During the process of oocyte maturation, CyclinA2(CycA2) controls the progression of cell cycle by activating different cyclin-dependent kinase (Cdk), which regulates the cell cycle through its expression and degradation. Eukaryotes contain 2 distinct types of CycA2 of A1 and A2. The transcription of CycA2 initiates in late G1, peaks and plateaus in mid-S, declines in G2 and totally disappears in metaphase. CycA2 is also involved in the G1/S and G2/M transitions. CycA2 is degradated by ubiquitin-proteasome system in mitosis. In recent study, it is reported that CycA2 also plays an important role in the meiotic division of female germ cells. It can promote entry into the first meiosis in mouse (Mus musculus) oocyte, furthermore, there is an unexpected role for its requirement for the sister chromatid segregation in the second meiosis. These results all suggest that CycA2 has an important role in oocyte maturation and embryo development. In order to investigate the effect of CycA2 on porcine (Sus scrofa) oocytes during in vitro maturation (IVM) in the present study, firstly, CycA2 gene was cloned from pig ovary by RT-PCR and constructed 2 eukaryotic vectors, the wild type pVenus-CycA2 and non-degradated pVenus-DN157CycA2, which were transfected into hela cells to confirm their expression and location by fluorescence microscopy observation and qRT-PCR. The cRNA of pVenus-CycA2 and pVenus-DN157CycA2 transcripted in vitro were microinjeted into porcine oocytes. Finally, the oocytes microinjected pVenus-CycA2 and pVenus-DN157CycA2 cRNA were collected for mature cultivation and the rate of the first polar body (PB1) extrusion were calculated. Besides, the CDK1 inhibitor roscovitine were used to treat the oocytes which were microinjected the DN157roscovitine cRNA and the rate of the PB1 extrusion was also examined. The results showed that the recombinant vector pVenus-CycA2 and pVenus-DN157CycA2 were successfully constructed. After transfection or microinjection, the fusion protein could express efficiently and localize accurately both in hela cells and porcine oocytes. This greatly facilitated the further study of CycA2, especially its role on oocytes maturation regulation. The rate of PB1 extrusion were decreased significantly compared with the control group(P＜0.01, P＜0.05). After treated with roscovitine, the oocytes microinjected pVenus-DN157CycA2 could extrude the PB1 and there was no significant difference in the rate of PB1 extrusion compared with the control group. In conclusion, CycA2 gene had an effect on the PB1 extrusion in a way associated with CDK1 during the porcine oocyte maturation, it provided a new direction in the research of in vitro maturation of oocytes and layed the foundation for the molecular mechanism of the CycA2 gene participates in the chromosome segregation. The study reveals an unexpected role for CycA2 in mediating PB1 extrusion in the first meiosis and also gives a platform to further explore the role of CycA2 gene in the process of the second meiotic division.

Mesenchymal stem cells (MSCs), which derived from stromal tissues of the body, are multipotent stem cells that can differentiate into a variety of cell types in vitro or in vivo including osteoblasts, chondrocytes, myocytes, adipocytes, even beta-pancreatic islets cells, cardiac cells and neural cells. MSCs therapy is becoming a novel therapeutic tool and resource for improving the quality of pets life and helping them free from painful conditions and diseases. Here, we reviewed the current state of MSCs therapy in pets clinical and discussed the problems in their application.

There is dramatic changing of histone modifications during in vitro maturating of oocytes, the changing of its expression pattern plays an important role on oocyte maturation and subsequently embryonic development. Suberoyl anilide hydroxamic acid (SAHA) acting as a anticancer drug is the potential inhibitor of histone deacetyltransferase, inhibiting the growth, differentiation and apoptosis of various types of tumor cell in vivo and vitro. To explore the effect of inhibitor of histone deacetyltransferase SAHA in vitro maturation and embryonic development potential of buffalo (Bubalus bubalus) oocytes, effects of maturing rate (first polar body discharge rate), cleavage rate of fertilized eggs, blastocyst rate and total cell number of blastocyst of buffalo oocytes were compared by different concentrations of (0, 2, 5, 10, 20 and 40 nmol/L) SAHA treatment. Results showed that maturing rate, cleavage rate, and blastocyst rate of group 10 nmol/LSAHA was significantly higher than control group ((77.07±1.79)% and (62.3±2.08)%, (86.81±1.93)% and (74.86±2.07)%, (27.56±2.86)% and (24.23±1.74)%, P＜0.05). As the concentration increasing, total cell number of blastocyst in each treatment group had a tendency to increase, but had no significantly difference with each other (141±10, 151±13, 165±17, 170±14, 147±20 and 185±22, P＞0.05). qRT-PCR tests revealed that the expression of (cAMP responsive element binding protein, CREB) binding protein gene (CBP), histone acetyltransferases 1(HAT1) gene and histonedeacetylase 1(HDAC1) gene in MⅡ stage oocytes of treatment groups were significantly reduced, though p300 was increased. In conclusion, histone deacetylase inhibitors SAHA was one of the most effective way to promote maturation of buffalo oocytes and embryonic development in vitro, this result will provid a theoretical basis for future study about gene regulatory mechanism of transgenic cloned embryonic-developmental.

