Invariant chain(Ii) is an important immunity molecule for vertebrate and plays a critical role in major histocompatibility complex(MHC)Ⅱ presenting antigen. To study the relation between chicken(Gus gallus) Ii and MHCⅡ molecules, we constructed 4 lentivirus vectors targeting to silence chicken Ii gene and compared their effect. First, we designed and synthesized 4 silencing chicken Ii gene sequences and obtained double-stranded (short hairpin RNA, shRNA) by one-step annealing method, then inserted them into lentivirus vectors pLL3.7 by a double enzyme digestion, respectively. The 4 recombinant vectors were named pLL-Ii-shRNA236, pLL-Ii-shRNA376, pLL-Ii-shRNA527 and pLL-Ii-shRNA539, respectively. Secondly,these vectors were transfected into 293T cells(human renal epithelial cell line transfected with adenovirus E1A (lethal infection gene)), respectively, and the green fluorescent protein expressed in these cells. When the packaged recombinant lentiviruses were injected into 293T cells, their titers were 2.2×107~5.1×107 TU/mL and their infection efficiency in chicken salvolar macrophage HD-11 was over 95%. Finally, a Real-time PCR was used to detect their effect on interfering mRNA of chicken Ii gene in HD-11 cells. The results showed that the interference efficiency of the 4 recombinant lentivirus vectors were 37%、52%、82% and 66%, respectively, compared with the control group of blank vector, in which pLL-Ii-shRNA527 showed peak efficiency. In conclusion, one recombinant lentivirus vector was selected from 4 vectors, which could most efficiently silence chicken Ii gene transfection in the chicken salvolar macrophage HD-11; these results suggested that the shRNA sequence determines the efficiency of recombinant vector in silencing specific gene. All these will provide experimental basis for studying the relationship between Ii and other immune molecules in presenting antigens.

Lipocalin, a family of diverse proteins that distribute widely in organisms, exhibits several advantages, compared with antibody. To construct a high-capacity and good-diversity ribosome display anticalin library for selecting small molecular anticalin with high affinity, bilin-binding protein gene(bbp) that is a member of lipocalin family from Pieris brassicae, was employed for the preparation of a random library. A DNA library, named bbp gene library, encoding amino acids of bbp possessing 16 randomly mutated residues, was generated using gene splicing by overlap extension PCR (SOE-PCR). Then, using the same method the elements (T and P fragments) that were necessary for efficient transcription and translation in vitro were introduced to bbp gene library to construct the ribosome display anticalin library. In conclusion, we successfully constructed a ribosome display anticalin library with a volume of 3.76×1023, high-capacity and good-diversity. This library could be served as an efficient selection system for antibodies of micro-molecular haptens.

Phytic acid is the main form of inositol and phosphorus in plant seeds, because of the body of monogastric animal lack of phytase enzyme, phytate phosphorus forms of phosphorus is difficult for monogastric animal absorption. Phytase has been widely used as feed additives, but the application research in food industry is just beginning. In this study, Aspergillus niger CICC2462 genomic DNA was taken as template, phytase gene(phy) fragments were amplified by PCR, and 5' and 3' homologous arms of saccharifying enzyme gene (glaA) were added on its ends. Ti plasmid of Agrobacterium gene replacement vector pSZHG-phy was constructed. In order to realize the high level expression of phy gene, phy gene replacement glaA gene homologous transformants were screened through A. tumefaciens-mediated transformation of A. niger. Results showed that 4 strains of homologous recombinant transformants were obtained, the rate of homologous recombination was 100%. Spectrophotometer was used to determined its highest fermentation of recombinant transformants, the result was 316.2 U/mL, 20.8 times as much as the starting strain. phy gene could express efficiently in A. niger glucoamylase production strains, and provide a basic data for the further development of food-grade phytase engineering bacteria.

