Rubber is a kind of important strategic material in China. There are important significance in rubber tree genomic study. In this study, the chromosome samples were prepared with the enzyme and low osmotic method using tender leaves of Hevea brasiliensis RY-7-33-97 as material. The preparation of chromosome was optimized. Chromosomes were successfully isolated by slender glass needles microdissection. The microdissected chromosomes was digested with proteinase K, and its DNA was extracted and digested with Sau3A, amplified by linker adaptormediated PCR(LA-PCR) for 2 rounds, a smear of DNA fragments ranging from 250 to 2 000 bp was generated. Amplified single chromosome production was verified by Southern blot. The results showed that the LA-PCR products were homogeneous with the H. brasiliensis genomic DNA, indicating that DNAs from the chromosome had been successfully amplified. The ninth chromosome was verified by fluorescence in situ hybridization (FISH), and constructed microcloning library of the ninth chromosome in H. brasiliensis. The library contains 48 000 clones. The inserts averaged 650 bp, and varied from 300 to 1 200 bp with no-load rate of 1%. In this study, construction of the single chromosome library of rubber tree, by the means of single chromosome microdissection and microcloning technology, will provide a new perspective for the study of rubber tree genome and a molecular assisting way for breeding of rubber tree.

G-protein coupled receptor 120 (GPR120) is the only highly expressed G protein-coupled receptor in adipose tissues, which plays a critical role in various physiological processes. DNA methylation is an important epigenetic modification to mediate tissue-specific gene expression that mostly occurs in CpG-rich regions (CpG islands). To study the effects of CpG island methylation for the expression levels of GPR120 in porcine (Sus scrofa)subcutaneous (backfat) and visceral (greater omentum) adipose tissues, this study used qRT-PCR to detect the relative expression levels of GPR120 in different adipose tissues of Jinhua pigs (6 months and 7 years old), using CpG island searcher online software to scan the CpG island distribution across the whole GPR120 DNA sequence (5 000 bp upstream of first exon to 5 000 bp downstream of last exon), and detected the methylation status of CpG island in different adipose tissues using Sequenom MassArray methylation approach, using bisulfite-sequencing PCR (BSP) to validate the MassArray results. Results showed that compared with greater omentum, subcutaneous adipose tissue had a higher adipocyte volume both at 6 months and 7 years (P＜0.01), and the relative transcript levels of GPR120 significantly higher expressed in subcutaneous adipose tissue than that of greater omentum (P＜0.01), which was consistent with adipocyte volume differences. The DNA sequence of GPR120 had abundant GC contents, and contained five CpG islands which were predicted by bioinformatics analysis successively located within different genomic locations, that were the 5' untranslated region, first exon, intron and 3' untranslated region. The methylation status of CpG island within second intron in subcutaneous adipose tissue was significantly higher than that of greater omentum (P＜0.01) both at 6 months and 7 years, which was confirmed by the BSP approach, while the methylation levels of other 4 islands had no significant difference between subcutaneous adipose tissue and greater omentum at the two time points. There was a positively correlation between the changes in methylation levels of CpG island within second intron and gene expression. This study presented an integrated analysis of gene-wide DNA methylation patterns in different tissues, and results indicated that GPR120 had abundant CpG islands, the tissue-specific different methylation of CpG island in gene body positively associated with GPR120 expression between subcutaneous adipose tissue and greater omentum. This comprehensive analysis provides a solid basis for exploring epigenetic mechanisms of GPR120 differentially expressed in tissues.

Perilipin gene(PLIN) plays a crucial role during lipid metabolism and fat deposition. In order to reveal the genetic effects of polymorphisms in PLIN on carcass and fatness traits in chicken, PLIN gene polymorphisms in 384 Luqin chicken (Gallus gallus)were detected by DNA sequencing and PCR-RFLP methods. Three single nucleotide polymorphism (SNP) loci, g.2272C＞T, g.2319C＞T and g.2467G＞A in PLIN gene were found, and their genetic effects on carcass and fatness traits were analyzed by sas6.12. Based on the 3 SNPs, 7 haplotypes were constructed by PHASE2.0. C-C-G was the major haplotype with the frequency of 71.94%, while the frequencies of C-C-A and C-T-A were lower than 1%. Eleven diplotypes were obtained from the 7 haplotypes, H1H1 was the dominant diplotype with the frequency of 52.60%. Statistical analysis showed that diplotype was extremely significantly associated with living body weight, carcass weight and leg muscle weight (P＜0.01), and significantly associated with abdominal fat weight and percentage of abdominal fat (P＜0.05), as well as breast intramuscular fat (P=0.094 9). The multiple-range test indicated that H1H3 chickens had the highest living body weight, carcass weight, leg muscle weight, abdominal fat weight and percentage of abdominal fat, followed by H4H6 chickens. H6H6 chickens were the lowest. The breast intramuscular fat content was the highest in H4H6 chickens and the lowest in H1H3 chickens. The results indicated that H6H6 was disadvantaged for chicken carcass and fatness traits, which should be removed during the breeding, while H4H6 was an advantaged diplotype which was suggested to be chosen to improve the carcass traits and meat quality of Luqin chickens. The research build the theoretical basis for the PLIN gene applied in poultry molecular breeding.