Although the isolation of porcine (Sus scrofa) embryonic stem cells (pESCs) has been reported, the self-renewal and differentiation potential of pESCs were limit. In this study, the isolation of pESCs from parthenogenetic embryos (PA), somatic cell nuclear transfer embryos (SCNT) and in vivo-produced embryos (IVP) based on the different culture media were researched. The culture condition and key pluripotent genes in PA and SCNT embryos were analyzed by the qRT-PCR, and the effects of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), inhibitors of glycogen synthase kinase 3β (CHIR99021), and mitogen-activated protein kinase 1 (PD0325901) on pESCs isolation were investigated. The results showed that PZM3 and NCSU23 media were suitable for PA and SCNT embryos development in vitro, respectively. The 4 octamer-binding transcription factor 4(OCT4) expression was significantly increased in PA and SCNT embryos compared with NANOG and SOX2 in PA and SCNT embryos. Basic fibroblast growth factor receptor(bFGFR) gene expression was also significantly increased, suggesting that growth factor bFGF was needed for pESCs isolation from PA and SCNT embryos. Furthermore, addition of bFGF in medium could elevate the efficiency of pESCs isolation and cloning from in vivo-produced embryos, which showed morphology of the epiblast-like stem cells (EpiSCs), expressed NANOG and SOX2 proteins by immunochemistry, and had alkaline phosphatase(AP) activity. The results may contribute to a new method to isolate pESCs.

Granulosa cells play an important biological role in ovary development. To reveal the characteristics of porcine (Sus scrofa) ovarian granulosa cells during the development, granulosa cells from porcine follicular fluid were isolated and cultured in vitro.The results of cell morphology and genes expression pattern showed that porcine granulosa cells retained some features as stem cells. The interaction between cumulus granulosa cells and oocytes inhibited the differentiation of cumulus granulosa cells. When granulosa cells and oocytes were cultured together, granulosa cells were under the undifferentiated state, but losing the inhibition of oocytes, the growth of granulosa cells were rapidly, and quickly became fibrosis and apoptosis. On the other hand, cumulus granulosa cells could promote oocyte maturation. Under the regular cell culture conditions, oocytes cultured in vitro could be activated by cumulus granulosa cells and developed to the morula stage. In this study, the primary cultured porcine granulosa cells were found to be able to form cell aggregates spontaneously, and showed the weak alkaline phosphatase staining. The result of culturing granulosa cells in the medium that was used for culturing pluripotent stem cells showed that granulosa cells could be well maintained and present the epithelial-like state. After several passages, granulosa cells still maintained a high growth efficiency. RT-PCR analysis showed that the cultured granulosa cells were expressed multiple pluripotent genes, including ctamer-binding transcription factor 4 (OCT4), SRY (sex determining region Y)-box 2(SOX2), Kruppel-like factor 4 (KLF4), cellular myelocytomatosis viral oncogene (c-MYC), and Tir na n-Og (NANOG), as well as granulosa cells pecific marker gene follicle-stimulating hormone receptor (FSHR). The results showed that OCT4 was highly expressed and showed the 2 spliceosomes. After low-voltage, long-time agarose gel electrophoresis, RT-PCR products of OCT4 were found to have the 2 bands, indicating that the granulosa cells were expressed OCT4 spliceosome OCT4A and OCT4B. Western blot analysis and immunofluorescence staining experiments further proved that granulosa cells were expressed OCT4 and retained OCT4 spliceosomes, and the translocations of OCT4 in granulosa cells were varied. In the primary cultured granulosa cells, OCT4 protein was found in the cytoplasm, however, in the passaged granulosacells, OCT4 protein was translocated into the nucleus. Western blot analysis showed that cytoplasmic proteins forming primary cultured granulosa cells had OCT4 protein, which was not detected in nuclear proteins. The mechanism of OCT4 distribution in granulosa cells remained to be investigated. In summary, this study showed that porcine granulosa cells retained certain characteristics to express pluripotency factors, which lays the foundation for the development of the ovaries.

