Chicken(Gallus gallus) is an important model organism and agricultural animal. Its sexuality is an important developmental trait in biological aspect and an economic character in poultry industry. However, the molecular mechanisms of sex determination and differentiation remain elusive in chicken. Despite the sex phenotype is determined by genes in a dichotomous ZZ/ZW sex chromosome, formation and differentiation of gonad and body gender is largely influenced by environmental factors, for instance hormone of estrogen. Regarding to the sexuality of chicken, there are two hypotheses that are referred as W-linked ovary determinant and Z dosage for testis determination, both of which are lack of convincing evidence. In this review, we have not only summarized the previous studies but also presented some new data on the epigenetics and cell autonomy in chicken sex determination and differentiation.

The genetic mechanism underlying yield traits is very complex, while QTL of rice yield traits which are uncovered in nitrogen starvation condition can provide a valuable reference to explain the genetic mechanism of yield traits, and facilitate fine QTL mapping and gene cloning, as well as screening of selective breeding of rice varieties tolerant to nitrogen starvation. In this study, 66 ILS population derived from the cross of Shuhui 527(Oryza. sativa ssp. indica) and Chujing 12(O. sativa ssp. japonica) which were served as the recurrent parent and donor parent; both marker-assisted selecting and high-generation backcrossing were used to detect QTL under the field experiment with normal and nitrogen starvation conditions by mapping with the single marker. A total of 24 QTLs were detected. Among of these QTLs, 20 QTLs were newly characterized, while qpn-1, qph-1, qby-4 and qby-2 were similar to the previous studies. qph-1a and qph-1b could affect the height of the site under the different treatments; we also found that both in the QTL of panicle number and grain yield per plant were linked with marker RM521 under nitrogen starvation condition; the additive effects of qpn-1b, qpn-2a and qpn-2b were less than 1. The results show that nitrogen starvation has strong negative effect on grain yield, panicle number, and biomass yield. Introgression lines population has strong ability for detecting micro effect QTL.

Oleosins are a type of small and basic protein covering the surface of oil body. Oleosin plays an important role in oil body stabilization and is often used as carrier for production of recombinant proteins in plants. The full length cDNA library of cultivated peanut(Arachis hypogaea L.) Luhua14 seeds was constructed and sequenced. From cDNA library we found 284 oleosin ESTs which could be classified into six groups, namely, AhOLEO-16.9，AhOLEO-17.7，AhOLEO-18.6，AhOLEO-22，AhOLEO-18.4 and AhOLEO-14.32 according to molecular weight of their coding proteins(GenBank Accession No: EG372527、EG373122, EG373716, EG372719, EE125019 and EE124234), in which AhOLEO-16.9，AhOLEO-17.7 and AhOLEO-14.3 family contained two members, the others contained one member, respectively, And the higher molecular weight oil oleosin gene AhOLEO-22 was first cloned in peanut. Peanut cDNA microarray and semi-quantitative RT-PCR results showed that the expression patterns of these six sub-family oleosin genes were similar in different organs and in seeds during different developmental stages. The expression of oleosin genes was very high in seed, while the expression of these genes was not detected in stems, leaves, flowers and roots. During seed development, the expression level of oleosin genes was low in DAP15 and gradually increased afterward. This research provides useful information for the study of peanut oleosin gene family as well as using of oleosin to improve peanut oil content and to produce foreign proteins.

To identify the purity of hybrid rape exactly, steadily and effectively, this study has screened 3 of 479 pairs of SSR primers which could distinguish a chemical hybriding male varieties of hybrid rape Qinyou 33 from its parental lines effectively and steadily based on the establishment of efficient simple sequence repeat(SSR) agarose electrophoresis standard analysis system through the primer screening. The single-plant in three high pure lines was verified by using one of the primer SA332. Results demonstrated that the three single-plant SSR map had high consistency with its corresponding standard maps, reaching 98%, 99.33%and 96.67%, respectively. The practical detection and the consistency of qinyou33 commercial seed compared with single-plant growing in field using the primer SA332 and SA85 were carried out at the same time. The result showed that the consistency compared with single-plant grow in field reached above 96%, 98.67% at most and the consistency between the primer SA332 and SA85 reached 0.67% and 2.67% respectively. The purity deviation between 150 strains of indoor small groups and the field test more than 300 strains only reached -2.19% to 3.76%. With the basically consistency of indoor SSR tests result and field actual purity, it is fully demonstrated that the high consistency SSR correspondence analysis is a powerful tool for identify hybridism purity of rape especially for chemical hybiding male varieties of hybrid rape which are no significant difference in morphology.

