Bacterial leaf spot blight and bacterial fruit blotch are the two main bacterial diseases in muskmelons (Cucumis melo). They always infect the muskmelons simultaneously with the similar symptoms, and it is difficult to use traditional methods to distinguish the two bacterial diseases. In this study, a rapid and simple immun ochromatography assay (ICA)for in-site detection of Pseudomonas syringae pv. lachrymas in muskmelons was developed using a new anti-Pseudomonas syringae pv. lachrymas monoclone antibody. In qualitative detection, the detection limit was determined as 2.5×103 cfu/mL. Specificity results showed that there was no cross-reactivity to Acidovorax citrulli which indicated the good specificity of the ICA. In the quantitative detection, the detection results of Pseudomonas syringae pv. lachrymas were scanned by a membrane strip reader, and a standard inhibition curve and calibration curve were established, the reading value ratio of T and C line had a remarkable linear relation with logarithm of Pseudomonas syringae pv. lachrymas concentration, the regression equation was y=0.3922x-1.263 8, R2=0.988 1. Therefore, the established high selective ICA was a very practical method for rapid, simple and low-cost detection of Pseudomonas syringae pv. lachrymas, and not only achieved in situ rapid detection of Pseudomonas syringae pv. lachrymas in the field, but also distinguished Acidovorax citrulli effectively, which was helpful for the development of melon planting industry.

Gene chip is a new biological technology based on the principle of nucleic acid hybridization, which shows important applications in the field of life science research. A method of simultaneously detecting Infectious laryngotracheitis virus (ILTV), Newcastle disease virus(NDV) and Infectious bronchitis virus (IBV) using DNA microarray and asymmetric PCR methods was developed. Primers based on specific conservative DNA fragments of the recombinant plasmid containing thymidine kinase (TK) and glycoproteins B (gB) genes of ILTV, fusion (F) and haemagglutinin-neuraminidase (HN) genes of NDV, membrane (M) and nucleocapsid (N) gene of IBV were designed and probe genes were amplified. Probes were purified using ethanol precipitation method and spotted on the amido-coating-glass slides to form a DNA microarray. Target genes were synthesized using fluorescent cy3-labeled primer by asymmetric PCR method and hybridized to microarray. Result of asymmetric PCR showed that single stranded of ILTV-TK, NDV-HN and IBV-N increased the most of all when concentration ratio of the restrictive and non-restrictive primer at 1∶10, and single product of ILTV-gB, NDV-F and IBV-M increased the most of all when the concentration ratio at 1∶20. The hybridization results showed the microarrays specific to the 3 poultry viruses could specifically hybridize with the corresponding sample, while any fluorescent signals could not be seen in negative controls. The sensitivity test showed that the concentration of DNA with 1.8×104 copies was still positive. Compared with the previous chip, hybridization efficiency and the hybridization signals increased significantly. Detection of 12 clinical samples, the results showed that the detection rate of DNA microarray and PCR method was consistent. This study showed that the developed microarray can simultaneously diagnosis 3 poultry disease of NDV-IBV-ILTV with fast, accurately and high-throughput, which can be applied to the detection a variety disease virus of chicken at intensive farming.

In order to investigate the transgenic efficiency of cytoplasm microinject mediated with SB (sleeping beauty), PB (piggyBac) and Tol2 transposons in mice (Mus musculus) and zebrafish (Danio rerio); We coinjected the plasmid of recombinant vector pT3-PST-CAG-GFP containing 3 transposons and the plasmids of SB, PB and Tol2 transposes expression vectors into the cytoplasm of mice and zebrafish zygote with a ratio of 1∶1 respectively, and detected the positive expression ratio of green fluorescent protein (GFP) by fluorescence microscope after cultivation in vitro. The results revealed that the highest transgenic efficiency among 3 transposons was PB (N=281), which reached 52% in 4 Ed (96 h) (Ed: embryo day) mice embryos, significantly higher than (P＜0.05) SB (47%, N=328) and Tol2 (36%, N=273). In the stage of 36 and 60 hpf (hour past fertilization) zebrafish embryos, the highest transgenic efficiency was Tol2, and the GFP expression rate was 36%(N=677) and 37%(N=685), respectively, significant higher than PB at 36 hpf (30%, N=691) and 60 hpf (32%, N=708), and SB at 36 hpf (28%, N=687) and 60 hpf (27%, N=675) respectively; The results indicated that high transgenic efficiency was achieved by using the cytoplasm microinjection technique mediated with transposons, and the transposition activity of different transposons displayed species difference, the highest gene transfer efficiency was PB transposon in mammal embryos, while in fish embryos, Tol2 transposon was the most efficient. This study provides basic reference for the improvement of animal transgenic efficiency.