Agrobacterium-mediated transformation technology has been widely used in japonica rice (Oryza sativa ssp. japonica) varieties, but so far inaccessible to indica rice(O. sativa ssp. indica) varieties, mainly attributed to calli browning in following regeneration, which has severely hindered the application of transformation in indica rice. In this study, cv. 93-11, a typical indica variety, was used and the mathematical model was established by central composite design combined with response surface methodology and the media component for callus subculture were optimized. Results showed that MS media was more suitable for primary callus induction than N6, B5, N6B and NMB, which callus browning was significant inhibited through observation and comparison. Based on MS media, the effects of single factors such as hormone, carbon source were analyzed through single factor experiment. It was found that the best 2,4-dichlorophenoxyacetic acid (2,4-D) was 3 mg/L, kinetin (KT) was 0.5 mg/L, sucrose was 15 g/L and maltose was 15 g/L. And then Box-Behnken design was used to obtain the best value. The determination coefficient R2 of the model was 0.9 476 and the optimal combination of media component for inducing yellow, compact and organized calli was 2,4-D 2.92 mg/L, KT 0.59 mg/L, sucrose 16.37 g/L and maltose 13.63 g/L. Histological and anatomical analysis revealed the remarkable morphological differences between normal calli and brown calli: The surface of normal calli was quite irregular and was full with distinctive globular nodules and the globular nodules were full with tightly packed cells. The somatic embryogenesis consisted of white, sheet-shaped non-embryogenesis structures could be descried from the yellowish opaque sectors of a calli. In contrast, the smooth surface of brown calli was due to the fact that nearly all cells distorted from round shape to flat shape and the inactivation cells were unlikely to occur globular nodules. qRT-PCR revealed that the transcript levels of artificial wound related gene auxin 1 gene (AUX1) and sucrose non-fermenting 1-related nrotein kinase 2 gene (SnRK2) had obviously difference between normal calli and brown calli, the mRNA accumulation of the AUX1 in normal calli with roughly a 5-fold increase compared to brown calli and SnRK2 transcription in brown calli was about 2.5-fold higher than in normal calli, while phenolic metabolism related gene phenylalanine ammonia-lyase gene (PAL) and polyphenol oxidase gene (PPO) could neither be detected in normal calli nor in brown calli, these expression patterns indicated that artificial wound was the main reason for calli brown. The results would be helpful to understand the mechanism of browning occurrence in plant tissue culture.

It was all known that Staphylococcus aureus was an infectious pathogen to human and animal. In order to study the protein of inhibitory activities to S. aureus, the protein from Bacillus subtilis UMBR1027 was extracted by ammonium sulfate precipitation, and separated by Sephadex LH-20 column chromatography as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and was also identified by ABI 4800 plus MALDI-TOF. Furthermore, the conditions of fermentation were optimized by the response surface method to increase the quantity of the bioactive protein from B. subtilis UMBR1027. The bacteriostasis activity against S. aureus was assessed by the filter paper diffusion method. The 3 key factors including the pH value of the buffer, the ionic strength in the solution and temperature of culture were evaluated respectively to optimize the conditions of fermentation by design Box-Behnken experiments. The results showed that the alkaline serine protease, subtilisin, endo-β-1,3-1,4 glucanase and alaph amylase were identified in the bioactive protein from B. subtilis UMBR1027. The optimization experiments were achieved by systematically adjusting the pH of the buffer, the ionic strength in the solution and temperature. The optimal culture conditions were the medium pH 6.95, salinity 10.38% and the temperature 34.95 ℃, respectively. The selected experiment under the culture conditions were that pH was 6.95, salinity was 10.38% and the temperature was 35 ℃, and the bacteriostatic circle diameter was 20.4 mm which was close to the predicted values. This paper identified antibactial protein from B. subtilis UMBR1027 and optimized ferment process by the response surface method. The results will provide theoretical basis to discover new natural drug molecules from marine.

In order to express capsid protein of porcine circovirus type 2 (PCV2) in insect cells, nuclear-localization-signal (NLS) free capsid protein gene of PCV2 Shandong strain was amplified from clinical samples, then, the gene was inserted into baculovirus transfer vector pFastBac-1, the resulted plasmid was transferred to competent Escherichia coli DH10-Bac cells, the recombinant shuttle Bacmid was transfected into sf9 insect cells and cytopathic effect was observed. Capsid protein expression was identified by PCR, indirect immunofluorescence test, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that a 614 bp-length fragment was amplified from suspected samples, the amplified capsid protein gene of PCV2 Shandong strain shared highest homology with PCV2d strains at nucleotide level (99.8%) and amino acid level(100%), and belong to PCV2d subtypes revealed by evolutionary tree. Comparing with vaccine strains (PCV2a or PCV2d) widely used in China, this PCV2d strain showed a different antigenic domain at 160~180 aa. pFastBac1-PCV2d- Cap and Bacmid-PCV2d-Cap were successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. The recombinated baculovirus induced cytopathogenic effect in sf9 insect cells, and successfully expressed a 24 kD protein, which could react with PCV2 antiserum and His-tag monoclonal antibody. The good immunogenicity of expressed capsid protein provides possible application in the development of porcine circovirus virus type 2d subunit vaccine and ELISA kit. This is the first report about PCV2d capsid protein expressed in baculovirus/insect cell expression system.