The purpose of this study is to identify cytoplasmic type of isonucleur allocytoplasm cytoplasmic male sterile lines with Aegilops kotschyi, Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines (90-110 and 8222) and to improve its directional genetic improvement in the research and application of hybrid wheat(Triticum aestivum L.). Sequence characterized amplified region(SCAR) molecular marker technique and amplified fragment length polymorphism(AFLP) marker molecular technique were used to analyze the wheat mitochondrial DNA. Three isonucleur allocytoplasm cytoplasmic male sterile lines with Aegilops kotschyi, Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines were selectively amplified using 64 primer combinations EcoRⅠ-NNN/MseⅠ-NNN, and the results showed that 113 out of 682 bands were polymorphic. Primer E-AGG/M-CTA was found to amplify a special 300 bp fragment in the cytoplasmic male sterile lines with Aegilops Kotschyi cytoplasm. Specific fragment was collected and sequenced, SCAR molecular markers were redesigned with Primer Premier 5.0, and SCAR marker(YW1) amplified a fragment in 3 isonuclear alloplasmic cytoplasmic male sterile lines and their maintainer lines. SCAR marker(YW2) was successfully designed and validated, and it did not amplify fragment in isonuclear alloplasmic cytoplasmic male sterile lines with Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines (90-110 and 8222), only one specific 198 bp fragment was amplified with YW2 in ms(Kots)-90-110 and ms(Kots)-8222 which showed sequence similarity was as high as 99%, and it was sequence of nicotinamide adenine dinucleotide(NADH) dehydrogenase gene (GenBank: EU534409.1). Oxidoreductase (complexⅠ) is the entry enzyme of mitochondrial oxidative phosphorylation, it was closely associated with the cytoplasmic male sterility in wheat. This SCAR marker can be used in molecular marker-assisted breeding for identifying wheat male sterile lines and provide theoretical and technical support for identifying cytoplasmic types of wheat.

Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) widely exists in various organisms and it is a key enzyme in process of glycolysis and gluconeogenesis. The genome walking method was applied with primers designed according to the 5'terminal sequence of GAPDH gene (GenBank accession No.: EF592180.1) in order to obtain its upstream sequence from wheat(Triticum aestivum L.), and a 1 071 bp upstream sequence of GAPDH was cloned. Sequence structure analysis and transcriptional regulatory elements prediction showed that the transcription start site was located at about 700 bp upstream of ATG, containing TATA-box, CAAT-box and several cis-acting elements such as MYB, MYC, WRKY, ABRE, HSE and GT-1, which were responsible to abiotic stress like ABA, drought and high salt stress. The CaMV 35S promoter of pBI121 was replaced by the upstream sequence of GAPDH to build a fusion plasmid in purpose to verify the promoter activity of the sequence. The results of Agrobacterium-mediated transient expression showed that the promoter exhibited obvious activity to drive GUS gene expression under ABA, low temperature, and especially under PEG6000. In conclusion, the promoter region of GAPDH gene was cloned by genome walking and the analysis showed that it was a strong inducible promoter.