Denaturing high-performance liquid chromatography (DHPLC) could be used to test the single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel) polymorphism markers, which would be applied for the rapid identification of diverse hybrid maize. In order to test the feasibility and reliability of DHPLC for molecular marker genotyping in maize (Zea mays), the DHPLC analysis on 10 hybrids and their parents were conducted. In this study, based on the information in maize genome database, two DNA fragments harboring multiple and informative SNPs were screened. PCR products of the two amplicons from all maize varieties were analyzed by DHPLC and DNA sequencing. We first analyzed the PCR product from C7-2 at the temperate predicted by WaveMarker software to obtain a homoduplex elution profile for the two fragments. The experimental results showed the software-predicted temperature was only suitable for the analysis of homoduplex DNA for AmpⅡ. By analyzing the PCR product from C7-2 in 0.5 ℃ decrements over the range of predicted temperature for AmpⅠ, the optimal condition appropriate for the analysis of homoduplex DNA and other genotypes was obtained. Our results showed under the optimal separation condition, the two screened DNA fragments were polymorphic across hybrids with distinct DHPLC elution profiles produced. AmpⅠexhibited 3 different DHPLC elution profiles and AmpⅡproduced 7 types of elution profiles. Different DHPLC profiles could be detected and distinguished easily due to their significant differences in peak number and retention time. For each of the two DNA fragments, some samples showed nearly identical profiles, which had the same peak number and similar retention times. The sequencing results showed that the samples exhibiting nearly identical elution profiles harbor common sequence variants, and each sequence variant presented a unique elution profile for each of the two DNA fragments. It confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in two DNA fragments could differentiated between 7 hybrids with at least a single profile pattern difference, except three varieties (C4, C87 and 6047×618) which had identical profiles. We also found that because hybrids displayed a characteristic pattern of DHPLC elution profile resulting from parental variation in allelic nucleotide sequence, the DHPLC pattern of the hybrid maize was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. We proposed it was practical to test genetic purity of F1 hybrid seeds by comparing their DHPLC elution profiles with that of a mixed sample of their two parents. Due to the advantages of high resolution, high through-put, simplicity and safety, DHPLC would be a useful technology for rapid identification of hybrids and seed genetic purity test.

Ralstonia solanacearum, the causal agent of bacterial wilt disease, demonstrates seriously pathogenic diversity. In order to understand the pathotypes of Ralstonia solanacearum strains from Fujian Province and its association to the composition of avirulence genes, the pathogenicity of 63 Ralstonia solanacearum strains isolated from different regions and host plants in the Fujian Province was detected using attenuation index (AI) method. The tested strains were divided into 3 pathogenic types which were virulent, interim and avirulent. The avirulence genes avrA, popP1 and popP2 of tested strains were also detected. Three avirulence genes had different distribution frequencies, avrA and popP1 showed the highest and lowest distribution frequency. It was 79.37% and 46.03%, respectively. The composition of avirulence genes of R. solanacearum strains differed in different host plants. The strains isolated from tomato (Lycopersicum esculentum) and pepper (Capsicum annuum) commonly contained 2~3 types of avirulence genes, but for the strains isolated from eggplant (Solanum melongena), two types of avirulence genes were the main distribution, and only avrA gene or no avirulence gene was detected in the strains isolated from peanut (Arachis hypogaea ). The composition of avirulence genes of R. solanacearum strains varied in different regions. Take R. solanacearum strains isolated from tomato host as an example, strains from Nanping City, Jianou County mainly distributed 3 and 2 types of avirulence genes, the proportion were 45.45% and 31.82%, respectively. Three avirulence genes were equally distributed in the strains from Ningde City, Pingnan County. Only avirulence genes avrA and popP2 were detected in the strains from Fuzhou City, Beifeng Town, and they were equally distributed. The strains from Fuzhou City, Yingkou Town had only popP2 gene, and the distribution frequency reached to 60.00%. Different pathogenic R. solanacearum strains had different composition of avirulence genes. For avirulent strains, 3 and 2 types of avirulence genes were the main distribution, while for interim strains 2 types of avirulence genes were detected, for virulent strains, only 1 type of avirulence gene was detected. Three avirulence genes showed positive correlation with the pathogenicity of R. solanacearum strains based on the Pearson correlation coefficient (PCC) analysis. The correlation coefficiency of popP1 gene to the pathogenicity of strains was the highest with the value of PCC 0.31, which reached to significantly positive. In conclusion, this work provides some important information for early warning and controlling the bacteria wilt disease.

