Unintended effects of genetically modified (GM) plants should be paid particular attention in the process of safety assessment of GM crops and their products, especially in regard to some long-term and potential food safety issues. To our knowledge, few research concerning on revealing possible unintended effects in underground portions of GM plants. In this study, with the aim to comprehensively investigate unintended effects of transgenic rice (Oryza sativa cv.) Huahui 1 (HH1), we performed DNA microarray analysis both in aerial and underground portions of HH1 and compared to its non-transgenic parent (Oryza sativa) Minghui 63 (MH63). The expression level of 8 genes located within 100 kb up and down-stream of insertion site of HH1 were not changed significantly. Considerable differentially expressed genes (DEGs) and significantly changed pathways were identified. The results showed that all these changes were not enough to cause obviously unintended effects both in aerial and underground portions of transgenic rice HH1. In addition, our study provides a clue to investigate unintended effects of GM plants in further safety assessment.

To analyze the association between mRNA expression levels of Spermadhesins gene (AQN1, AQN3, AWN, PSPⅠand PSPⅡ) and sperm motility inhibitor gene (SPMI) in ejaculated sperm with sperm capacitation and acrosome reaction status, swine (Sus scrofa) sperm capacitation and acrosome reaction status were induced in vitro. The expression levels of Spermadhesins and SPMI genes in sperms were analyzed by semiquantitative RT-PCR (sqRT-PCR). The results showed that the percentage of sperm capacitation and acrosome reaction was 58.41% and 35.34%, respectively. Most Spermadhesins mRNA expression levels were increased with capacitation time prolonged. AQN1 and PSPⅡ mRNA levels were increased a little with capacitation for 4 h, while significantly increased with capacitation for 8 h (P＜0.05). The difference between 4 and 8 h was not significant (P＞0.05). AQN3 and PSPⅠmRNA level demonstrated the similar expression rythme, with no significant change at 4 h capacitation or significant higher at 8 h capacitation than 4 h and control. AWN mRNA level was decreased with capacitation time prolonged, but the difference was not significant (P＞0.05). SPMI mRNA level was decreased at 4 h capacitation and increased at 8 h capacitation, but no obvious rythme with no significant difference among groups (P＞0.05). AQN1, AQN3, PSPⅠ, PSPⅡ and SPMI mRNA level were all increased after acrosome reaction induced by progesterone ( P4) except AWN, among which PSPⅠand PSPⅡ were increased significantly (P＜0.05), and AWN was decreased significantly (P＜0.05). The results showed that Spermadhesins might play a positive control on sperm capacitation, or take part in the interaction of oocytes and sperms. SPMI gene might not work on sperm acrosome reaction and capacitation. This study will be helpful greatly to further research the mechanism of sperm action, capacitation and acrosome reaction.

Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is an infectious porcine (Sus scrofa) respiratory tract disease causing severe economic losses worldwide in the swine industry. Aiming at developing a safe attenuated vaccine against multiple serotypes, the APP serotype 7 isolates WF83 strain (APP-7-WF83) based recombinant transfer vector pBSLNKAR was constructed, which contained the kanamycin resistance gene (Kan) as a resisitance screening gene replacing complete toxin Ⅱ activate gene C (apxⅡC), with the N-terminal and C-terminal fragment squence of toxinⅠstructural gene A (apxⅠA) of Actinobacillus pleuropneumoniae RTX-toxin (Apx) gene family from APP serotype Ⅰ isolates shope4074 strain inserted. The vector pBSLNKAR was electroporated into the parent strain APP-7-WF83 to build mutant strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC. The antibodies expressed by N-terminal fragment squence of toxin Ⅰstructural gene A (apxⅠAN) neutralized the toxinⅠso that the mutant had no hemolysis compared with APP-7-WF83 by hemolytic detection. PCR identification showed that WF83ΔapxⅡC/apxⅠAN/apxⅠAC lacked about 377 bp of apxⅡC and was inserted about 377 bp of apxⅠAC and 1 188 bp of apxⅠAN compared with APP-7-WF83. With 0.5 mL of trypticase soy broth (TSB) (blank control), the parent strain APP-7-WF83 group and the mutant WF83ΔapxⅡC/apxⅠAN/apxⅠAC group inoculated mice (Mus musculus) with 1.1×109 cfu/mL, respectively. The results showed that the mice of parent strain groups died of 100%, the mice of mutant group had no death. The results confirmed that the gene deletion composite attenuated strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC was successfully constructed, which supplies basic data for further research on gene deleted attenuated vaccine.