The copy number of exogenous gene in transgenic plants is an important factor influencing the expression and genetic stability of transgene. To detect the copy number of exogenous target gene RNAi sequence of lipoxygenase gene (Loxi) in 6 transgenic lines of wheat (Triticum aestivum) from 2 populations (3 transgenic lines in from each population), and analyze the activity of lipoxygenase in seeds and obtain transgenic wheat lines with low lipoxygenase activity and stable inheritance of Loxi, TaqMan Real-time quantitative PCR was performed to estimate the copy number of Loxi in wheat transgenic TF5 lines, using an endogenous single copy gene Pinb (puroindoline b) as a reference gene. The lipoxygenase activity was determined using the linoleic acid as the substrate, and statistical analysis was performed simultaneously. The results showed that correlation coefficients of standard curve for the reference gene and the exogenous gene both were close to 1, and the amplification efficiency of Pinb and Loxi genes were appropriately, which was 107.7% and 104.5%, respectively. The copy number of Loxi in 6 transgenic wheat lines were different, the minimum and maximum copy number was 1 and 8, respectively. The lipoxygenase activity in the seeds of 2 parent cultivars Shanyou 225 and Xinong 889 were (860±28.20) and (1 132±59.80) U, respectively. Compared to that of their parent materials, the lipoxygenase activities in the seeds of transgenic lines were decreased by various degree (approximately from 21% to 95% of their parents), in which 5 of them decreased significantly. For 3 transgenic lines of Xinong 889, their exogenous gene copy number was 2, 6 and 8, respectively, and their corresponding lipoxygenase activity was 32.76%, 26.39% and 21.05% of their parent, respectively. For 3 transgenic lines of Shanyou 225, their exogenous gene copy number was 1, 3 and 6, respectively, while their corresponding lipoxygenase activity was 95.47%, 51.06% and 37.73% of their parent, respectively. The copy number of exogenous Loxi in the transgenic wheat lines was negatively correlated with the lipoxygenase activity in its the seeds. The results showed that the activity of lipoxygenase in these transgenic wheat lines could be suppressed effectively by RNAi. In this study, the transgenic wheat lines with low lipoxygenase activity in their seeds were developed, and their exogenous gene copy number was analyzed. These transgenic wheat lines with very low lipoxygenase activity in their seeds can be used as parent marterials or new germplasm in wheat breeding program.

Lux operon codes a series of proteins that mediate the autofluorescence of luminous bacteria by catalyzing long-chain fatty acids molecules through the oxidation reduction reactions. Methods for constructing luminescent Escherichia coli transformed with lux operon were introduced to study distribution and survival of E. coli in vitro and in vivo. In this study, we transformed the pL1217 plasmid carrying with lux operon into E.coli DH5α strain, and performed the biological characteristics of E. coli growth curve assay and fluorescence spectrum analysis of lux operon. Meanwhile, the bioluminescent assay of E. coli transfected with lux operon were performed by infrared video data imaging system (IVIS) kinetics to test whether the luminous bacteria could inherited stably in vivo. The E. coli cells with lux operon at the log phase were collected and injected into the cecum of ICR mouse (Mus musculus). 24 h after infection, fluorescent bacteria from mouse small intestine occurred and the number of the bacteria was counted. The results showed that the E. coli transfected with lux operon in log phase produced strong blue/green fluorescence that last more than 16 h in vivo, and indicated that E. coli cells were capable of colonizing in small intestine and maintaining fluorescence without disturbing its normal activities. The study of E. coli cells colonizing in mouse bowel provides an experimental model that can be used to monitor the pathogenic E. coli activities in animal bowel in vivo and investigate the pathogenesis enteral infections by bacteria.

Tomato mosaic virus (ToMV), a type species of the genus Tobamovirus, severely affects the yield and quality of crops. ToMV-N5 infection caused Lycopersicon esculentum Mill. main cultivar Hezuo 903 systemic necrosis. It is essential to obtain its viral infectious clone for analyzing the mechanism of ToMV-N5 pathogenesis. In this study, the full length of ToMV-N5 genomic RNA was cloned into pCB301 vector which carried duplicated 35S promoter and ribozyme and then pCB301-ToMV-N5 was transformed into Agrobacterium tumefaciens GV3101. Agroinfiltration results indicated that the seedlings of Nicotiana benthemiana and L.esculentum infected by pCB301-ToMV-N5 appreared the same phenotypic caused by ToMV-N5 viral particles. Vector pCB301-ToMV-N5CP45GFP was constructed by replacing the ORF of viral coat protein gene (CP) with that of green fluorescent protein gene (GFP) and the data showed that the vector could highly express GFP in leaves of N. benthemiana in the presence of gene silencing suppressor p19. Even in leaves infiltrated with A. tumefaciens cells diluted 1∶500 from a starting optical density (OD600) of 1.0, most cells of the infiltrated zone expressed GFP at 7 days post-infitation(dpi). Due to the lack of CP, pCB301-ToMV-N5CP45GFP could not move to upper leaves of host plants. pCB301-ToMV-N5CP45GFP-CPU5 was obtained by inserting CP (nt 5 498~6 145) of Tobacco mosaic virus (TMV) U5 strain and the phenotype of pCB301-ToMV-N5CP45GFP-CPU5 on N. benthamiana suggested that the recombinant ToMV-N5 could move to non-inoculated leaves and GFP the whole plant expressed in the whole plant at 10 dpi. While the results from the seedlings of L.esculentum inoculated with pCB301-ToMV-N5CP45GFP-CPU5 suggested that there was only some weak GFP expression on inoculated leaves under the microscope. The successful construct of Agrobacterium-mediated infectious clone and its expression vector provides basic data for dissecting the molecular mechanism of ToMV-N5 replication and movement.