In order to isolate the bone marrow mesenchymai stem cells (BMSCs) of dog (Canis lupus) and induce the BMSCs to differentiate into osteoblastic cells, the BMSCs were isolated in vitro by whole bone marrow adherent method. After proliferation into full confluency, the BMSCs were subcultured. During proliferation, cell growth curve assay was performed in different passages. Pluripotent markers (SRY-related high-monility-group (HMG)-box protein-2 (SOX2), octamer-binding transcription factor-4 (OCT4) and NANOG) and osteoblast specific markers (osteocalcin (OCN) and osteonectin (ONN)) were defected by qRT-PCR in undifferentiated cells and cells induced differentiation for 10 and 21 d. Alkaline phosphatase (ALP) and alizarin red were defected by staining in undifferentiated cells and cells induced differentiation for different days. The results showed that the growth curve of undifferentiated cells in p5 and p10 were similar. However, the proliferation rate of cells in p20 was significantly slower than that of cells in p5 and p10. Before differentiation, the pluripotent genes such as OCT4 and SOX2, but not NANOG expressed in the BMSCs. The low expression levels of OCT4 and SOX2 could also be detected after induced differentiation for 10 days. However, after induced differentiation for 21 days, there was no expression of OCT4 and SOX2 in differentiated cells. For osteoblast specific markers, the expression levels of OCN and ONN increased along with induced differentiation. Alkaline phosphatase activity of differentiated cells significantly increased after induced for 7 days. Calcified nodules could be observed and stained red after induced for 21 days, and the number of calcified nodules increased along with induced differentiation. These results indicated that BMSCs of dog could be isolated and induced differentiation into mature osteoblasts successfully in vitro, which offer a new approach to the clinical treatment of severe fractures and osteodystrophic arthropathy of the pet dogs.

PolⅡ promoter-derived short RNAs can interfere with cellular mRNA for nuclear export and may create conditions for enhancing heterologous gene expression mediated by plant virus RNA-based vectors in plant. To test this hypothesis, we synthesized Arabidopsis thaliana U6-1 RNA gene and the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b gene in vitro and constructed PolⅡpromoter-directed U6 RNA and CMV-2b plant expression vector respectively. The vectors were co-inoculated on Nicotiana benthamiana plants with Tobacco mosaic virus (TMV)-based plant expression vector through agroinfiltration. The expression levels of green fluorescence protein (GFP) in inoculated Nicotiana leaves were then examined by Western blotting and ELISA respectively. All data were collected for the assessment of the effect of PolⅡ promoter-synthesized sRNA on plant virus RNA-based expression vector system. The results showed that expression of GFP mediated by TMV-based expression vector was significantly enhanced in the presence of PolⅡ promoter-derived short RNAs (approximately 2.5-fold higher than the?control levels). And the accumulation of pathogenesis-related protein 2 (PR2) induced by TMV-based vector was dramatically decreased in the presence of PolⅡ promoter-derived short RNAs (approximately eight times?lower than?the?control levels). We speculated that the competition of nuclear export resulted in decreased protective antiviral mRNAs export into the cytoplasm and so far enhanced GFP gene expression in the infiltrated plant tissues. Furthermore, co-expression with PolⅡ promoter-derived sRNA also significantly increased the expression of GFP to levels similar to those induced by CMV-2b suppressor. The results provides an alternative to improve recombinant protein expression in plants from agroinfection-compatible plant virus expression vectors.