PR domain containing 1, with ZNF domain(PRDM 1) is of great importance in the formation of primordial germ cell (PGC). A lot of reports focused on its function in the development of embryonic stem cell (ESC) to PGC, but it is unclear whether it has effects on the induction differentiation of adult stem cells or not. In this study, PRDM1 gene was cloned(GenBank No. JX154081.1) of mouse(Mus musculus), Lentivirus expression vector pCDH-PRDM1 was constructed and prepared Lentivirus particles using 293T cells, then, human umbilical cord mesenchymal stem cell (hUC-MSC) were transduced with PRDM1 gene-carrying virus. Results showed that PRDM1 could overexpress in hUC-MSC and upregulate the expression of some germ cell associated genes including stage-specific embryonic antigen-1(SSEA-1), developmental pluripotency associated 3 Dppa3(STELLA), stem cell growth factor receptor(C-KIT) and sex determining region Y-box 2(SOX2). The research provides important basis for further study of PRDM1, and is helpful to the research on induced differentiation of hUC-MSC towards germ cells.

Oocyte-specific linker histone(H1foo), the member of histone families, is located specificly in mammalian oocytes and primary embryos. There exists fast replacement between H1foo and its counterpart during the process of fertilization and nuclear transfer of mouse (Mus musculus) , cattle(Bos taurus) and porcine(Sus scrofa), which do help to open the chromosome in donor nuclear and achieve complete reprogramming. Induced pluripotent stem cell(iPSC) technology is another reprogramming strategy different from somatic nuclear transfer. Many reports showed that an open chromosomal structure facilitates somatic reprogramming and increases the induction efficiency of iPSCs. Therefore, in order to investigate whether H1foo has an effect on the induction efficiency of iPSCs, a transient expression of H1foo was conducted in the induction of porcine iPSCs. Two consecutive infections were conducted to the porcine fetal fibroblasts (PFF) by adding doxycycline(DOX)-inducible Lentiviral vectors expressing octamer-binding transcription factor-4(OCT4), SRY-related high-mobility-group(HMG)-box protein-2(SOX2), Kruppel-like factor-4(Klf4) and cellular homologue of avian myelocytomatosis virus oncogene(c-MYC). Vector pVenus-H1foo was electro-transferred into the OSKM-PFF and the cells transferred pVenus were acted as negative control. After several days, the obtained colonies were identified by IF and AP staining as iPSCs. By counting the number of iPSCs colonies, the results showed that the number of AP-positive colonies obtained in PFF with pVenus-H1foo or pVenus was almost the same. It was the first exploration about the effect of H1foo on induction of iPSCs which turned out there was no evident effect. The results above make basis data for the researches on mechnisms of reprogramming including somatic cell nuclear transfer and iPSCs.

Superovulation is one of important methods for obtaining oocytes, zygotes and embryos of different developmental stages. In order to investigate the effect of superovulation at different starting time on course of ovulation, fertilization and embryonic development, Kunming mouse(Mus musculus) in proestrus were superovulated by injecting pregnant mare serum gonadotropin(PMSG) at 12:00, 16:00, 20:00 and 24:00, respectively, followed by injecting human chorionic gonadotropin(hCG) 48 h later. And then, superovulated mice mated with male overnight, and vagina plugs were detected at the next morning. The results of oocytes, zygotes and embryos collected in different time after injection of hCG showed: For mouse superovulated at 12:00, 16:00, 20:00 and 24:00, the opportune times to collect oocytes from oviduct were about 16.23, 14.02, 15.93 and 12.98 h after injection of hCG, respectively; The opportune times to collect zygote were 24.62, 23.60, 26.53 and 20.03 h after injection of hCG, respectively; The opportune times to collect cleavage embryos were 42.76, 42.39, 41.85 and 40.47 h after injection of hCG, respectively; The opportune times to collect 4-cell embryos were 56.87, 57.84, 57.31 and 56.92 h after injection of hCG, respectively; The opportune times to collect 8-cell embryos were 66.89, 67.39, 66.20 and 66.07 h after injection of hCG, respectively; The opportune times to collect morulae were 76.31, 76.21, 76.29 and 75.11 h after injection of hCG, respectively; The opportune times to collect blastocysts were 100.65, 97.14, 93.91 and 96.86 h after injection of hCG, respectively; The opportune times to collect hatched blastocysts were 114.57, 112.34, 112.11 and 110.28 h after injection of hCG, respectively. In conclusion, the starting time of superovulation had no significant effect on course of ovulation, fertilization and embryo development. By regulating superovulation starting time, the appropriate time to collect oocytes, zygotes and embryos in different developmental stage could be selected. Mouse could be superovulated at a reasonable starting time to satisfy different research requirement. This study provides technical support for research involving in mouse oocytes, zygotes and embryos in accurate developmental stage.