To explore the pluripotent maintenance and update the functional influence of pluripotent genes cNanog and cPouV in chicken (Gallus gallus) embryonic stem cells(cESCs), cESCs were transfected by the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed and screened early in our lab. Interference efficiency was detected with Real- time PCR and reached more than 65% at 96 h (P＜0.05). With downregulation of cNanog or cPouV gene expression, cESCs showed differentiation and growth rate of these cells slowed down. Domed colony of these cells gradually disappeared when the edge of colony became irregular. At 96 h after transfection, alkline phosphatase(AKP) and stage-specific embryonic antigen-1(SSEA-1) expression in cESCs were not be detected if cNanog gene was interfered but still stayed a part of expression in cells that cPouV gene was interfered. While downregulation of cNanog plus to cPouV gene expression, the remaining expressions of AKP and SSEA-1 were all disappeared. Results showed that cPouV and cNanog gene played an important role in maintaining pluripotency and self-renewal in chicken embryonic stem cells, and cNanog gene was dominant. These results may provide insights into the molecular regulation mechanism of avian during development and the difference between avian and mamal.

Nitric oxide(NO) produced by macrophages plays a crucial role in the process of resisting and killing pathogenic microorganisms. In order to demonstrate the effect of mammalian target of rapamycin(mTOR) signaling pathway on NO in the process of host macrophages infected by Mycobacterium tuberculosis, BALB/c mouse (Mus musculus) macrophage cell line RAW264.7 was infected by Bacillus Calmette-Guérin (BCG), which were cultured in the presence of mTOR specific inhibitor rapamycin. The production of NO and the mRNA and protein expression of inducible nitric oxide synthase (iNOS) were detected by the methods of Griess, qRT-PCR and Western blot, meanwhile the lipopolysaccharide (LPS) was used as reference. The results showed that the production of NO was significantly increased in the RAW264.7 cells which were treated by BCG and LPS for 6, 12 and 24 h compared with the untreated?control?group, the expression levels of mRNA and protein of iNOS were extremely significantly increased (P＜0.01). The production of NO and the expression of iNOS were extremely significantly inhibited by 10 nmol/L rapamycin after BCG infection for 6 and 12 h (P＜0.01) compared with the control group; however, after infection for 24 h, the inhibition effect by rapamycin was not significant (P＞0.05), but the expression of iNOS was extremely significantly inhibited (P＜0.01). Moreover, after the RAW264.7 cells were treated by LPS for 6 and 24 h, 10 nmol/L rapamycin could extremely significantly inhibit the production of NO and the expression of iNOS (P＜0.01); the production of NO was significantly inhibited after the treatment for 12 h (P＜0.05). Further more, when the cells were treated by LPS for 6 h, the inhibition of rapamycin on the expression of iNOS was not significant difference (P＞0.05), but extremely significant difference after treatment for 12 and 24 h (P＜0.01). The present findings showed that rapamycin has important regulation effect on the production of NO in the process of Mycobacterium tuberculosis infection of host macrophages; these data may provide an important theoretical basis for the role of mTOR signaling pathway in the genesis and development of the tuberculosis.

To identify single nucleotide polymorphisms (SNPs) and candidate genes affecting growth traits of Jinghai yellow chicken(Gallus gallus), a kind of chicken 60K SNP chip was used to study 396 Jinghai yellow chicken for the 15 SNPs, and the associations were analyzed between the 15 SNPs and 12 growth indexes. Linkage disequilibrium (LD) and haplotype analysis of the significant SNPs were also performed. The results showed that 9 of the 15 SNPs were identified to be associated with at least one growth trait of Jinghai yellow chicken (P＜0.05), and seven of the 9 SNPs were identified to be associated with more than one growth trait(P＜0.05). Among these SNPs, rs15497877, rs13972304, rs13553164, rs14917647 and rs13973515 were associated with body weight and average daily weight gains on both early (2~8 weeks) and late growth (10~16) stage (P＜0.05); rs316142388 and rs14917305 only had effects on the late growth stage (12~16 weeks) (P＜0.05), rs314214528 and rs13553485 only had effects on the early growth stage (2~6 weeks) (P＜0.05). The genetic parameters indicated that the average polymorphism information content (PIC) was a bit small and continue selection of Jinghai yellow chicken was possible and necessary. The result of LD and haplotype analysis showed that SNPs in the region of 173.5~175.0 Mb on chromosome one was in strong linkage disequilibrium and had high research value. Moreover, haplotype TTTCCTCAC should be valued as it accounted for 32.4% of total haplotypes. The functions of a few proximal genes including karyopherin alpha 3 (KPNA3), deleted in lymphocytic leukemia 7(DLEU7) and forkhead box O1 (FOXO1) were discussed and they were found to affect chicken growth by different ways. All these represent results not only promote the researches on SNPs having effects on growth traits of Jinghai yellow chicken, but also can benefit the utilizations for marker assisted selection (MAS) on Jinghai yellow chicken and even Chinese chickens.