Shanghai white pig (Sus scrofa) has many outstanding economic traits, such as high prolific, high disease resistance, high resistance to crude feed and heterosis. Revaling the molecular genetic architechture of these complex traits is an important issue for genetic study. With the development of genetic study and biotechnologies as well as upgarating of study methods, the requirement to reveal the genetic mechanism of these complex traits in the genome level rathter than in the single or several genes level is becoming more intense. However, up to date, no studies performed genetic variation analysis for Shanghai white pig on the genome-wide. What's more, the genetic mechanism for their outstanding traits is still unclear. Therefore, this study will be performed as follows: 1)Next generation sequencing technology was used to detect the genome-wide of genetic variations using . The genetic variants of 99 Shanghai white pigs, 252 pigs from Taihu pig breeds (Meishan, Erhualian, Mi, Fengjing, Shawutou and Jiaxing Black) and 156 foreign pigs were genotyped according to the genotyping by genome reducing and sequencing (GGRS) protocol (http://klab.sjtu.edu.cn/GGRS/). Briefly, high molecular weight genomic DNA samples were extracted from ear tissue, digested with Ava Ⅱ, and then ligated with a unique adapter-barcode. Next, the samples were pooled and enriched to construct a sequencing library. Finally, the sequence libraries (fragments ranging from 300~400 bp, including the adapter-barcode sequence) were sequenced on an Illumina Hiseq2000 (the sequencing process is given in detail by the manufacturer, Illumina) instrument with a paired-end (2×100 bp) pattern. The SNPs of more than 75 (30% ) samples were genotyped and an average sequencing depth greater than 5× was achieved. In total, 100 120 SNP were identified in the genomes of the current pig population. These results were helpful to know the genome information of the Shanghai white pig and enrich the databse of genetic markers in the Chinese pig breeds. 2) Applied the detected genetic markers for conducting the genetic diversity and population structure analysis to provide suggestions for breeds classification and future conservation. Genetic diversity and population structure were analysised. The breed or sub-type classification of a population can bring a large effect for its protection and conservation. The genetic diversity and population structure of these pig breeds were investigated using whole-genome SNP data. Firstly, the allelic richness (AR), proportion of polymorphic markers (PN) and expected heterozygosity (He) were used to investigate the genome-wide genetic variability within these pig breeds. As a result, Shanghai white pigs exhibited the highest level of genetic diversity within population compared(PN=0.731, AR=1.709, He=0.221) to Taihu pig breeds and western pig breeds. Additionally, the genetic differentiation between Shanghai white and Taihu pig breeds (0.237±0.007) was greater than the one between Shanghai white and western pig breeds(0.219±0.008). What's more, (Neighbor Joining, NJ)-tree, principal components analysis(PCA) and STRUCTURE analysis showed that all the populations could be well distinguished from each other. In conclusion, The rationality of the current breed classification for Shanghai white pig were confirmed based on genome-wide genetic markers. The study results also lay a necessary theoretical basis for effectively conserving and utilizing the special genetic resource.

G protein coupled receptors (GPCRs) constitute the biggest transmembrane receptor superfamily which mediates the signal transduction and generally exists in animals, plants and microbes. The genes encoding GPCRs and their genomic locations were obtained and identified by searching the genome database of Setosphaeria turcica with the HMMER 3.0 software based on hidden markov model. The MEGA 5.0 software was employed to analyze the systemic evolution. Genetic structure conservative sites of GPCRs were analyzed online through generalized sample data system (GSDS), InterPro and SMART. It was finally confirmed that there were 9 GPCR genes in the genome of S. turcica. Three of them belonged to putative nitrogen sensors, while there were 2 pheromone receptors and 2 carbon sensors respectively. And the remaining 2 were classified as cyclic adenosine monophosphate (cAMP) receptor-like protein and Fungal opsin. GPCR superfamily genes distributed in the genome randomly, and its structure was complicated. All members consisted of N-terminal, seven transmembrane domains, three intracellular loops, three extracellular loops and C-terminal. Each transmembrane domain comprised 20 to 25 amino acids. Finally, relative expression quantities of GPCR genes in the invasive structure formation process were detected by using the qRT-PCR technology. Transcriptional pattern analysis showed that in appressorium mature period 12 hours post inoculation (hpi), all genes were significantly downregulated (P＜0.05). Except StRtc1 and Stfdd123, trends of other genes all showed a downregulated/flat (3 hpi)-upregulated (6 hpi)- downregulated (12 hpi)-upregulated (24 hpi). Especially, they were significantly upregulated (P＜0.05) during the invasive mycelium formation (24 hpi). In appressorial formation period (6 hpi), only StSte2p and StRtc2 significantly upregulated. The expression levels of StRtc1 and Stfdd123 in each stage were not significant or significantly downregulated. This study provides a theoretical basis for in-depth analysis about the function of GPCR superfamily in plant pathogenic fungi.