Paired-liked homodomain transcription factor 2 (PITX2) is an important transcription factor of Wnt/β-catenin signaling pathway, and plays an important role in cell proliferation, differentiation, organogenesis, pituitary development and embryonic development. Different tissues of Saanen dairy goats (Capra hircus) were collected as the experimental materials in this study. The PITX2 gene was cloned and analyzed using reverse transcription-polymerase chain reaction (RT-PCR), TA cloning and bioinformatic methods, meanwhile the mRNA expression profiles of this gene was revealed using quantitative Real-time PCR (qRT-PCR). The results showed that the coding sequence of PITX2 gene was 978 bp, encoding 325 amino acids, which shared 99%, 95%, 90%, 90% and 93% homology with Bos taurus, Sus scrofa, Mus musculus, Rattus norvegicus and Homo sapiens, respectively. The PITX2 gene expressed in varied tissues of rams and ewes, which showed a broad spectrum of gene expression. The expression of PITX2 gene was the highest in pancreas of ram, followed by small intestine, lungs and muscles, and the lowest in spleen. Moreover, the expression levels in pancreas, lungs, small intestine and muscles were significantly higher than that in other tissues. The expression of PITX2 gene was the highest in liver of ewe, followed closely by breast, lungs, and the lowest in adipose tissue. Moreover, the expression of breast was significantly higher than that in heart, kidney, muscle and adipose tissues, which suggested that this gene might be related with lactation performance. The results will lay a foundation for further study of the gene function in the regulation of lactation.

The pteridine pathway regulates the synthesis of yellow to reddish pteridine pigments in zebrafish (Danio rerio). GTP cyclohydrolase 1 (Gch1) is found as the rate-limiting enzyme of this pathway. Koi carp (Cyprinus carpio var. koi) is one of the most widespread cultured ornamental fish with various color patterns. In order to study the function, conservation and expression pattern of Gch1 in different colors of Koi carp, the present study amplified the full-length Gch1 cDNA (GenBank No. KP056544) based on the assembled transcriptome of common carp (Cyprinus carpio). The protein consistsed of 251 amino acids with a conserved GTP-cyclohydrolaseⅠdomain. Gene Ontology annotation indicated that Gch1 was involved in the hydrolase activity, nitrogen compound metabolic process and amino acid metabolic process. Homolog search found it's high conservation across vertebrates. Adaptive evolution analysis revealed that the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) of Gch1 between Koi carp and zebrafish was only 0.06, indicating that it was under negative selection. These results indicated that Gch1 participated in the pteridine pathway in Koi carp. Differential expression analysis revealed significantly higher level (P＜0.05) of Gch1 in red skin than in white skin, indicating that the high expression of Gch1 maight turn the skin color to red. This study revealed the different expression of Gch1 between red and white skin in Koi carp and found the conservation of Gch1 across vertebrates, which may be helpful to the study of pigmentation mechanism in other fish.