In order to find a simple and effective method for enucleation in mouse(Mus musculus) oocytes, present study improved a enucleation method of metaphaseⅡ(MⅡ) oocytes under the condition of Piezo-electricity generation device assisted micromanipulation, and compared the enucleation efficiency and further development of nuclear transfer reconstructed embryo between improved and conventional enucleation method in sucrose-free or sucrose-containing enucleation manipulation solution. In addition, after enucleation by improved method, the further development of reconstructed embryo was compared in manipulation solution containing different serum concentration. The results demonstrated that the rates of visible nuclear of MⅡ oocytes were 98.4%(184/187) and 98.7 % (153/155) in manipulation solution without sucrose and with 3% sucrose, respectively. By adjusting the object lens distance to visualize spindle region of MⅡ oocyte, there was no significant difference (P＞0.05). In enucleation manipulation solution without and with sucrose, by using improved enucleation method, that was enucleation pipette pass through the zona pellucida, aspirated the MⅡ spindle region and pulled out from oocytes, the enucleation rates were 84.6% and 88.2% respectively. The enucleation rates by using conventional enucleation method in these manipulation solutions were 83.1% and 88.6%, respectively, there was no significant differences between them (P＞0.05). However, the oocyte cytoplasm extracted was significant less in improved method than that in conventional method, indicating the advantage of improved method. After enucleated using improved method in manipulation solution without sucrose, the reconstructed embryo developmental rates of 8-cell embryo, morula and blastocyst were the highest among these groups. The developmental rates of morula(16.3%) and blastocyst(5.8%) by using improved method in manipulation solution without sucrose were significantly higher than in manipulation solution with sucrose(5.1% and 0), and conventional method in manipulation solution without or with sucrose(3.3% and 1.2%)(P＜0.05). Moreover, blastocysts were obtained only using improved method in manipulation solution without sucrose. The blastocysts rate in manipulation solution containing 20% fetal bovine serum(FBS) group was significantly higher than 10% FBS group (10.9% vs 4.9%)(P＜0.05). Conclusion was that oocytes enucleated by improved methods in manipulation solution containing 20% FBS was effective condition for supporting the development of reconstructed embryos. Present study made a helpful exploration in improvement of somatic cell nuclear transfer efficiency by improving the nuclear visualization and enucleation methods for mouse oocytes.

The mature oocyte cytoplasm have a role in the reprogramming of donor cells, maturation of oocytes become the key factors in the process of nuclear transplantation. In order to study the effect of the removal of cumulus cells around germinal vesicle breakdown(GVBD) on porcine(Sus scrofa) oocyte in vitro maturation, porcine cumulus - oocyte complexes (COCs) were cultured in vitro and got samples per 5 h. Chromosomal morphological changes of porcine oocyte in vitro maturation were examined by aceto-orcein staining technique, and established the timetable of the oocytes chromosome developmental process. Then the cumulus cells were carried off from COCs in 21, 27 and 42 h, respectively. The effects of cumulus cells on oocyte maturation were investigated by counting the number of excreted polar body. From 2~17 h, most of the oocytes were in GV stage (88.30%~52.38%); 22 h, most of the oocytes were in germinal vesical breakdown~premetaphaseⅠ(GVBD~pre-MⅠ)stage(63.25%); 27 h, most of the oocytes in metaphaseⅠ(MⅠ)stage(42.25%); Until culturied for 32 h, the majority oocytes stayed in anaphaseⅠ(AⅠ)(29.90%); 37 h, telophaseⅠ(TⅠ) oocytes were the most (40%); And by 42 h, most of the oocytes reached metaphaseⅡ(MⅡ)stage (72.81%). We carried off cumulus cells at 21, 27 and 44 h and set these oocytes as 3 groups. The result showed that the maturation rate of 21 h group was 25.56%, lower than that of 27 h group, which reached 34.33%, the 42 h group was the highest that reached 84.33%. These results illustrate the importance of cumulus cells on oocyte maturation in vitro oocyte maturation. This study is conducive to further exploring and analyzing the methods of porcine oocytes matured in vitro, improving the culture system for enhanced maturation rate of porcine oocytes in vitro, and lay the foundation for the quality of embryos cultured in vitro.