4-coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. The catalytic activity of 4CL towards sinapate got a lot of attention at present. Only Arabidopsis thaliana At4CL4 and soybean (Glycine max) Gm4CL1 have catalytic ability toward sinapate so far. There is a conserved valine which is located in the substrate binding pocket by sequence comparison of rice (Oryza sativa subsp. japonica) Os4CL5 with At4CL4 and Gm4CL1. The existence of valine between Pro336 and Leu338 may eliminate the activity of Os4CL5 towards sinapate. In this paper, the expression vectors of wild-type Os4CL5 and mutant Os4CL5 with the deletion of Val337 were constructed. Then the recombinant plasmids were transformed into Escherichia coli BL21(DE3) respectively. The recombinant cells were grown and induced by isopropyl-β-d-thiogalactoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the induced protein was about 57.0 kD consistent with the predicted value. After purification by affinity chromatography, the enzymatic properties of wild-type Os4CL5 and mutant Os4CL5 were primarily characterized. The results showed that wild-type Os4CL5 had different enzymatic activity towards the different substrate. The rank in order of turnover rate for different substrate was coumarate＞ferulate＞caffeate. It should be noted that sinapate was not accepted as a substrate under experimental conditions. The deletion of Val337 from rice Os4CL5 results in increased activity towards different substrate and the generation of new substrate specificities towards sinapate. This study provides a theoretical basis for the regulation of lignin biosynthesis by genetic engineering.

Association analysis is an effective method for the germplasm resources studying which is fundamental in plant breeding. All 115 tea (Camellia sinensis) were tested in several traits related to tea quality based on association analysis using 67 EST-SSR markers. All 67 EST-SSR markers were polymorphic with different diversity among 115 tea germplasm. All 161 alleles were amplified with 2.4 per markers on an average. The polymorphic information content (PIC) ranged from 0.03 to 0.68 with the average of 0.36, TM096 was highest, while TM099 was the lowest. The observed heterozygosity (Ho) varied from 0.03 to 1 with the average of 0.45. The expected heterozygosity (He) varied from 0.03 to 0.72 with the average of 0.43. Linkage disequilibrium (LD) is the basis of association analysis. So the analysis of LD was carried out. The results demonstrated that 2 211 pairs of loci were detected, 259 of which were in high level (D'＞0.5). And then 115 tea germplasm was divided into 4 groups through the method of population structure. Based on the Q-value (K=4) of each individual as covariant, association analysis between quality-related traits with EST-SSRs was performed. The results showed that 19 EST-SSR markers associating with these traits were detected in all, 5 for the content of water extract, of which TM066 with the highest explanation of phenotypic variation 13.32%; There were 5 markers associated with the content of ployphenols, of which TM092 with the highest explanation of phenotypic variation 13.68%. Six markers for the content of total free amino, TM083 explanted 7.6% phenotypic variation. Three markers for the content of caffeine, TM111 markers had the highest explanation of phenotypic variation 7.8%. Furthermore, the results showed that some markers were associated with two traits, such as TM066, TM092 and TM074-2 associating with the contents of water extract and polyphenols, TM086-1 associating with the contents of polyphenols and total free amino; and TM088, TM111 and TM124 associating with the contents of total free amino and caffeine. The findings can be recommended for improvement of tea quality and early identification of varieties in tea plant through the further research on these markers.

Isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrum identification and bioinformatics were applied in this study to analyze the molecular mechanisms of weak embryos of duck (Anas platyrhynchos domestica), and select differentially expressed proteins that could be used in promoting hatchability. All 136 differentially expressed proteins were identified, and 76 proteins were significantly up-regulated and 60 proteins were significantly down-regulated in helped breaking eggs compared with that in normally hatching eggs. Gene Ontology (GO) and pathway enrichment analyses showed that the most of the differentially expressed proteins were related to glucose metabolism, oxygen transport, stress response, redox reaction and cytoskeleton. Four enzymes in glycolysis, three enzymes in cellular respiration pathways were significantly up-regulated (P＜0.05), and 7 proteins in actin filament dynamic process were up-regulated, whereas 3 hemoglobins in oxygen transport and 3 heat shock proteins in stress response were significantly down-regulated (P＜0.05) in helped breaking eggs compared with normally hatching eggs. The qRT-PCR was used for verifying the mRNA expression of cytoplasmic aconitate hydratase (ACO1), fructose-bisphosphate aldolase B (ALDOB), glyceraldehyde-3-phosphate dehydrogenase 1 (G3P1) and heat shock cognate 71 kD protein (HSPA8). Only the expression level of ACO1 were consistent in mRNA and protein. The results indicated that weak embryo might be related to glycometabolism, respiratory metabolism and some other biological processes, and there was lower energy metabolism in helped breaking eggs. This study provides theoretical basis of proteomics for molecular mechanism of weak embryos in duck.

Male germline stem cells (mGSCs) present low differentiation degree, with the ability of self-renewal and differentiating into sperms, are the foundation of males producing sperms. The study of regulation factors and mechanism of mGSCs self-renewal, proliferation and differentiation is of great significance to the understanding of spermatogenesis. E-cadherin is an important regulation factor of the microenvironment of stem cells, which plays a complex role in the mGSCs regulation, including stem cells self-renewal, proliferation and differentiation, etc. E-cadherin helps mGSCs anchores in the niche, mediating cell-niche signaling transmission, at the same time providing polarizing signals guiding stem cells undergo symmetric cell division or asymmetric division, affecting mGSCs continuous, long-term self-renewal. Ensuring that only undifferentiated, functional stem cells are kept in the niche, stem cells in differentiating lose the competitive advantage and leave. The microenvironment can regulate the self-renewal and differentiation of mGSCs. Continuous research of germline stem cell niche is expected to reveal the regulatory mechanisms between self-renewal and differentiation of mGSCs. Sequence alignment found that the mRNA sequence and the amino acid sequence of E-cadherin were highly conserved in a variety of species. In order to confirm the E-cadherin expression status in dairy goat (Capra hircus) testis, immunohistochemical staining found that as a cell membrane protein, E-cadherin widely expressed in dairy goat testis. Addationally, E-cadherin expression quantity of cells at the basement of seminiferous tubules, where mGSCs usually localized, was obviously higher than that of other cells in the seminiferous tubules. Those results indicated that E-cadherin, as an important component of dairy goat mGSCs microenvironment, played an important role in maintaining the microenvironment of male spermatogonial stem cell niche. For further research, the E-cadherin-GFP mammalian over-expression vector was transfected into mGSCs-I-SB, a dairy goat male germ stem cell cell line we had established, to explore the effects of E-cadherin on mGSCs. Gene expression changes of E-cadherin and other marker genes associated with cell proliferation and pluripotency were tested by qRT-PCR, Western blot and immunofluorescence staining. The result showed that E-cadherin expression quantity increased obviously, and the expression of octamer-binding transcription factor 4 (OCT4), integrin alpha-6 (CD49f), glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRa1), promyeloeytie leukaemia zinc-finger (PLZF) and proliferating cell nuclear antigen (PCNA), genes associated with cell self-renewal, proliferation and pluripotency maintenance were also raised in E-cadherin overexpression (0CDH1) dairy goat mGSCs groups compared with control groups (Con). Immunofluorescence staining detection found that those genes colocalized with E-cadherin-GFP. These results indicated that E-cadherin promoted dairy goat mGSCs self-renewal, proliferation and pluripotency maintenance. The study of the influence of E-cadherin on dairy goat mGSCs will provide a molecular basis for further study of cell self-renewal, proliferation and differentiation processes, which will be useful for establishing stable male germline stem cell lines.

Asymmetric cell division and nonrandom sister chromatid segregation are the hotspot in the field of life science. Asymmetric cell division includes morphological asymmetric division and functional asymmetrical division. Asymmetric cell division is very important for sustainable life and organizational integrity. In recent years, in the field of germ cell and stem cell research, studies on the phenomenon of asymmetric cell division made some new progress, these developments will be helpful to understanding and knowing the formation of asymmetric cell division and its regulation mechanisms. Mammalian oocytes maturation asymmetrical division (form a larger volume of eggs and small volume of polar body) is a representative of the morphology of equal division; stem cells proliferation induced by tissue injure is functional representatives of asymmetrical divisions, a process that although two offspring produced by cells form approximation, but have different fate and function, one stay in micro environment to maintain the original stem cell properties, and the other is to differentiate into functional cells to replace damaged cells function. Asymmetric cell division is rooted in nonrandom sister chromatid segregation (optional). The testis tissue of Drosophila melanogaster can be microscopic observation in vivo in vitro of spermatogonial stem cells and in the asymmetric division and sister chromosome separation can be real-time observation. In recent years, asymmetric cell division and sister chromosome separation results mainly from fruit flies. According to the reports of nonrandom sister chromatid segregation of Drosophila melanogaster spermatogonial stem cells in recent years, the review will explain the hypothesis and mechanisms of asymmetric cell division, and try to assume the future development of the field. It can provide some ideas for the future development of the field.