To research the effect of breeds and feeding patterns on chicken meat flavor, and screening of inosine monophosphate acid (IMP) and intramuscular fat (IMF) candidate genes, Chengkou chicken、Daninghe chicken、Qingjiaoma chicken were selected as the study populations, to analyze IMP content in breast muscle by high performance liquid chromatograph, extrac the IMF in breast muscle by soxhlet extraction. The expressions of 5 candidate genes related to IMP and 9 candidate genes related to IMF were detected with Real-time quantitative PCR. The results revealed that the IMP content of Chengkou chicken was higher than that of Daninghe and Qingjiaoma chickens, the IMP content of scatter-feed chicken was higher than that of henhouse-feed; the IMF content of Chengkou chicken was lower than that of Daninghe and Qingjiaoma chickens, the IMF content of scatter-feed chicken was lower than that of henhouse-feed. The expression levels of IMP candidate genes, aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase (PURH) and adenosine monophosphate deaminase1 (AMPD1), and the IMF candidate genes, heart fatty acid binding protein (H-FABP), leptin receptor (LEPR), fatty acid transport protein 1 (FATP1) and uncoupling protein 3 (UCP3) were correlated with the contents of IMP and IMF in breast muscle, respectively. The results showed that both breed and feeding pattern had significant influence on IMP, IMF contents and expression of their candidate genes. The expression levels of candidate genes were closely related with IMP and IMF content. The results of this research will provide some theoretical basis for high quality poultry breeding.

Owing to the lack of Cymbidium ensifolium genomic resource, the development and application of molecular markers have been limited. In order to obtain more efficient markers, we sequenced C. ensifolium transcriptome deeply to search simple sequence repeat (SSR). Here, high-quality reads were assembled into 101 423 unigenes. The unigenes were used to explore genic-SSR derived from gene (genic-SSR), which resulted in 17 793 genic-SSRs. The location of the SSR was estimated according to the open reading frame (ORF) and untranslated region (UTR) within the unigenes. A total of 3 065 genic-SSRs was located within 5'UTR；5 235 was within 3'UTR; 2 907 was within coding region; 6 586 was undetermined. A total of 80 was chosen from genic-SSRs sets, based on their motif, size and location. The primers were designed for 80 genic-SSRs using software "PRIMER 3.0", and then tested for their amplification. Of 80 genic-SSR primers, 61 succeeded in PCR amplification and 39 showed polymorphism among 12 C. ensifolium accessions. Among 39 polymorphic markers, the polymorphism information content (PIC) averaged 0.348, ranging from 0.141 to 0.746. The 39 polymorphic markers identified 108 alleles among 12 C. ensifolium accessions, and the allelic number was 2.77 per locus, ranging from 2 to 6. The SSR26 was the most polymorphic marker, with PIC of 0.746 and 6 alleles. The Nei genetic distance was estimated for each pair of the 12 C. ensifolium accessions, which ranged from 0.010 to 0.475, with an average 0.260. In addition, unigenes containing genic-SSR were annotated on the basis of BLAST similarity searches. The unigenes containing the 61 genic-SSRs were searched against non-redundant (Nr), gene ontology database (GO), eukaryotic orthologous groups (KOGs) and kyoto encyclopedia of genes and genomes (KEGG) database, respectively. Most of them were annotated as crucial genes that were associated with important biological function. There were 52 unigenes hit Nr sequences, of which 38 were classified into 25 GO terms, 18 were assigned into 9 KOG categories, 15 were involved in 14 KEGG pathways. Thus, the 61 genic-SSRs having PCR product were also endowed with the corresponding biological information. These polymorphic genic-SSRs enriched the types of C. ensifolium molecular markers. They are very useful to analyze genetic diversity and reveal population structure in C. ensifolium. They could be also used to explore gene with linkage mapping and population genetics-based approaches, such as association mapping. The annotation of genic-SSR is extremely helpful to understand associations between these markers and the interesting traits. All of the information will in turn accelerate research on genomics, and functional genomics of C. ensifolium.