Pluripotent stem cells (PSCs), which are regarded as important cell resources, have become the research focus around the world in recent years. Acquisition and acquaintance of these cells are of significance in medical research and biotechnology application. Pig(Sus scrofa) is considered to be the most important domestic animal in PSCs establishment, because of its similarities in physiology and metabolic to human(Homo sapiens). For now, however, pig PSCs establishment are still facing a lot of problems. Combined with the successful experiences of the PSCs research of mouse(Mus musculus) and human, we summarized cultural conditions, regulatory factors, signaling pathways, and gene expression profiling of those reported porcine PSCs. We provide useful information for the establishment of defined embryonic stem cells(ESCs) and iPSCs of pig.

As porcine body size, physical structure, and metabolism are similar to that of humans, pig(Sus scrofa domesticus) is suitable to be human disease and regenerative medicine research model. Porcine induced pluripotent stem cells (iPSCs) have been generated by using a cocktail of defined transcription factors, however, the inserted gene copy is different. Here, we generated porcine secondary fibroblasts which contained tetracycline operator(TetO)-FUW-OSKM and FUW-M2rtTA and it could reprogram in the presence of the doxycycline(DOX) without further viral infection. The lentiviruses (TetO-FUW-OSKM and FUW-M2rtTA) infected porcine embryo fibroblasts(PEF) were reprogrammed into primary iPSCs in the iPSCs medium with DOX. The primary iPSCs were positive for alkaline phosphatase (AP) staining and only could be passaged with leukemia inhibitory factor(LIF) and basic fibroblast growth factor(bFGF) on the Matrigel. The fibroblasts were differentiated by embryoid body formation and termed TetO-PEF. The TetO-PEF contains TetO-FUW-OSKM and FUW-M2rtTA which the inserted gene copy was the same. Without further viral infection could the TetO-PEF be reprogramming in the iPSCs medium with DOX. The iPSCs obtained in this way were termed the secondary iPSCs. Reprogramming of iPSCs from the TetO-PEF was more efficient and faster than that from fibroblasts. The secondary iPSCs were characterized by AP staining and with expression of OCT4, SOX2,SSEA1,SSEA4 and TRA-1-60 and without expression of TRA-1-81. A system was set up for reprogramming of porcine somatic cells to pluripotency only with DOX, which will provide cell for the optimization of porcine iPSCs culture medium.

In order to explore the fertility characteristics of Triticum timopheevii cytoplasmic male sterility(T-CMS), and to furtherly provide the theoretical and technical support for taking advantage of T-type sterile lines to breed hybrid wheat (Triticum aestivum L.) by using molecular assisted breeding, restorer and sterile DNA pools were established using the extremly sterile and fertile plants among the F2 population of ms(S)Aikang 58/R113, respectively. A hundred and nity six sets of SSR markers which were located on the 1st and 6th chromosome groups were screened for polymorphism between the 2 parents. The results showed that 3 markers which located on the chromosome 1AS and 4 markers which located on the chromosome 6BS were found to be amplified stably polymorphic bands between the parents and the 2 gene pools. Linkage analysis indicated that the microsatellite locus Xgwm136, Xgpw7062 and Xgdm33 located on chromosome 1AS were found to be linked to the restorer gene Rf1 with the estimated genetic distance of 4.8, 9.6 and 13.7 cM, respectively, with an order of Xgdm33, Xgwm136, Rf1 and Xgpw7062. Xgpw1079, Xgwm193, Xgpw7011 and Xgwm508 located on chromosome 6BS were found to be linked to the restorer gene Rf4 with the estimated genetic distance of 3.4, 6.8, 13.7 and 21.5 cM, respectively, with an order of Xgpw7011, Xgpw1079, Rf4, Xgwm193 and Xgwm508. The result also showed that the fertility of T-CMS restorer R113 in wheat was mainly influenced by 2 major fertility restorer genes(Rf1 and Rf4) and some minor enhance genes of fertility. The breeding for new fertility restorer lines of T-CMS(or S-CMS) in wheat would be facilitated by using the seven SSR markers，which can effectively improve the selection efficiency of the corresponding restorer.

