results indicated that treatment of AMSCs with CPA at different concentrations affected cell cycle progression in a dose-dependent manner. CPA resulted in cells arrested at G0/G1 stages. After adipogenic induction and osteogenic induction, no obvious morphological differences was observed in the formation of adipocytes and osteoblasts. However, the Real-time PCR detections of the induced cells showed that both peroxisome proliferator-activated receptorγ(PPARγ, critical adipogenic gene) and runt-related transcription factor 2(Runx, critical osteogenic gene) were significantly changed during adipogenic induction or osteogenic induction. PPARγ expression was significantly increased after 4 d treatment compared to the control (P＜0.01), Runx expression was lower in treatment group than the control (P＜0.01). These results suggested CPA induced AMSCs arrested at G0/G1 stages and CPA treatments of AMSCs affected adipogenic and osteogenic induction in gene expression level. This experiment provides a basis for the study of hedgehog signaling pathway in AMSCs proliferation and differentiation.

To investigate the effectiveness of ayu (Plecoglossus altivelis) artificial gynogenesis by different inducing methods, in this paper, the development of ovum was triggered by inactivated ayu sperm, then cold shock, heat shock and hydrostatic pressure treatment were carried out to retain the second polar body, respectively. The genetic feature of the offspring was analyzed by simple sequence repeats (SSR) and sequence-related amplified polymorphism (SRAP). Our data suggested that the fertility rate, eyespot rate and hatching rate of clod shock group were (39.6±2.5)%, (25.3±3.5)% and (6.7±1.2)%, respectively. The fertility rate, eyespot rate and hatching rate of heat shock group were (61.0±2.0)%、(34.7±2.1)% and (11.0±2.0)%, respectively. And the fertility rate, eyespot rate and hatching rate of hydrostatic pressure group were (80.3±2.1)%、(66.3±1.5)% and (57.0±2.0)%, respectively. These data showed that hydrostatic pressure treatment was the optimum approach to induce ayu gynogenesis. And the hatching rate was significantly higher than that of cold shock and heat shock with P＜0.01. The survival rate of 15-day-old offspring of hydrostatic pressure group was (15.2±8.2)%, while that of clod shock group was less than 1% and no survival was found in heat shock group. The results of SSR suggested ayu gynogenesis could be induced by hydrostatic pressure treatment through retaining the second polar body. In tested gynogenesis offspring samples, 14 samples in 16 were detected without male-specific bands. The gynogenesis rate of offspring was 87.5%. In the locus of Pag-003, the pattern showed that the gynogenesis offspring was all homozygote. While, in the locus of Pag-051 and Pag-076, the pattern showed that the recombination rates were 28.6% and 93.3% respectively between centromere and SSR locus. The result of SRAP showed that 5 bands in the female parent and 4 bands in the male parent were amplified respectively including 4 female-specific bands and 3 male-specific bands by the K-group primers. In the offspring, The dominant and recessive ratio of the female-specific band a4 was 1∶1(P=1.000), and that of a1, a3 and a5 were the same with each other and approximately followed Mendel’s law (1.33∶1)(P=0.705). One band in the female parent and 8 bands in the male parent were amplified respectively including 7 male-special bands by the P-group primers. Male-parent specific bands were detected in No.7 and No.14 (12.5%), which implied the sperms, triggering the ovum development of No.7 and No.14, were incompletely inactivated. Comparing with SSR, SRAP could be directly used to analyze multi-locus genetic feature each time and provide more genetic information. Our data suggest that hydrostatic pressure treatment could be used to induce ayu artificial gynogenesis. The results are of enormous significance in theoretical direction and practical application for ayu selective breeding. Our data suggest that hydrostatic pressure treatment could be used to induce ayu artificial gynogenesis. The results are of enormous significance in theoretical direction and practical application for ayu selective breeding.

Erianthus arundinaceus is not only an important sugarcane breeding germplasm resources, but also a potential energy grass plants, and the genetic diversity is the premise of utilization of E. arundinaceus. In this experiment, we analyzed genetic diversity of 45 accessions of E. arundinaceus collected from natural habitats in seven Chinese provinces using 20 sequence-related amplified polymorphism(SRAP) primer pairs (PPs). Results showed that: 1) 20 PPs generated 434 bands and 69% was polymorphic bands. The Nei's genetic similarity(GS) coefficient of the tested accessions ranged from 0.703 to 0.986, and the average Nei's GS coefficient was 0.842. There was rich genetic diversity among the tested wild resources of E. arundinaceus. 2) The tested accessions were clustered into 3 groups and 7 subgroups by unweighted pair-group method with arithmetic means(UPGMA) dendrogram and principal component analysis. The cluster results demonstrated a strong geographic effect on molecular variation of the local E. arundinaceus, and there was no significant relationship between genetic distance and geographic distance among accessions. 3) The genetic diversity and cluster results were affected by geographic landforms and environments, the gene flow was blocked by Ocean and mountains, and was promoted by river. A high level of genetic variation of E. arundinaceus and main impact factors of genetic variation are presented, this conclusion will be highly valuable in genetic improvement in the species per se and likely in sugarcane.