Puerarin (Pue), an isoflavone extracted from the root of Pueraria lobata of leguminous plant, has an anti-inflammatory effect. The aim of this experiment was to investigate anti-inflammatory mechanism of puerarin in the inflammation model of bovine(Bos taurus) mammary epithelial cells (bMECs) induced by lipopolysaccharide (LPS). In this study, the bMECs were primary cultured with the collagenase digestion and both cytokeratin-18 (CK-18) and β-casein of the cells were identified by immunofluorescence technique and Western blot, respectively. The cell counting kit-8 (CCK-8) method was used to determine the suitable stimulating does of LPS used for inducing inflammation of the bMECs. The enzyme-linked immunosorbent assay (ELISA) kits was used to detect the changes of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) content in bMECs culture supernatant. The bMECs were divided into control group, inflammatory model group, puerarin group and dexamethasone positive control group. The protein expressions of p-IκB-α, IκB-α, p-p65 and p65 were detected by Western blot in each group of cells. The results showed that the bMECs were proved with the presence of specific CK-18 in cytoplasm and could express β-casein. There was a significant difference in the contents of TNF-α and IL-1β between the cells treated with 10 μg/mL LPS for 24 h and the cells in the control group (P＜0.05). The data above demonstrated the establishment of inflammation model in the bMECs was successful. The results indicated that the p-IκB-α protein was obviously decreased (P＜0.05) in the puerarin group compared to that in the inflammatory model group. Meanwhile, p-p65 protein and the contents of TNF-α in the cell culture supernate of the puerarin group were significantly lower than that in inflammatory model group (P＜0.01). There was no significant difference between the puerarin group and the dexamethasone positive group. Taken together, our date indicate that the puerarin acted as an anti-inflammatory via blockading the activation of nuclear transcription factor-κappaB(NF-κB) signaling pathway and inhibited TNF-α expression. This study laid the foundation for the development of clinical anti-inflammatory agent and therapy of dairy cow mastitis.

Expansin is a protein unique to the plant cell wall. It plays a key role in the expansion and relaxation of cell wall and decides the cell growth, to a great extent, during the process of plant growth. Both α-expansin (EXPA) and β-expansin (EXPB) are two larger expansin superfamilies. There are at least 34 α-expansin genes in rice (Oryza sativa). In this paper, we explored the effect of OsEXPA8 on cell division and growth of suspension cells from rice calli by transgenic techniques. Using the flow cytometry to analyze the difference of cell cycle between wild-type and transgenic suspension-cells for different culture time, we found that 35S::OsEXPA8 (overexpression) transgenic cells mainly remained in G2/M stage, while OsEXPA8-RNAi (knock-down) transgenic cells remain in G1 stage. Furthermore, the difference of cell number between wild-type and transgenic cells for different culture time were investigated by a hemocytometer, and the result showed 35S::OsEXPA8 overexpression cell＞wild-type cell＞OsEXPA8-RNAi transgenic cell number. In addition, the cell size was measured, and the result demonstrated 35S::OsEXPA8 overexpression cell＞wild-type cell＞OsEXPA8-RNAi transgenic cell. Finally, the callus was measured, and the result indicated 35S::OsEXPA8 overexpression callus＞wild-type callus＞OsEXPA8-RNAi transgenic callus. These results demonstrated that OsEXPA8 promoted suspension cell division and growth, and increased the cell number and size in rice. Therefore, OsEXPA8 plays an essential role in regulating the suspension cell growth and development in rice. This study would improve our understanding of the mechanism underlying the cell wall relaxation during plant cell growth, and can have signi?cant agricultural potential for improving crop growth by genetic modification.

The transcription factor Nanog plays a core role to maintain the self-renewal and pluripotency of stem cells. In order to study the regulatory function of pig (Sus scrofa) Nanog in pluripotent networks, the recombinant Nanog protein was used to generate the mouse anti-Nanog polyclonal antibody. Firstly, the CDS of Nanog cloned from porcine kidney was connected into pET32a vector to construct the recombinant expression vector pET32a-Nanog. Next, the pET32a-Nanog was transformed into Escherichia coli Rosseta (DE3) and Nanog expression was induced by isopropyl-β-d-thiogalactoside (IPTG). After optimizing the induction time, temperature and IPTG concentrations, the recombinant Nanog protein (57 kD) was obtained. The Nanog protein was purified and then used as the antigen to immunize C57BL/6 mice (Mus musculus). After 6 weeks, the positive antiserum was collected, and the antibody titer and specificity were detected by Western blot and immunofluorescence assay. The results showed that the recombinant Nanog could be highly expressed under the induction conditions of 28 ℃, 8 mmol/L IPTG and 2.5 h; the generated polyclonal anti-Nanog antibody could react specifically with Nanog generated from porcine induced pluripotent stem cells, suggesting that the polyclonal antibody can provide a powerful tool for porcine pluripotent stem cell research.