Cultivated peanut (Arachis hypogaea) is one of the important oil crops, and the planting area stabilize at about 4.7 million hectares in China in recent years. In general, the oleic acid content of kernels is about 36%~67% in cultivars currently grown in China, and the ratio of oleic acid to linoleic acid (O/L) ranges from 0.78 to 3.5. Increasing the O/L of peanut cultivars significantly improves the nutritional quality and prolongs the shelf-life, and has become one of the major goals of researchers in the area of plant lipid modification. Peanut ??12 fatty acid desaturase genes(AhFAD2A and AhFAD2B) are the key genes controlling the content of oleic acid and O/L in the peanut kernels, and the mutations at both AhFAD2A and AhFAD2B loci exist in the high oleic acid peanut varieties. In view of the G/A variation in AhFAD2A 448 nt and the A base insertion-deletion (InDel) variation in AhFAD2B 442 nt in the low-oleate and high-oleate peanut germplasm, several molecular markers and detection methods including cleaved amplified polymorphic sequence (CAPS), allele-specific PCR(AS-PCR), and so on, have been developed and applied. In this study, the primers, probes and detection protocol of the TaqMan q-PCR for high-throughput detection of the two SNP allelic variations were developed, and a number of samples including some varieties and individual plants from the backcross group BC2F1 and BC1F2 were detected. It was found that this method was highly efficient and accurate, and the detection results were in accord with the sequencing results. Furthermore, in order to confirm the results of genotype discrimination, their oleic acid and linoleic acid contents were detected by gas chromatography or near infrared reflectance spectroscopy. Kompetitive allele specific PCR (KASP) was a homogeneous, fluorescent, endpoint genotyping technology, which offered the simplest, most cost-effective and flexible way to determine both SNP and insertion/deletion genotypes. An efficient KASP method for detecting these two allelic variations was also developed in this study. Comparison with the method of TaqMan probe, the KASP was more economic due to applying the universal fluorescence-labeled probe. Using the TaqMan qPCR method and the KASP method, the genotypes of AhFAD2A and AhFAD2B gene in 14 peanut varieties and the seeds of the backcross group BC2F1 and BC1F2 were identified. The consistency rates of the two methods for G/A SNP and A/-InDel were 71.7% and 96.7%, respectively. The lower accuracy for G/A SNP by KASP results from the interfering of the G at 448 nt of AhFAD2B and the most similarity of sequences between AhFAD2A and AhFAD2B. Based on the different fluorescent probes for G/A or A/-InDel allele variation, these two genotyping assays could distinguish three different genotypes (the homozygote, heterozygote of wild-type and mutant-type) in each reaction. In this paper, the advantages and disadvantages of different methods including CAPS, AS-PCR, sequencing, TaqMan probe and KASP were compared. Among these methods, TaqMan probe and KASP with the obvious detection accuracy and stability were high-throughput detection methods, and need a higher requirement for the instruments, such as the q-PCR equipment or laboratory of the government chemist (LGC) SNPline XL PCR equipment. Finally, the application prospect of the new high-throughput method was also discussed. These high-throughput methods will greatly improve the accuracy and efficiency of selecting for high oleic acid and effectively accelerate the breeding course of peanut varieties with the target traits.

Loop mediated isothermal amplification (LAMP) is one of isothermal nucleic acid amplification methods that have been widely employed in many areas. In this study, a new closed-tube colorimetric method for monitoring LAMP with a novel metal indicator was developed. Firstly, acid chrome blue K (ACBK), the metal indicator was evaluated in the initial LAMP reaction combined with various combination of reaction ingredient, such as reaction buffer, dNTPs mixture, primers mixture or Mg2+, respectively. This procedure demonstrated that the solution color of the LAMP reaction with ACBK could be changed from red to blue, as the concentration of Mg2+ decreasing in the reaction solution. Subsequently, the concentration of ACBK and Mg2+ in the LAMP detection system was selected and optimized. Finally, selected 2 μL 0.04% ACBK and 6.0 mmol/L Mg2+ in the 25 μL LAMP detection system. The LAMP with ACBK for detection of TNOS was optimized and established. Further, the specificity of the new established colorimetric assay employing ACBK in LAMP reaction for TNOS was tested with diverse transgenic events from different crops, and the sensitivity threshold of the assay was absolute 100 copies for transgenic rice(Oryza sativa) genomic DNA and 50 ng 0.1% DNA from rice, soybean (Glycine max), rape (Brassica napus), and maize(Zea mays), respectively. Finally, the applicability of the LAMP assay was further validated successfully using practical maize samples. All the detection results can be easily discerned either via naked-eye or using UV-vis spectroscopy. The established method can accelerate the speed of detection of genetically modified ingredients as well as provide new idea for judgment of the isothermal nucleic acid amplification.