In order to identify simple sequence repeat (SSR) markers which associated with yield or quality characters, this study was conducted to investigate polymorphism analysis of locis、cluster analysis based on unweight pair method using arithmetic averages、population structure analysis based on mathematic model and linkage disquilibrium analysis among 91 wheat (Triticum aestivum L.) cultivars and breeding lines using 88 wheat SSR markers distributed throughout the wheat genome. A total of 883 alleles were detected, with 2 to 30 alleles per locus and a mean genetic richness of 10.03. The average genetic diversity index was 0.703, with a range from 0.160 to 0.932. The mean PIC value was 0.667, ranging from 0.148 to 0.929. These results indicated that the genetic diversity of the 91 Chinese winter wheat cultivars was low. Further analysis showed that B genome had the highest genetic diversity and D genome had the lowest genetic diversity, significant differences were observed between these 2 genomes (P＜0.01). The collection of winter wheats could be divided into 3 and 4 subgroups based on UPGMA (unweight pair method using arithmetic averages) and STRUCTURE analyses, respectively. The results of clustering had no obvious relationship with the geographic eco-type, but partly correlated to some extent with pedigrees. The results of clustering and population structure analyses could complement and confirm each other, making the results more reliable. Results of AMOVA (analysis of molecular variance) indicated that the majority genetic variation occurred in different individuals within a population. Furthermore, gene flow proved that there was some high frequency of gene intercommunication among different subpopulations. The baseline of linkage disequilibrium (LD) decay for genome was r2=0.028 7 and the mean LD decay distance of the whole genome was 4.3 cM, while the mean LD decay distance of A, B and D genome was 3.7, 1.0 and 4.1 cM, respectively. The LD decay distance and rate of B genome was shorter and faster than A and D genomes. The results of this study were expected to provide valuable information for future association analysis. Evaluating the genetic diversity, population structure and LD of Chinese winter wheat will provide useful information support for enhancing the efficiency of further breeding, molecular assisted selection (MAS) and association analysis.

Platelet-derived growth factor receptor (PDGFRα) is the member of tyrosine kinase receptor family and has a potential role of regulating lipid metabolism. The aim of this study was to clone cDNA of PDGFRα and analyze its mRNA expression level in different tissues and different periods of pig (Sus scrofa). The specific primers were designed based on the published nucleotide sequence of human (Homo sapiens), bovine (Bos taurus) and mouse (Mus musculus) in GenBank, and the whole cDNA of porcine PDGFRα gene was cloned using RT-PCR and RACE. The cloned full-length cDNA of PDGFRα gene was 6 360 bp (GenBank accession: KP329568) containing an open reading frame (ORF) of 3 270 bp, 2 964 bp 3' untranslated regions (UTR), 126 bp 5'UTR, encoding a protein of 1 089 amino acid. This gene spaned approximately 148 Mb on the genome, and contained 23 exons and 22 introns. The homology analysis of porcine PDGFRα gene indicated that the nucleotides and amino acids had high similarity with human, bovine, sheep (Ovis aries) and mouse. Ten N-glycosylation sites and 75 phosphorylation sites were predicted in the translated PDGFRα protein, as well as one signal peptide. The protein structure analysis showed that PDGFRα protein had 2 transmembrane helices. The N-terminal of the PDGFR protein was high hydrophobic, but the C-terminal showed high hydrophilism. Quantitative Real-time PCR (qRT-PCR) results suggested that PDGFRα mRNA expressed in heart, liver, spleen, lung, kidney, small intestine and skeletal muscle. PDGFRα mRNA expressed abundantly in spleen, followed by kidney and lung. Six kinds of muscle, including soleus muscle, supraspinatus muscle, biceps femoris muscle, psoas minor muscle, gastrocnemius muscle and loin muscle, were selected. The qRT-PCR results showed that the expression of slow muscles represented with soleus muscle was extremely higher than that in fast muscles represented with loin muscle (P＜0.01). Different growth stages expression analysis showed that expression of porcine PDGFRα gene was extremely higher in the primary stage than that in weaning and adult stage (P＜0.01). In conclusion, the research obtained the full-length cDNA of porcine PDGFRα gene, the rule of tissue expression and relative mRNA expression of PDGFRα gene in different muscles. The results provide a basic data for further understanding of the function of PDGFRα gene.