Calpatatin (CAST) is the endogenous inhibitor of the calpain system, which has been found to affect the postmortem tenderization of skeletal muscle. This experiment was designed to study the effects of polymorphism of 5' flanking regulating of CAST gene on meat quality traits in broiler (Gallus gallus), F、D、Y、B and S3 5 strains of quality broiler lines were used in the present study. The genetic polymorphisms of calpastatin (CAST) was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP), and analyze the association of polymorphisms sites with quality broiler lines which included the PH value, Vitamin B1, muscle color, water holding capacity, intramuscular fat content and density of muscle fiber. In the present study, the 5' regulatory region of CAST gene of 300 broilers was amplified and a uniform fragment of 210 bp was obtained. The results indicated that one SNP site was detected in 5' flanking regulating of CAST gene. The 210 bp PCR product was digested with restriction endonuclease BMPI. Three genotypes (AA, AG and GG) were found at G-198A mutation locus at a distance of CDS-198 locus in 300 broilers by digesting the PCR product with BMPI, and three frequency of AA, AG and GG genotypes was 0.3, 0.2, 0.5, respectively in Y strain (0,125, 0.1, 0.775, respectively in S strain; 0.2, 0.375, 0.425, respectively in F strain; 0.05, 0.35, 0.6, respectively in D strain; 0.1897, 0.3966, 0.4138, respectively in B strain.). The fitness test showed that all quality broiler lines at three mutation locus were in Hardy-Weinberg equilibrium. The relationship between CAST gene and meat quality traits in quality broiler was analyzed by least squares linear model. The individuals with AA genotype had significant effect on the intramuscular fat content and density of muscle fiber(P＜0.05), and has no significant effect on the other meat quality traits(P＞0.05). AA was the most favorable genotype for meat quality traits in broiler. The results preliminarily indicate that allele A of G-198A polymorphic site of CAST gene is a potential DNA marker for improving meat quality traits in broiler. The information found in the present study is very important for improving meat quality in broiler by marker assisted selection.

Haemophilus parasuis is a common bacterial agent of porcine (Susscrofa domestica), and the infection of this pathogen is characterized by fibrinous to fibrinopurulent polyserositis and polyarthritis. Cytolethal distending toxin (CDT) is proposed as an important virulent factor of H. parasuis, but some biological and immunological characteristics of CDT are still to be studied. Highly purified CDT holotoxin was prepared through co-refolding of 3 subunits by dialysis at 4 ℃ into a native buffer and purification by Ni-NTA affinity chromatography and gel-filtration chromatography. Cytotoxicity of CDT on both Marc145 and porcine peripheral blood lymphocyte (PBLC) was studied. Moreover, neutralization effect of specific antibody of CDT subunits against its cytotoxicity was measured. It showed that stable ternary complex were formed following co-refolding with 3 recombinant CDT subunits, and the formation of triplex CDT was confirmed by immunoprecipitation assay with purified IgG against CdtC subunit. The CDT holotoxin could induce cell distention by 2~4 fold of the original size and nuclear enlargement on Marc145, while any one or 2 of the 3 subunits could not induce typical cytotoxicity. Both CdtB and CdtC anti-serum could inhibit cytotoxicity of the assembled CDT holotoxin, and CdtC anti serum showed the best neutralization effect with the titer of 1∶16, while no neutralization effect of CdtA anti-serum was observed. The unassembled recombinant subunits mixture was significantly more susceptible to the neutralization effect of both CdtB and CdtC anti-serum than the assembled CDT holotoxin (P＜0.05). Furthermore, H. parasuis CDT blocked proliferation of mitogen activated PBLC, and the proliferation of PBLC was significantly suppressed when applied 20 or 10 μL holotoxin to ConA or phorbol-My-ryslate-acetate (PMA) activated cells, respectively (P＜0.05). When cultured in vitro, virulent and non-virulent H. parasuis secreted proteins showed the same cytotoxicity effect with the titer of 1∶512; The result of immunoprecipitation assay with purified IgG against CdtC subunit showed that CDT in the secreted proteins was present as ternary complex. Hence, H. parasuis CDT recombinant subunits could form holotoxin in vitro, and the assembled holotoxin exhibited effective bio-activity; Humoral immune responses against CdtB and CdtC could completely block the cytotoxicity caused by the holotoxin; Holotoxin showed significant proliferation suppression effect on mitogen activated PBLC cells; H. parasuis secreted proteins of 2 strains with great variation in virulence showed the same cytotoxicity, and CDT in the secreted proteins was presented as triplex holotoxin. H. parasuis CDT exhibited potent immunosuppressive effect, and it was possible to play important roles in colonization and causing systemic infection in the host, while it was not responsible for the extensive virulence variation between strains. This study provides novel information for understanding the pathogenesis of H. parasuis.