Influenza in birds is caused by infection with viruses of the family Orthomyxoviridae placed in the genus influenza virus A. Many species of birds have been shown to be susceptible to influenza A viruses. Influenza A viruses are classified into subtypes on the basis of their haemagglutinin (HA) and neuraminidase (NA) antigens. Traditional diagnostic methods of the subtype H5 influenza are laborious, time-consuming or insensitive, such as virus isolation, haemagglutination, neuraminidase inhibition and RT-PCR. So, more sensitive and convenient methods are in demand. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers the advantage of omitting thermocycling, enabling RNA amplification at constant temperature, and is used most widely among the isothermal amplification technologies. In order to develop a detection method of subtype H5 Avian influenza virus (AIV) with RT-LAMP, 3 pairs of primers specifically directing to 8 recognition sites on the hemagglutinin (HA) gene of H5-AIV were designed, and then mixed into the other ingredients as recommended in the instruction of the RT-LAMP kits with or without freeze-dried protecting agents. The recombinant plasmid with the whole genome of subtype H5N1-AIV was extracted, purified and diluted to make each reaction system of RT-LAMP and RT-PCR contain 106, 105, 104, 103, 102, 101 or 100 copies. The sensitivities of the RT-LAMP before and after lyophilization, and the difference on sensitivity between RT-LAMP and RT-PCR by turbidity monitoring and agarose gel electrophoresis analysis, respectively, were determined and compared. The specificities of RT-LAMP and RT-PCR with subtypes H1~H15 AIV, Newcastle disease virus(NDV) and Infectious bronchitis virus (IBV) were tested. The feasibility of the visualization of RT-LAMP was tested by adding the color developing solution (mixture of SYBR Green I and hydroxynaphthol blue) into the isothermal amplification systems after reaction for 1 h at 63 ℃. Finally, totally 326 clinical samples with the established RT-LAMP and RT-PCR, and the percentages of the positive samples were compared between the 2 methods. The results of sensitivity tests showed that, at least as 102 copies of the recombinant plasmids with the target gene could be detected by RT-LAMP after reaction for 1 h at 63 ℃, with a sensitivity of about 10-fold higher than that of the RT-PCR. Moreover, the sensitivity of RT-LAMP won't be interfered by lyophilization with 5% trehalose, 1.25% mannitol and 1.25% bovine serum albumin, or by the color developing agent added when the amplification reaction finished. The specificity tests indicated that all the virus except subtype H5 AIV showed negative results both by RT-LAMP and RT-PCR. Among 326 clinically collected, 38 and 31 swabs were confirmed as positive by RT-LAMP and RT-PCR, respectively. All the 31 positive swabs by RT-PCR were also positive by RT-LAMP, and 7 negative swabs by RT-PCR were detected as positive by RT-LAMP. So, the sensitivity of RT-LAMP was preferable to the RT-PCR assay. The above results suggested that the RT-LAMP had higher sensitivity and good specificity, and could be used as a point-of-care (POC) test product since it is convenient for storage and very easy for the veterinarians to use in the farms.

The new variety of ayu (Plecoglossus altivlis) named “Zhemin No.1” was selectively bred from wild fish population which was captured in Fuxi stream, Ninghai, Zhejiāng province in 2002. A population selection process was performed by using growth traits and shape as the main selection index. After successive seven-generation of mass selection, a novel breeding line of ayu termed "Zhemin No.1" was successfully established. Compared with common ayu cultured under the same conditions, the body length and weight of Zhemin No.1 at 9-month old were 11.36 % and 25.92% higher, respectively. During 2012~2014, the pilot-scale culture of Zhemin No.1 reached 45 000 m2 freshwater. The survival rate of Zhemin No.1 at 9 month old was 30.00% higher than common ayu. The deformity rate of Zhemin No.1 at 9 month old was close to zero. The average revenue increased by 38.98%. The breeding process, germplasm characteristics, qualified traits and pilot test of Zhemin No.1 were detailedly described in this paper. This article will be beneficial for the breeding of other aquatic species and the expanding aquaculture of Zhemin No.1.