Actinomycetes, as important sources of naturally occurred antibiotics, are strains for discovery of natural anti-infective agents. In order to isolate and purify the endophytic actinomycetes from mangrove plants in Beilun Estuary National Nature Reserve, Guangxi Zhuang Autonomous Region, explore their potential antimicrobial activities against pathogenic bacteria and fungi, and preliminarily clarify taxonomic status of bioactive strains, all 15 samples from different parts of 5 mangrove plants were cultured separately in 14 different liquid media for 4 weeks，and the enriched cultural media were then spreaded on the corresponding agar media with standard serial dilution plating method to isolate and purify the colonies. A total of 200 isolates were purified from samples including 62 strains from Avicennia marina, 43 strains from Kandelia candel，38 strains from Rhizophora stylosa, 29 strains from Clerodendrum inerme and 28 strains from Excoecaria agallocha, respectively. After removing the duplicate isolates by comparison of their cultural and morphological characterizations, 106 purified endophytic strains were tested for their antimicrobial activities against 6 strains of bacteria and 20 strains of Candida albicans by point planted method and disk diffusion method. A total of 52 isolates showed positive results and the positive rate was 49.1%. Among them, inhibitory rates against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteusbacillus vulgaris, Hemolytic streptococcus and Klebsiella pneumoniae were 16%, 15.1%, 4.7%, 7.5%, 11.3% and 3.8%, respectively; Inhibitory rates aganist drug-sensitive strains of Candida albicans and drug-resistant strains of Candida albicans were 16.0% and 29.2%, respectively. The bioactive strains were identified preliminarily as Streptomyces (21 strains), Nocardiopsis (26 strains), Pseudonocardia (2 strains), Agrococcus (2 strains) and Isoptericola (1 strain) by phylogenetic analysis based on 16S rRNA gene sequences. Among them, the similarities of both strains UMBR1016 and UMBR1034 with Nocardiopsis alba DSM 43377T were 98.27% and 98.37%, respectively; the similarity between strain UMBR 1008 and Nocardiopsis flavescens SA6T, and between strain UMBR 1028 and Streptomyces pratensis ch24T, was 98.36% and 98.49%, respectively. The results that similarities of 16S rRNA gene sequences were less than 98.6% indicated 3 strains including UMBR1016, UMBR1034 and UMBR 1008 were potential new species of Nocardiopsis genus and UMBR 1028 was potential new species of Streptomyces genus. The 38 and 35 isolates were found to show antimicrobial activity against at least one tested bacteria and one strain of Candida albicans, and the inhibitory rates were 35.8% and 33.0%, respectively. Abundant endophytic actinomycetes, including new species and potential antimicrobial activities found in the study, indicated that endophytic actinomycetes from mangrove plants in Beilun of Guangxi province could be a potential source for the discovery of new species and natural products and deserved further development in next step. The study will improve the conservation of mangrove plants in our country and further utilization of endophytic actinomycetes as important sources of drug discovery.

Promoters of resistance genes play important roles on interactions between the pathogen of Xanthomonas and the host plant. Bacterial spot caused by Xanthomonas is a disease that severely threats tomato production. Current data suggest that the recognition of the pathogen race T3 to the resistance gene Rx4 might be associated with the promoter of the gene. Therefore, a 2 149 bp upstream sequence of the resistance gene Rx4 was obtained from the genomic DNA of Solanum pimpinellifolium accession PI128216 by PCR amplification in this study. Comparing to the database of Plant Cis-acting Regulatory DNA Elements (PLACE) revealed that the sequence contained the basic core promoter elements, gibberellin, abscisic acid, and ethylene related cis-elements, dehydration, salt, and cold response-related cis-elements, large number of pathogen-related transcription factors such as WRKY and MYB binding elements, and resistance-related elements W-box, G-box. A series deletion fragments of the promoter sequence were fused with the GUS gene and transiently expressed in tomato (Nicotiana benthamiana) cotyledon and tobacco leaves. Quantitative analysis of GUS activity indicated that the activity was very weak using a 297 bp fragment from -338 to -41. The remaining three fragments of 511 bp from -552 to -41, 1 244 bp from -1 285 to -41, and 733 bp from -1 285 to -552 showed increased activities but the differences were not remarkably. These results suggested that the region from -1 285 to -338 had an important role on promoter activity of the Rx4 gene. This finding provides useful information for further investigating the promoter types as well as the interaction between the resistance gene Rx4 and the race T3 pathogen of bacterial spot in tomato.