Thyroid-stimulating hormone receptor (TSHR) may play important roles in process of seasonal breeding in mammals mediated by photoperiod, but the hereditary character and molecular mechanism in sheep (Ovis aries) are still unclear. Therefore, the T481C locus on exon 10 of TSHR in our unpublished chip date was chosen as candidate SNP, polymerase chain reaction-single strand conformation polymerase (PCR-SSCP) method was used to detect genotypes in six sheep breeds which have differences in reproductive characteristics (Altay, Chinese Merino, Suffolk, Duo Lang, Hu and Small Tail Han Sheep). The results showed that the T-C point mutation was detected in 481 bp of exon 10 of TSHR gene in sheep, which was a silent mutation, three genotypes (TT, TC and CC) were found, TT genotype at T481C locus was preponderant genotype in non-seasonal breeding sheep flocks (Small tail han sheep and Duo lang sheep), while the lowest percentage of the genotype in seasonal breeding sheep flocks(Altay, Chinese merino and Suffolk) is only 8%. Analysis of variance suggested T481C was significantly different between the allele frequencies in seasonal and non-seasonal breeding sheep breeds(P＜0.01). These results suggest that there is a big difference in the SNP distribution between seasonal breeding and non seasonal breeding sheep populations, and the SNP can be used as an ideal molecular marker in non-seasonal reproduction sheep breeding.

Bolting and flowering time are important agronomic characters for Chinese cabbage (Brassica rapa ssp. pekinensis), in which the yield and quality of harvested product are influenced by premature bolting. Chinese cabbage-cabbage (Brassica oleracea var. capitata) translocation lines of AT4 series added No. 4 chromosome fragments from cabbage in Chinese cabbage background were used as materials in this experiment. Commercial Chinese cabbage varieties Hanchun58, Yangchun for late bolting and Beijingxiaoza56 were used as control genotypes for bolting identification. All 45 late bolting lines and 15 early bolting lines were identified by phenotypic observation. In late bolting group, all 36 lines were extremely late and 9 lines were late. In early bolting group, 7 lines were early and 8 lines were extremely early. Late bolting lines and early bolting lines with 4 different vernalization controls were displayed by cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis. A total of 126 differential expressed transcript-derived fragments (TDFs) were identified, including the presence of bands in late bolting lines and without bands in early bolting lines, the presence of bands in early bolting lines and without bands in late bolting lines, expression increased or decreased with vernalization time increased in late bolting lines, expression increased or decreased with vernalization time increased in early bolting lines. All 74 TDFs were obtained by sequenced. BLAST and alignments showed that 61 TDFs shared the highest levels of similarity with homologous sequence, including 41 TDFs shared the highest levels of similarity with genes of known function and 20 TDFs with genes of unknown function, and 13 TDFs with novel expressed sequence. The TDFs of known function were involved in genes encoding enzymes working in metabolism, energy metabolism, cellular transport, signal transduction, regulation of transcription, DNA modification, cell cycle, etc. Among the 61 TDFs shared the highest levels of similarity with homologous sequence, 5 of them shared the highest levels of similarity with genes of cabbage, 2 of them shared the highest levels of similarity with genes of Penaeus monodon, 1 of them shared the highest levels of similarity with genes of Anas platyrhynchos, 1 of them shared the highest levels of similarity with genes of uncultured cyanobacterium, and 52 of them shared the highest levels of similarity with genes of Chinese cabbage. The acquisition of late bolting Chinese cabbage-cabbage translocation lines will provide useful materials for breeding new varieties program. The difference in expression of TDFs between late and early bolting lines will lay the foundation of understanding key genes and their regulation mechanism of bolting and flowering in Chinese cabbage.