Adipose-specific phospholipase A2 gene(AdPLA) is a major regulator of adipocyte lipolysis, and in order to construct the prokaryotic expression system of cattle AdPLA gene, the coding sequence of AdPLA gene was obtained by Overlap PCR method from cattle(Bos taurus) genomic DNA and cloned into pGM-T vector, the positive clone sequencing result was same as the sequence in GenBank (GenBank No. NM_001075280). The positive plasmid was digested with BamHⅠ and HindⅢ, then the fragment was ligated with the expression vector pET28a(+), the recombined plasmid was transformed into Escherichia coli BL21(DE3) and induced with IPTG. SDS-PAGE result showed that the fusion protein His-AdPLA was expressed in E.coli, which indicated the prokaryotic expression system of recombined vector pET28a(+)-AdPLA was constructed successfully. This study provides a good foundation for further research of cattle AdPLA gene and lays a pathway for developing the AdPLA protein product in the future.

The traditional diagnostic method for Pseudorabies virus(PRV) has the defect of cumbersome procedures, long period, high cost and poor specificity. Thus we built a sensitive and specific loop-mediated isothermal amplification(LAMP) method for the detection of PRV, and used hydroxy naphthol blue(HNB) as indicator to judge the reaction result. The highly conserved gB (glycoprotein B)gene was selected to design primers. The detection could be finished within 45 min after the reaction system was established. Both LAMP and PCR were highly specific to PRV，but he LAMP-based assay, whose detection limit was 102 copies of the target sequence，was 10 times more sensitive than that of the PCR assay. In HNB test, when the ultimate concentration of HNB was 150 μmol/L, the color contrast of the LAMP products between positive samples and negative control was obvious and the sensisity of LAMP reaction was the best.The detection of clinical samples showed that the LAMP reaction had the similar detection rate with the PCR assay. In this experiment, the LAMP method is a highly specific and sensitive detection technology, which can be used instead of traditional PCR method.

There is not synchronized between nuclear maturation and cytoplasmic maturation of in vitro maturated oocytes. In order to solve this problem, we use butyrolactone I (BL-Ⅰ), cdc2 kinase inhibitor, to prevent porcine oocyte nuclear maturation in vitro. Porcine oocytes were cultured for 44 h in the medium containing different concentrations of BL-Ⅰ(0, 10, 20, 40 and 80 mol/L, respectively). With the increase of BL-Ⅰ concentration, the inhibition efficiency on maturation of porcine oocytes was increased. When treated with BL-Ⅰ in 20, 40 and 80 mol/L, respectively, the oocytes reached 25%, 47% and 67%, respectively, at germinal vesicle(GV) stage, and 38%, 9% and 0% at MII stage, which were both significant difference with control group. In addition, After removed BL-Ⅰand incubated for additional 44 h in ooycte maturation media, these oocytes at MⅡ stage were significant increased and reached 51%, 47% and 5%, respective, which showed the BL-Ⅰinhibition to maturation of oocytes is reversible. Additionally, oocytes incubated with 20 μmol/L BL-Ⅰ for 20 and 24 h without BL-Ⅰ (20 h＋+24 h-treatment group), showed low percentage at MⅡ stage compared with the control group (culture for 44 h without BL-Ⅰ) (P < 0.05, 65.6% vs 70.1%). When the matured oocytes from 20 h＋+24 h- treatment group were parthenogenetic activated, the cleavage rate, blastocyst rate and blastocyst total cell number had increased, but no significant difference compared with the control group. When the matured oocytes were used for nuclear transfer, the cleavage rate, blastocyst rate and blastocyst total cell number were better than that of the control group, but only significant difference in cleavage rate (P <0.05, 69% vs 62%). These results indicate that BL-Ⅰ can significantly inhibit the porcine oocytes maturation, and that the inhibition can be reversed by removing BL-Ⅰ. These results also show that matured oocytes treated with the proper concentration of BL-Ⅰ can slightly improved the development ability of parthenogenetic and nuclear transfer embryos.