In order to analyze genetic polymorphisms of Allium fistulosum CMS (cytoplasmic male sterile) lines and maintainer, 3 mitochondrial gene probes ATP synthase F0 subunit 6 (atp6), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 3 (cox3), combined with 2 restriction enzymes HindⅢ and BamHⅠ were used to detect the polymorphism of mitochondrial genomes. The Southern blot results indicated that mitochondrial genome had polymorphism between the CMS lines and the maintainer lines. By using the combination of HindⅢ/nad5 in the RFLP (restriction fragment length polymorphism) experiment, 5 CMS lines showed 1 specific band of 10 kb，and the 4 maintainer lines showed another specific band of 7 kb. In summary, RFLP (HindⅢ/nad5) could be used to identify the type of cytoplasm in Allium fistulosum. These results may help to reveal the molecular mechanisms underlying the CMS and will be useful for mitochondrial gene cloning studies of the Allium fistulosum.

α-1,2-fucosyltransferase gene (FUT1) is the important candidate gene of piglets' resistance to Escherichia coli F18. This study aimed to probe into the structures and important mutation loci of FUT1 gene. Based on the previous detection results which implied the transcription activity of FUT1 gene upstream sequence by dual-luciferase reporter, we screened the -1150 to 50 bp CpG islands, the promoter and the transcription factor binding sites of the FUT1 gene upstream sequence which showed promoter activity. Then, in this study, FUT1 gene upstream sequence was analyzed by cloning and bioinformatics, and the polymorphism in wild boar and 11 different pig breeds were investigated by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results showed that there were 3 CpG islands which located at -1104~-886 bp, -796 ~-619 bp and -316~-130 bp; the analysis of characteristics of the promoter and transcription factor binding sites showed there were Sp1, NRF-2, c-ETS and GATA-1 existing; the results of PCR-SSCP analysis showed that there existed only a T(-726)C point mutation and AA, AB and BB genotypes were detected. Difference of genotype distribution between domestic pigs and foreign was significant. Genotype AA was undetectable in the Chinese native pig breeds such as Erhualian pigs, Fengjing pigs, Huai pigs and Xiang pigs,, and frequency of genotype AA was low in other Chinese native pig breeds (0.012~0.058), genotype BB was dominant in wild boar and Chinese native pig breeds except Xiang pigs; but the frequency of genotype AA was higher in 3 western commercial breeds (Duroc, Yorkshire and Landrace) and 50% Duroc descent Sutai pigs and genotype BB barely existed (0.021~0.112). This study further confirmed that the genetic basis which resistance to E. coli F18 was significant differences between Chinese native pig breeds and western pig breeds, which might be related to the T(-726)C point mutation in FUT1 gene promoter. This result provides a theoretical foundation for screening and determining stable genetic marker, as well as conducting the analysis of regulatory mechanism of swine FUT1 gene.

This study sought to assess mitochondrial DNA (mtDNA) diversity and origin of Jiangxi Anyi Tile-like Chickens(Gallus gallus domestiaus). Blood samples were collected from 30 chickens. The complete sequence of the mtDNA D-loop was PCR amplified and subsequently sequenced (Genbank accession No.KP893677~KP893682.). DNA sequence of mtDNA D-loop of five red jungle fowl subspecies were collected from the GenBank and used as reference sequences. The results showed that, the D-loop region of Anyi Tile-like Chickens was 1 231~1 232 bp in length. The nucleotide diversity was (0.006 13±0.000 14), and the haplotype diversity was (0.837±0.027). The 6 haplotypes were observed based on 18 mutant site and clustered into three clades (A, B, and C), of which clade C was dominant. Three haplotypes belong to clade C, two haplotypes are in clade B, one is in clade A. The clade A and clade B were clustered with Gallus gallus spadiceus, clade C was clustered with four subspecies of red jungle fowl. These results show a high mitochondrial D-loop diversity and indicate multiple maternal origins for Anyi Tile-like Chickens. The results in this study will be a reference for conservation and utilization of Anyi Tile-like Chickens.