To enable industrial Saccharomyces cerevisiae ferment xylose into ethanol by the expression of XylA and overexpression of XKS1 and PPP genes TAL1、TKL1、RKI1 and RPE1, the promoter FBA cloned from S. cerevisiae was ligated with xylose isomerase gene (XylA) and the ligation product was inserted into the shuttle expression vector of pYES2, then the expression vector pYES2-FBA-XylA was constructed, and the promoter TPI cloned from S. cerevisiae was inserted into the vectors pUG6 and pUG72, respectively, and then the vector pUG6-TPI and pUG72-TPI were constructed. The vector pUG6-TPI contained the selection marker of KanMX and pUG72-TPI contained the selection marker of ura. Different primer which contained homologous sequences were designed. Taking pUG6 and pUG72 as template, the fragments which enhanced expression of XKS1 and PPP genes TAL1、TKL1、RKI1 and RPE1 were obtained by PCR. The fragment was integrated into S. cerevisiae by the LiAc chemical method. Then the fragment with different selection marker was integrated into the allele loci of S. Cerevisiae. These two different markers were removed by inducing recombination enzyme expression after vector pSH65 were transformed into S. Cerevisiae. The TPI promoter were inserted into the rest 4 loci of the genes and removed the markers in the same way. Then the PPP enhanced recombinant strain was obtained. The expression vector pYES2-FBA-xylA was transformed into the PPP enhanced recombinant, and the recombinant of pXI-T1308U- was constructed. Expression levels of XKS1 and PPP genes between the original strain and recombinant strain were analyzed by qRT-PCR, and the results showed that the transcription levels of TAL1、TKL1、 RKI1、RPE1 and XKS1 of recombinant strain were increased to 1.62, 3.98, 17.36, 4.17 and 4.08-fold, respectively, compared with original strain. XI (EC 5.3.1.5) activity in the cell extracts of the strains were determined, and the recombinant strain exhibited specific XI activity as high as 0.39 U/mg protein whereas no XI activity could be detected in the original strain. The co-fermentation of glucose and xylose of recombinant strain was studied, and fermentation samples were analyzed by HPLC, results showed an increased consumption rate of xylose about 17.54% compared with its original strain. And the ethanol yield was about 0.23 g/g xylose. In conclusion, the recombinant industrial strain could ferment xylose by enhancing expression of the key gene XKS1 and PPP gene under the expression of XI. This work provides a new promising reference for further development of xylose-fermenting yeast for industrial lignocellulosic ethanol production.

Progesterone receptor membrane component 1(PGRMC1), one member of membrane-associated progesterone receptor (MAPR) protein family, is involved in anti-apoptotic processes of progesterone within mammalian granule cells and luteal cells, and plays important roles in promoting oocyte maturation and maintaining ovary function. But the effects of PGRMC1 on reproduction traits in sheep (Ovis aries) have not been reported. In this study, sheep PGRMC1 cDNA was cloned and its expression pattern in tissues was analyzed by semi-quntitative PCR. On this basis, taking 'Duolang' sheep (non-seasonal breeding) and Chinese 'Merino' sheep (seasonal breeding) as the research object, the expression of PGRMC1 mRNA levels in hypothalamus-pituitary-ovary (HPO) axis in estrus and anestrus were analyzed and compared by. Results showed that the partial cDNA of sheep PGRMC1 was 1459 bp (GenBank: KM114208) and included a whole open reading frame of 585 bp encoding 194 amino acids. The encoding protein contained a Cyt-b5 conserved domain. PGRMC1 mRNA were widely expressed in the tested tissues, in which the expression level of ovary, uterus, liver, kidney and hypothalamus was much higher than that of the others. The real-time PCR results showed that the expression level of PGRMC1 in uterus, ovary and pineal gland in estrus than that in anestrus in the same breed. The expression level of PGRMC1 existed some differences in gonadal axis tissues between different breeds. Whether in estrue or in anestrue, the expression level of PGRMC1 in uterus, ovary and pineal gland in 'Duolang' sheep were all higher than that in Chinese Merino sheep. The results suggested that PGRMC1 might play important roles in maintaining uterus and ovarian function, regulating hormone secretion and estrus behavior and so on. To provide a certain theoretical basis for further understanding the biological function of PGRMC1.

