Seedborne bacteria, including Pseudomonas viridiflava (Pv), P. syringae pv. lachrymans (Psl), P. syringae pv. syringae (Pss), Acidovorax citrulli (Ac), Xanthomonas cucurbitae (Xc) and X. melonis (Xm), are important diseases in melon crops production. By using three sets of multiplex PCR and a chromogenic macroarray system, it was allowed us to detect multiple target bacterial species in bacterial suspension, naturally infected or artificially infected seed samples simultaneously without the use of agarose gel electrophoresis. The average qualitative detection limit could be reach N×103~N×104 CFU/mL with pure bacteria culture. Target bacteria could be detected from artificial infected watermelon(Citrullus vulgaris), melon(Euphorbia meloformis) and cucumber(Cucumis sativus) seeds and naturally infected A. citrulli watermelon seeds. The new method is efficient, high-throughput and practical, and gets rid of the dependence of fluorescence scanner. This study provides a method for the simultaneous detection of plant seedborne pathogens.

Due to significant advances in DNA sequencing technology, human genome sequencing is becoming a powerful tool in recent medical practice. The 1000 Genomes Project and ENCODE Project have provided a comprehensive variation and functional annotation of human genome, and such information will greatly benefit the medical application of sequencing in the long run. Nowadays, the sequencing approach is very successful in identifying the unknown mutation that leads to rare Mendelian diseases. In addition, sequencing cancer genomes comprehensively characterizes genome variations and leads to a better classification and more targeted treatment. The application of genome sequencing in common diseases and the bacteria/virus infection is still limited but promising. More importantly, the genome information not only facilitates the treatment of disease, but also provides guidance on disease prevention such as fetus genetic screening. The whole society still needs to overcome the challenges such as cost, high demand of computational power to process sequencing data and immature communication channel to take full advantage of sequencing in clinical setting. However, these issues are likely to be resolved by new technology, cloud computing solution and better education in the near future. This review focused on how recent applications of genome sequencing lead to better disease treatment and prevention.

It has been widely suggested that the incompletely reprogramming of donor cell was the major reason of the inefficiency of nuclear transfer. The present study was to investigate the effect of histone deacetylase inhibitor(Scriptaid) on the reprogramming of yak(Bos grunniens) fetal fibroblast cells, and the in vitro developmental capability of yak-bovine(Bos grunniens) interspecies somatic cell nuclear transfer(iSCNT) embryos after donor cells were treated with Scriptaid. The yak fetal fibroblasts were treated with 0, 500, 1 000, 1 500 and 3 000 nmol/L Scriptaid for 24 h, respectively. Then observed cell morphology, accounted the survival rate, analyzed cell cycle by flow cytometry, detected acetylation level of H3K9ac by immunofluorescence, and evaluated the developmental competence of iSCNT embryos, which showed that the cell morphology was changed significantly, the cell became flatten and the area of nuclear was larger after Scriptaid treatment; cell proliferation was inhibited gradually and most cells were arrested at G0/G1 stage after Scriptaid treatment for 24 h; the acetylation level of H3K9ac was enhanced along with the concentrations of Scriptaid; the cleavage and blastocyst formation rate of iSCNT embryos improved significantly when donor cells treated with 1 000 nmol/L Scriptaid for 24 h(P<0.05). The results suggested that Scriptaid can significantly improve the histone acetylation level of yak fetal fibroblast cells, pre-treated donor cells with 1 000 nmol/L Scriptaid was helpful for improving in vitro development of yak-bovine iSCNT embryos. It was concluded that Scriptaid enhances the reprogramming in terms of high histone acetylation of donor cells, and it is useful for studying reprogramming and a novel way to improve the efficiency of yak iSCNT.

In order to screen the differentially expressed protein and explore the mechanism of hepatotoxicity induced by copper nanoparticles in rat(Rattus norvegicus), the differential proteins were separated and identified by two dimensional gel electro-phoresis (2-DE) and MALDI-TOF-TOF MS, and then verified by Real-time PCR, and analyzed by bioinformatics. Total 43 spots of differentially expressed proteins were found，of which a spot 1006 associated with hepatotoxity was identified as phosphatidy lethanolamine-binding protein 1(PEBP1)(accession No. IP1002309), the results of Real-time PCR was the same as that of 2-DE. Bioinformatics analysis showed that PEBP1 was located in the cytoplasm, and had no signal peptide, it may belongs to non-secretory protein, and contain phosphatidy lethanolamine-binding protein family signature 64YTLVLTDPDAPSRKDPKFREWHH86; the random coils and extended chains were its main secondary structural elements. The homology analysis showed that PEBP1 had a high homology between rat and other nine species, and the phylogenetic tree of PEBP1 was constructed by Neighbor-joint method. The expression of PEBP1 protein was reduced in poisoned rats so as to liver cell mitochondrial dysfunction, which may be a pathway of copper nanoparticles to exert the hepatotoxic effects.