Pepper (Capsicum) is an economically important genus of the Solanaceae family that also includes tomato and potato. Pepper has the same chromosome number as tomato and potato, but there is a huge difference in genome size between pepper and tomato/potato. Pepper genome expansion occurred after the pepper-tomato divergence (~36 Mya), which made it perfect model for genome evolution studies. Developments on research technology have led to the achievements of exciting investigation results in this field in the last decade. Pepper genome expansion, the types of chromosome rearrangements, whole genome duplication, fruit development biology, evolution of genes involved in capsaicin synthesis, and molecular footprints of artificial selection have been elucidated successively. In this paper, we review the research advances in genomics of pepper and make some suggestion for future investigation and provide a resource for genetic improvement and breeding programs.

A quick, simple, in situ, cheap and accurate method for lipid content determination in microalgae is important to microalgae biodiesel research. In this study, Chlorella vulgaris was cultivated in nitrogen depleted media, and total lipids were extracted by Soxhlet extraction at different growth stages, and the result showed lipids content increased with the increase of cultivation time. Lipid content of the biomass was highest (12%~13%) at the 4th days of cultivation. At the same time, we used three staining methods (Nile red, Sudan Black B and Oil Red O dye methods) for quantification the relative lipid levels in various stage strains of C. vulgaris. The results showed that there was a linear correlation between the lipids level and the light absorb value of stained microalgal cells. The content of microalgal cell lipids (y) was related to the absorbance(x) of cells and can be expressed with a regression equation of the form y =kx+b. The regression equation of Nile red method was y=0.009543x+3.087(fluorescence value at 580 nm), and the correlation coefficient (R2) was 0.9577(P＜0.001). For Sudan Black B method, the equation was y=30.06x+3.705(optical density at 645 nm), and R2 is 0.9603(P＜0.001). In Oil Red O dye manner, the regression equation was y=72.83x-27.87(optical density at 600 nm), and R2 was 0.8878(P＜0.005), but optical density at 600 nm ranged only within 0.4~0.6. These results indicated that three staining strategies were suitable for estimating the lipid content of C. vulgaris cells. The Nile red method involved incubating live cells in the dye and then reading fluorescence with a spectrophotometer, and application of this method was simple, high sensitivity, rapid but high cost. Sudan black B method had relatively complicated steps, but this strategy had lower cost and equipment. Oil Red O dye manner also could be used to test lipids content, but its most significant disadvantage is its low sensitivity and large error. The research results provide guidance for testing lipids content according to experimental conditions.

Gene targeting is a new molecular biology technique, which introduces heritable genetic modification on the basis of DNA homologous recombination. Compared with classic approaches, homologous recombination using a promoter trap vector could yield higher targeting frequencies. It is very important for the correct expression of exogenous gene. In this study, neor gene was amplified by PCR technology from plasmid pIRES2-EGFP and BamHⅠ and BglⅡ restriction enzyme sites were added to the upstream and downstream, respectively; then the PCR fragment was cloned into pMD19-T simple Vector, named pMD-neo. Plasmids pMD-neo and pEGFP-C1 were digested by BamHⅠ and BglⅡ, and ligated together to obtain intermediate plasmid pEGFP-neo. Then pEGFP-neo and pIRES2-EGFP were digested by Bsp1407Ⅰ and SfiⅠto constitute another intermediate plasmid pIRES-EGFPneo. At last, multiple cloning sites (MCS) were added to pIRES-EGFPneo after digestion of pIRES-EGFPneo and pMD-MCS with BamHⅠand MluⅠ, and the final gene trap vector pMCS-IEN was constituted. To test the expression of green fluorescence protein (GFP) and G418 resistance gene, the PGK promoter was added to the upstream of MCS of the pMCS-IEN and the reconstitute plasmid was transfected to Hela cells. GFP and G418 resistance colonies were obtained after screening by microscopy and G418 selection. When the homologous arms were inserted into the basic vector pMCS-IEN and formed the promoter trap vector，it could be used to target the objective gene. Therefore, this vector will become a useful tool to generate gene modification animals.