Reference molecule is a recombinant plasmid which contain endogenous and exogenous genes. In order to provide a reference material for genetically modified organisms detection, the reference molecule which included six genes(Cauliflower mosaic virus(CaMV 35S)、Bacillus thuringiensis cry1Ab(cry1Ab)、sucrose phosphate synthase(SPS)、fiber-specific acyl carrier protein(fsACP)、nopaline synthase(NOS)、neomycin-3'-phosphotransferase (NPTⅡ) was constructed and confirmed by PCR, restriction analysis and sequencing. The repeatability, uniformity, homogeneity, stability and quantification method were also analyzed. The results shown as following: The quantitative accuracy of reference molecule which quantified by digital PCR was highest, followed by the PicoGreen method, and the quantitative accuracy of reference molecule which quantified by spectrophotometry was last; The repeatability, uniformity and homogeneity of reference molecules were very well, and the reference molecule could be used as the candidate of reference material; After one month stored at low temperature, the stability of reference molecule shown well. But the results of stability at high temperature shown that the reference molecule could not be stored. The reference molecule could be used for quantitative analysis of transgenic materials. The methods of construction, preparation and correctness verification of reference molecules can provide a reference example for the research of standard plasmid. Besides, this study provides a scientific basis and an example for the analysis and storage of reference molecules.

Lipopolysaccharide-binding protein(LBP) is key factor in identifying endotoxin of gram-negative bacterium and starting immune response. The aim of this study was to understand LBP gene codon use features and provide foundation for selecting appropriate receptor animals and expression systems. In this research, pig(Sus scrofa) LBP gene(GenBank Accession: NM-001128435.1) based on electronic cloning sequence was analyzed by Codon W, CHIPS, and CUSP programs online, and then compared with eight disease resistance-related genes, genomes in model organisms and LBP genes from 14 animal species. The results showed that most of pig LBP genes were bias toward the synonymous codons with G and C at the third codon position. There were twenty-seven biased codons (relative synonymous codon usage(RSCU)＞1), whose biased strongly codons were GCC, CAC, CTG and TCC (RSCU≥2). However, all of eight disease resistance-related genes were bias toward codons with G and C, and had twenty-three biased codons, whose biased strongly codons were GCC, ATC, CTG and GTG. The comparative analysis results of LBP genes from 14 animal species also showed that gene expression level was general in 14 animal species，and all of the LBP genes were bias toward the synonymous codons with G and C at the third codon position, which was possible due to special functions of these genes. Clustering analysis showed that pigs belonging to artiodactyla and animals belonging to carnivora were together for a class, which disagreed with taxonomic relationship. The differences in codon usage frequency between pig LBP gene and genome of Mus musculus were less than other two kinds of model organisms' (Escherichia coli and Saccharomyces Yeast) genome. Therefore, Mus musculus expression systems might be more suitable for expression of LBP gene. This study can provide certain theoretical basis for selecting the appropriate receptor animals in animal genetic improvement, selecting appropriate expression systems and improving the expression level of LBP gene.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection would lead to suboptimal innate immune responses and a weaker adaptive immune response, thus increasing the susceptibility to secondary infection and weakening the body's resistance to diseases. The signaling pathway of nuclear factor-κappaB (NF-κB) plays an important role in the viral infection and diseasees development by regulating host innate immune response and cell proliferation, differentiation and apoptosis. It has been proved that some non-structural proteins (Nsps) of PRRSV can inhibit the activation of NF-κB signaling pathway in early infection, host innate immune response would be suppressed when the transcriptional expression of genes regulated by NF-κB did not happen or reduced, such as proinflammatory cytokines, cell adhesion molecules, chemokines and acute reactive protein. All above revealed that the NF-κB could play a key role in PRRSV infection. In this paper, we make a brief summary of the NF-κB signaling pathway activation and function and the role of NF-κB in the antibody-dependent enhancement (ADE), innate immune response, apoptosis of alveolar macrophages, and pulmonary injury PRRSV infection caused, also the non-structural proteins (Nsps) influences on NF-κB signal pathway were expounded. This paper can provide some new ideas for control strategies and vaccine researches of PRRSV.