23.5 kD small heat shock protein is an important anther differentially expressed protein between male sterile line and its conversion line in wheat BNS(Bainong sterility). In order to validate the relationship between this protein and BNS male sterility, the differentially expressed mRNA of this protein's gene, hsp23.5, was examined by means of fluorescent Real-time quantitative PCR taking the anthers of BNS as materials at three key developmental stages(tetrad phase, uninucleated period and binucleated period)．The results showed that the expression quantities of the hsp23.5 mRNA in three developmental stages were continuously rised in conversion line, but down-regulated significantly in sterile line. The amount of down-regulated in sterile line was as many as the times of 3.8, 20.0 and 4.6 in the three developmental periods comparing to that in conversion line, respectively. The result of sequencing indicated that the numbers of bases of hsp23.5 cDNA segment cloned by RT-PCR were 82.56% homologous wheat sequences from NCBI. By BLAST alignment and phylogenetic tree analysis, there were the biggest segment sequence identities of 94%, and the nearest genetic distance of <0.05, between the BNS hsp23.5 and common wheat(Triticm aestivum) hsp23.6 ，and between the BNS hsp23.5 and cone wheat(T. turgidum) hsp23.5. It was also found that the mRNA expression quantity of the gene was up-regulated in sterile line in binucleated period because of higher atmosphere temperature appearance in this period. These results suggested that the hsp23.5 gene expressed constitutive in BNS conversion line, and inducible by higher temperiture in sterile line. It may be the characteristic of sensitive-temperature that regulated the down-regulated expression of the gene in sterile line. It also implied that hsp23.5 may be one of the important relation sterile gene.

The recent studies discovered bovine leukocyte interleukin-32 gene (IL-32) is a new type of inflammatory cytokine and has many biological functions. It can induce the immune cells to product immune factors and chemical factors and play an important role in autoimmune/inflammatory diseases. And IL-32 can promote cell apoptosis, inhibite the growth of mycobacterium tuberculosis and other mycobacterium's infections, and improve the ability of resistance to disease of IL-32γ. In this study, we cloned the bovine IL-32β and IL-32γ genes, which were alternative splicing isoform of IL-32β, from Qinchuan cattle(Bos taurus) spleen. The result showed that the IL-32β gene was located on bovine chromosome 25th. There were five bases difference between the cloned bovine IL-32β and the published sequence in NCBI, but the differences did not affect the protein secondary structures, suggesting that these nucleotide differences were the SNPs of IL32 gene in Qinchuan cattle. The bovine IL-32γ had an additional sequence fragment from the second intron of IL-32 gene comparing with IL-32β, and that sequence encoded additional 47 amino acids. In order to study the function of IL-32γ, we constructed the eukaryotic expression vector of pIRES-IL32γ-GFP, which was stably transfected into mouse(Mus musculus) macrophages RAW264.7. After selection, the IL32γ overexpressing mouse macrophage cells were obtained. At the same time, the macrophages that were stably transfected with human IL-32γ gene were used as a positive control. Real-time PCR analysis showed that the bovine IL-32γ gene had similar effect as that of human IL-32γ to regulate the expression of cytokines, such as IL-1β, IL-6, MIP2 and TNFα. This study provided the crucial information for further study on the biological functions of bovine IL-32γ gene.

The heat shock protein(HSP) is closely related with thermal-resistance of organisms. This study was conducted to investigate the expression of HSP90 mRNA in examined tissues from Shaoxing duck under the different treatments and during the recovery period after heat challenge, in hope of elucidating the molecular mechanism of HSP90 gene. HSP90 cDNA were cloned and characterized from the livers of Shaoxing duck (Anas platyrhyncho) using RT-PCR. The full-length of HSP90 consisted of an open reading frame of 2 211 bp encoding a putative protein of 736 amino acids (GenBank accession No. JQ837244). Comparison of amino acid sequence of HSP90 revealed that Shaoxing duck shared 92.6%~99% amino acid identity to that of domestic poultry and shared 80%~88% amino acid identity to that of some mammalian. Fluorescent Real-time quantitative PCR was applied to determined HSP90 expression after exposure to different thermal shock. Long-term treatment at 30℃ up-regulated HSP90 transcription in pituitary; long-term treatment at 35℃ significantly enhanced HSP90 expression in heart, liver and pituitary; acute treatment at 40℃ increased HSP90 level at maximum in heart, liver, kidney, pituitary and pancreas, respectively. During the recovery period, HSP90 mRNA in liver and heart reached their highest levels at 1 h post challenge and then both decreased to pre-challenge levels from 3 h post challenge. We concluded that levels of HSP90 mRNA in heart, liver and pituitary were significantly associated with the intensity of heat shock. The study provided basic data for the prevention and control of heat shock in Shaoxing duck using HSP90 mRNA expression as an index.