From the composite hybrid rice (Oryza sativa L.) F2 population of breeding materials Guanghui128, japonica rice Kitaake, Shuhui955 and Shuhui881 found a mutant named TS (top-spikelet-two-grain mutant). The most significant variation of TS is the top of the first and secondary branch of spikelet of the TS, which clusters for 2~3 grains together. In order to specify the genetic mechanism of clustered gene ts and its utilization value in rice breeding, we performed morphological observation and genetic analysis of clustering traits in F1 and F2 segregating population derived from cross between TS and indica G2480B, analyzed the application prospect of ts by using 3 stable maintainer lines with ts bred by backcross and selfing for many generations. Morphological observation showed that F1 population was completely dominant and segregation ratio of F2 population fitted to a pair of gene separation mode (3∶1), which demonstrated that this trait was controlled by a single recessive gene and could stablely inherit. Gene ts was mapped onto the long arm of the Chr.6. and furtherly located between RM20323 and RM6298 according to the data from Gramene web, with genetic distance of 5.1 and 5.6 cM, respectively. L332, one of the 3 bred maintainer lines, kept clustered traits and its yield no significant differences, compared with one of the parents (G2480B), with a better cooking and eating quality. The expression of ts gene had no significant correlation with the effective panicle number, grain number per panicle, 1 000-grain weight and single plant yield of rice, and no correlation with seed setting rate. The above results provide the theoretical basic data for the use of ts gene in rice breeding.

Leaf senescence affects the yields and fiber quality of cotton (Gossypium hirsutum L.), while ethephon is an important plant growth regulator, which plays important role in promoting the senescence of plant tissues. DNA methylation is an important component of the epigenetic regulation, and plays a very important role in the regulation of gene expression in higher plants. The methylation sensitive amplified polymorphism (MSAP) technique was used to assess epigenetic variations of cotton cotyledons under ethephon treatment, which would help to isolate and identify methylation regulating genes during cotton cotyledons senescence. The results showed that the cotton cotyledons genomic DNA methylation ratios were 32.99%, 35.45% and 37.49%, respectively, lower than that of the control group (37.92%), when they were treated with 300, 500 and 700 mg/L ethephon. In contrast with the control group, treated with 300, 500 and 700 mg/L ethephon, methylation and demethylation loci ratio of cotton cotyledons genomic DNA were 2.71%, 3.63%, 4.88% and 10.66%, 9.84%, 9.23%, respectively. The ratios of DNA methylation and demethylation increased gradually accompanied with the enhancement of ethephon treatment. Seventeen MSAP different fragments were identified, and NCBI Blast results showed that these different fragments were homologous to functional genes, including senescence-associated genes (such as pectin methylesterase gene), abiotic stress-related genes (such as cytochrome P450), signal transduction-related genes(such as alcohol dehydrogenase gene, cytoskeleton-related genes and actin depolymerizing factor), and protein synthesis-related genes(such as translation elongation factor 1A-1), which were associated with senescence of cotton cotyledons under ethephon treatment. In conclusion, in the ethylene-induced senescence course of cotton cotyledons, DNA methylation played an important role in the regulation of cotton cotyledons senescence, which provides a theoretical basis for regulation mechanism of cotton senescence from the genomic level.

Porcine aminopeptidase N (pAPN) was the receptor protein of a series of virus, such as Transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV). So this study aimed to probe into the structures and important mutation loci of pAPN gene and provide basis for screening out effective genetic markers of the resistance to viral diarrhea in Meishan pigs (Sus scrofa). The functional regions and important mutation loci were screened out by bioinformatics analysis of the protein structure through PCR, cloning, sequencing and bioinformatics tools. The results showed that the cDNA sequence of pAPN gene (GenBank accession No. KF280271) of Meishan pigs was 2 886 bp in length, including 20 exons, which encoded 961 amino acids. Compared with standard pAPN gene (Accession No. NM_214277.1) sequence in the Genbank database, there were 10-amino acid mutations and 2-amino acid deletions. Six mutations of Phe82Asn, Leu107Phe, Leu108Ile, Ser330Pro, Trp399Arg, and Glu465Gly occurred in the catalytic activity area of APN enzyme. One mutation of Gln747His and 2 deletions of 748Tyr and 749Ser occurred in APN virus combined area. Prediction of protein structure and functional analysis of encoding product showed that pAPN protein was unstable and hydrophilic, without signal peptide, including 1 transmembrane helice (transmembrane region between 12th and 34th amino acid). Secondary structure of pAPN protein mainly contained alpha helix and beta folding. Encoded product was mainly responsible for cell envelope, central intermediary metabolism and biosynthesis of cofactors. One mutation of Gln747His and 2 deletions of 748Tyr and 749Ser might associate with the combination ability of aminopeptidase N and virus. This study analyzed the function and screened out the mutations of pAPN gene, which provides theoretical basis for selecting effective genetic marker of the resistance to viral diarrhea in future.