Taimen (Hucho taimen) is a rare cold-water fish that mainly distributes in the Irtysh River and Heilongjiang River. Studies on the cryopreservation of H. taimen sperm and establishment of cryobank are of great values in many aspects, such as the preservation of resources, artificial reproduction and breeding of fish, rearing of the larval, and also the recovery of the population. In the present study, 9 types of sperm diluents (MPRS, RS, Hanks, ELS, EG1, EG2, Stein, SS-2 and D15) that consisted of the basic liquid medium plus inorganic salt, glucose, and fetal bovine serum (FBS) were prepared and used for the cryopreservation of H. taimen spermatozoa. The motility of spermatozoa, in these diluents with or without the cryoprotectant ((dimethyl sulfoxide, DMSO) or MeOH), and also after the freezing treatment with liquid nitrogen, was detected. The results showed that the sperm motility was relatively high (73.00±2.65)%~(96.67±2.08)% in each diluents mentioned above, however declined after the addition of the DMSO, and only possessed a quite low activity (1.21±0.00)% or (1.1±0.00)% with ELS or D15 after the freezing treatment. Furtherly, a suitable concentration of MeOH or DMSO was screened out with ELS and the results were as follows. It was found that the sperm motility remained at (79.00±6.52)%~(88.80±7.89)% after a 20 min pre-freezing treatment with MeOH at the concentrations of 4%~12%. The sperm motility was significantly (P＜0.05) decreased after the freezing treatment compared with the fresh semen, and a relatively high ratio had been to (18.33±10.61)% with 6% of MeOH. On the other hand, the motility of the perm had ranged of (43.75±11.09)% ~(81.67±2.89)% after the pre-freezing treatment with DMSO (4%~12%) for 5 min, and was tremendously declined (3.25±0.30)%~(45.00±5.77)% after the treatment for 20 min, but still kept a rather low activity (1.00±0.00)%~(2.75±1.50)%(P＜0.05) after the freezing treatments. D15 was used as the basic diluents to screen out the concentrations of glucose. The results showed that the sperm motility was relatively high (17.80±2.59)% after the freezing treatment with glucose at the concentration of 30% (D30), but still was significantly (P＜0.05) lower than the fresh semen. In addition, a short-term preservation results conducted with 4 diluents (D15, D30, Stein and ELS) indicated that the sperm had the longest survival time (45 h) with Stein diluents, moderate with D30 (15 h). In conclusion, the H. taimen sperm cryopreserved with ELS and D30 plus with 6% MeOH had a certain degree of motility. Our results will lay the foundations for the cryopreservation of H. taimen sperm on a large scale and furtherly provide the basis for the establishment of the sperm cryobanks.

Glucocorticoid receptor (GR) is a member of the conservative nuclear receptor superfamily. It belongs to nuclear transcription factor. It plays an important role in maintaining the stability of the body and animal breeding and metabolism. The aim of this experiment was to clone the cDNA full length of GR gene of pigeon (Columba livia domestica) and analyze its expression profile. The specific primers were designed based on the published necleotide sequence of chicken (Gallus gallus domesticus) . The sequencing results showed that GR gene containing 2 313 bp CDS, 102 bp 5'UTR and 18 bp 3'UTR, encoding a protein of 770 amino acids(GenBank accession No: NM_001037826.1). The sequence homology analysis showed that the pigeon GR gene shared 94%, 85% and 75% homology of the amino acid sequence with chicken, crocodlie (Alligator mississippienses) and human (Homo sapiens), respectively. The amino acid sequence analysis of Chicken, crocodile and human found the GR amino acid sequence had a very conserved DNA binding domain (DBD), and a higher conserved hormone recognition domain (LBD), in addition to a GR specific region. qRT-PCR results suggested that GR mRNA were expressed in the different organizations. And the expression quantity from high to low were testis, pancreas, lung, ovary, abdominal fat, liver, heart, skeletal muscle, kidney, spleen, oviduct, stomach, hypothalamus, brain, intestine and craw successively. The expression of GR gene in the testis was significantly higher than other tissues (P＜0.05), followed by the pancreas and lung (P＜0.05). In this study, we obtained the full length of GR gene cDNA and the expression of GR gene, this results provide a basic data for further study on the function of GR gene.

Rice (Oryza sativa) is a staple food crop for more than half of the world population. Photoperiod-thermo-sensitive genic male sterility (PGMS) is widely used in two-line hybrid breeding. Recently, a great progress has been made in rice PGMS. A number of sterile genes of PGMS were mapped on rice chromosomes, and 3 of them pms 3, p/tms12-1 and tms 5 were cloned. At the present review, we summarized the advancements on mechanism of fertility transformation, related-gene mapping and cloning, mechanism of fertility transformation, and molecular regulation in PGMS, and furtherly discussed the epigenetic role in the study of PGMS. These findings in this review had significant implications in further studies.