Growth traits of pearl oyster(Pinctada martensii) is quantitative traits. Genetic diversity of 90 individuals sampled from the fourth generation selected line of pearl oyster were evaluated by 50 EST-SSR markers, and then a correlation analysis between the markers and total weight, shell length, shell height and shell width of the selected line was performed using a GLM model of SPSS 16.0 software. The results found that total of 204 alleles were detected and the number of alleles(Ne) were between 2~9 at each locus, with 4.080 number of alleles on average. The effective number of alleles(Na) was 2.574. The mean values of observed heterozygosity(Ho), expected heterozygosity(He) and polymorphism information content (PIC) were 0.534, 0.543 and 0.491, respectively. Totally, 15 loci were significantly related with the growth traits (P<0.05). There were 7 loci significantly related with total weight (P<0.05), 6 loci significantly related with shell length (P<0.05), 6 loci significantly related with shell height (P<0.05) and 4 loci significantly related with shell width (P<0.05). Favorable genotypes for each growth traits were obtained by a multiple comparison among the loci. The correlation obtained in this study between growth trait and EST-SSR markers is benefit to further study in the marker assisted breeding of this species.

Wheat leaf rust, caused by Puccinia triticina, is an important epidemic disease of wheat in China. The objective of this research was to understand the molecular genetic diversity and genetic relationship of P. triticina populations in China. In the research, all the Chinese P. triticina samples were collected from Hebei, Henan, Shandong and Sichuan Province in 2009 for analyzing by SSR marker. For the leaf rust populations, the average observed number of alleles (Na) per locus was 1.75, the effective number of alleles (Ne) was 1.40, Shannon's information index (I) was 0.35, Nei's gene diversity (H) was 0.23, and percent of polymorphic was 75.29%. The result showed that the populations of P. triticina were considerably genetic diversified and the genetic diversity level of P. triticina in Hebei, Henan and Shandong was higher than that in Sichuan. UPGMA cluster analysis showed that 4 populations were clustered into 2 groups at similarity coefficient 0.96, Henan, Shandong and Sichuan clustered in group 2, Hebei clustered in group 1, and the closest genetic relationships were found between Henan and Shandong. Genetic differentiation was investigated by analysis of molecular variance(AMOVA). There were about 8.93% of the total variations among populations and about 91.07% of the total variations within populations. The Nm was 6.10. The result showed that the populations of P. triticina possessed relatively high levels of genetic diversity. There was high genetic similarity among populations, and the genetic relationships were associated with graphical distribution. The main genetic variation came from the same population. The migration of pathogen among the regions was presented, and this conclusion will help to determine the epidemic regions and dispersal routes of wheat leaf rust.

A total of 73 tomato(Lycopersicon esculentum) samples exhibiting stunting, yellowing and leaf-curling symptoms of tomato yellow leaf curl disease (TYLCD) were collected in Jingyang, Shaanxi Province. Using degenerate primer pair PA and PB of geminiviruses and specific primer pair TYT-F and TYT-R of Tomato yellow leaf curl virus (TYLCV), geminiviruses and TYLCV were detected in 70 samples by PCR. The DNA-B and satellite DNA molecules of geminiviruses were not detectable from all these samples with their universal primers respectively. Using rolling circle amplification PCR (RCA-PCR), cloning and sequencing techniques, the complete nucleotide sequence of isolate SX8(GenBank Accession No. JN412854)was determined to be 2 781 bp and has highly identity of nucleotide sequences (99.9%) with TYLCV-[SD2] (GenBank Accession No. GU199587)isolated from tomato in Shandong Province. The phylogenetic analysis indicated that TYLCV-[SX8] shared high relativity with those previously reported isolates of TYLCV in China. These results suggest that TYLCD in Shaanxi Province is caused by TYLCV which may originate from DNA-A molecules of TYLCV infecting tomato in Shandong Province.