A large scale of cold water fish germplasm resources extensively distribute in Irtysh River of China, the burbot, Lota lota (Gadidae, Telestei) is as one of cold water groundfish, and the establishment of sperm cryopreservation technology and cryobank is of great significance to its germplasm resources preservation. In the present study, 7 kinds of sperm diluents were prepared with the basic liquid including inorganic salt, glucose, sucrose and fetal bovine serum(FBS). The sperm were diluted at a ratio of 5∶1 (extender: sperm), the motility were observed and recorded under the microscope. The results indicated that the sperm expressed certain motility in the SS, D15, and CM ((13.33±2.89)%~(8.33±2.88)%). On this basis, 22 kinds of gradient dilutions (SS-00~SS-10 and D15-0~D15-7) were prepared, and then diluted sperm, observed motility as in the same way. The results indicated that the motility were relatively higher in the SS-0(56.67±5.77)%, SS-2(63.33±5.77)%, SS-5(53.33±5.77)% and D15-7 (63.33±5.77)%, closed to the fresh sperm motility (76.67±5.77)% (P＜0.05). Mixed cryoprotectants and DM were prepared with dimethyl sulfoxide (DMSO) and MeOH at a ratio of 3∶2. And sperm were diluted by diluents selected, SS-5 and D15-7 at a ratio of 5∶1 (extender: sperm), then added 5%~12%DM, respectively, for sperm cryopreservation. Before cryopreservation, the sperm was activated with 2 ℃ freshwater, observed and recorded the motility under microscope. The results indicated that the motility of 5% DM within the SS-5 and D15-7 was (73.33±5.77)%, there were no significant difference with fresh sperm (P＞0.05), but the sperm motility almost was 0 with the rised DM concentration to of 10%~12%. After cryopreservation, the frozen sperm motility results showed that with the DM concentration of up to 6% in the SS-5, the sperm motility were relatively higher of (26.67±5.77)% (P＜0.05). In addition, when the DM concentration was 7% in the D15-7, the motility was also relatively higher (26.67±5.77)% (P＜0.05). The present study results provide the theoretical and technical basis for cryopreservation of spermatozoa of the burbot from Irtysh River of China.

Thyroid hormone plays an important role in the metamorphosis of flatfish and its biological effects are mediated by thyroid hormone receptors (TRs). In this study, the full length of TRβ had been sequenced in Cynoglossus semilaevis and its biological activities were analyzed. The nucleotide sequence of TRβ was 2 345 bp, including 5' terminal UTR of 454 bp, 3' terminal UTR of 703 bp and complete open reading frame of 1 188 bp which encoded 396 amino acid residues. Compared to the amino acid sequences of TRs in different species, we found a insertion of 9 amino acids in the hinge region which was unique to TRβ of teleosts. We found that the expression level of TRβ in liver was significantly higher than that in other tissues using Real-time PCR. We speculated that TRβ had diverse functions in tissues of adult Cynoglossus semilaevis, especially might play an important role in liver metabolism. We also found that TRβ gene transcript increased rapidly in the beginning of metamorphosis, reached peak in metamorphic climax. Moreover, larvae were exposed to the thyroid hormone(T4) and goitrogen thiourea(TU) and the expression levels of TRβ were detected. The expression levels of TRβ in control and T4-treated group rapidly increased as metamorphosis began. On the contrary, the expression levels of TRβ in TU-treated group still remained in a low level. In conclusion, TRβ gene played an important role in the metamorphosis of Cynoglossus semilaevis. The research provides a theoretical basis and reference for molecular mechanism research of metamorphosis of Cynoglossus semilaevis.