Cry2Ab toxin of Bacillus thuringiensis is a toxic protein, which is wildly used in controlling lepidopteran pest in agricultural production. In this research, the cry2Ab gene (1 914 bp) was amplified from total DNA of B. thuringiensis WB9 strain by a pair of primer designed by the full-length sequence of published cry2Ab gene. Then, cry2Ab was ligated with linearized pGEX-KG vector by restriction enzyme BamHⅠand XhoⅠfor the construction of cry2Ab-pk expression vector. The soluble Cry2Ab-GST fusion protein (approximately 90 kD) was obtained after transferring Cry2Ab-PK expression vector into Escherichia coli BL21 (DE3) and then inducing by 0.8 mmol/L IPTG at 16℃ for 36 h. Total soluble protein was purified by batch purification and GST tag was removed by the use of prescission protease to obtain soluble Cry2Ab protein (approximately 65 kD). Polyclonal antibody against Cry2Ab was produced by immunizing the purified Cry2Ab to New Zealand white rabbit(Oryctolagus cuniculus) after three times of intramuscular injection and one time of intravenous injection. The titter of antibody was over 1∶150 000, measured by indirect ELISA. Specificity of the prepared antibody was determined by Western blot, showing that the polyclonal antibody against the Cry2Aa or Cry2Ab protein was positive and the antibody against Cry1Ab or Cry3Aa protein was negative. These results indicated that antibody against Cry2Ab protein can specifically identify Cry2A protein but cannot identify other three domains Cry protein including Cry1Ab and Cry3Aa. These results will provide technical support for further study of Cry2A toxins mechanism and the interaction between Cry2A toxins and its receptors.

microRNA-200b(miR-200b) has similar functions in regulating body size in mammals, while its exact roles has not been examined in mammals. To study the expression of miR-200b during the embryo development stage, and investigate the important roles of miR-200b and its target gene in regulating the body size of mouse, in this study, precursor miR-200b was amplified from the mouse(Mus musculus) genome DNA, RNA folding sofware was used to construct the secondary structure of miR-200b; potential target genes were predicted with bioinformatic software, and validated through Dual-luciferase reporter assay system; the expression profiles of miR-200b and its target gene Fog2 were detected using Real-time PCR in different tissues of adult mouse and embryos at the stage of 10.5~15.0 days after coitus. The results showed that the miR-200b precursor had the typical stem-loop structure of microRNA, and the Fog2 gene was predicted and tested to be a target gene of miR-200b, which showed negatively related expression to miR-200b in the embryos dpc10.5~15.0; qRT-PCR results showed that the miR-200b was expressed in almost all the tissues of female mouse, with higher expression in muscle, kidney and uterine, and lower expression in heart, spleen and lung tissues. From the expression profile and target gene testing results, we can conclude that miR-200b may play important roles in regulating mouse embryo development through acting with its target gene Fog2.

The accuracy, reliability and comparability of genetically modified (GM) detection result can not be ensured due to the lack of GM reference material. In this paper, the selected genetically modified (GM) maize(Zea mays) line NK603 and its recipient were grinded and the particle size was about 100 μm. Three different levels of matrix reference materials were gravimetrically prepared. The homogeneity and stability were checked by quantitative real time PCR. The result showed the matrix reference materials were homogenous and stable under the 4℃ during one year's stock. The property values determined by gravimetric method and their expanded uncertainties(k=2) of three matrix GM reference material were (0.50±0.10)%, (1.00±0.35)% and (5.00±0.35)%, respectively. Seven different laboratories were chosen to validate the property values of the GM maize reference materials and the result showed that it was agreed well with the value determined by the gravimetrical method for each level. The uncertainties of the GM reference materials were comparable with those international GM reference material. Thus, the GM maize reference materials can be applied for the GM maize quantitative detection to ensure the detection result accurate and reliable.

This paper discovered the growth improvement of red carp transferd with the myostatin propeptide gene from Larimichthys crocea . Myostatin (MSTN) propeptide gene from Larimichthys crocea and the internal ribosome entrysite(IRES)-enhanced green fluorescence protein gene(EGFP) fragment from the pIRES-EGFP plasmid were cloned by RT-PCR and PCR, respectively. After confirming their sequences, the recombinant Tol2 transposon donor plasmid pT2AL200R150G-MSTN propeptide-IRES-EGFP was constructed. Three different gene transfer methods, sperm-mediated gene transfer (SMGT), electroporation (E) and gene gun (G) were used to obtain transgenic red carp (Cyprinus carpio). In SMGT, common sperm storage buffer (S1) and hypotonic dilution (S2) were used, respectively. In electroporation, 1 500 V (E1) and 2 500 V (E2) were applied, respectively. The rates of EGFP expression were as follows: S1, 38%; S2, 48%; E1, 47%; E2, 53% and G, 2% in larvae after 72 of hatching, respectively. The positive rates of reverse transcript-polymerase chain reaction (RT-PCR) for the transgene were as follows: S1, 35%; S2, 45%; E1, 45%; E2, 55% and G, 1.8% in larvae after 10 d of hatching, respectively. After 75 d of hatching, the body length and weight of transgenic fish increased 21.31% and 27.59% in comparison with the control, respectively. The results showed that gene transfer efficiency could be improved by treating sperm with hypertonic and hypotonic dilution, and electroporation.