The objective of this study was to find a more efficient transfection method between a new transfection method and the traditional transfection methods. Three types of bovine donor cells (fetal fibroblast cells, fetal oviduct epithelial cells and cumulus granulosa cells) that commonly used for nuclear transfer were transfected with plasmid DNA of green fluorescent protein (GFP) gene using the nucleofection, electroporation and lipofection. We firstly determined optimal transfection parameters for nucleofection and electroporation, then transfection experiments were carried out to compare efficiency of optimized nucleofection and electroporation with lipofection under identical conditions, and green fluorescence ratio and cell survival were assessed 48 h after transfection. The results showed that the optimization program of nucleofection for fetal fibroblasts, fetal oviduct epithelial cells and cumulus granulosa cells were V013, T023 and U023; In electroporation, when the electric field was 90 V/mm, pulse duration was 10 ms, fetal fibroblasts and cumulus granulosa cells got the highest efficiency, while parameters of 80 V/mm and 5 ms were good for fetal oviduct epithelial cells; Finally, after transfection comparision experiments, the nucleofection demonstrated the highest transfection efficiency of (98.78±0.30)%、(88.43±2.10)% and (71.31±0.77)% respectively in all three types of cells, were significantly higher than electroporation and lipofection; and lipofection yielded the highest survival rate, electroporation got the lowest. The results demonstrate that nucleofection can be a more efficient alternative to produce transgenic cattle when combining with somatic cell cloning technology.

In order to find stronger linked microsatellite markers, 9 polymorphic EST-SSR loci were selected in German Merino grading crossing offspring F2 with Chinese Merino sheep, then the genetic polymorphism of group and the correlation between microsatellite markers and wool traits were analyzed. The results showed that the loci had rich polymorphism and 39 alleles was detected among 9 loci in the group and 6 of them had significance differences between genotypes. There were 5 loci related with natural length, 4 loci related with stretched length and 3 loci related with the diameter. The results of F test for the relativity of EST-SSR markers with wool traits showed that 33176918 and 33176988 loci had significant genetic effects in the natural wool length and average diameter, respectively (P<0.05). The study suggests that 33176918 locus has significant marker effect on natural length and CD genotype is the favorable genotype; 33176988 locus has significant marker effect on fibre diameter and BC genotype is the favorable genotype. These two loci probable have high linkage with genetic locus of wool traits.

Erwinia amylovora causes fire blight on many plants of the Rosaceae family such as apple and pear. The Twin-arginine translocation(Tat) pathway plays a crucial role in transporting the proteins which are related to virulence. In this study, a tatC disruption mutant EaΔtatC was successfully constructed by homologous recombination as well as the complement strain EaΔtatC(pME-tatC). The results showed that the tatC mutant EaΔtatC displayed the reduced virulence, extracellular polysaccharide production, chemotaxis, motility, flagella assembly and growth in vitro. However, compared to wild type strain NCPPB1665(Ea1665), the induction of hypersensitive response in nonhost tobacco, biofilm synthesis and cellulose production of the tatC mutant EaΔtatC didn't show significant difference. These findings demonstrate that tatC gene in Erwinia amylovora is involved in growth, and motility and play an important role in virulence.

Mycoparasitic chitinases play an important role in resisting plant diseases for their strong capacity of degrading chitin. In order to clone and study chitinase gene(tfchi1) fromTalaromyces flavus, a pair of degenerate primers were designed according to the conserved sequences of chitinase genes in other fungi. The full-length DNA and cDNA of tfchi1 were cloned by using RT-PCR, 3'-RACE and 5'-TAIL (GenBank: GU361769, GU361770). The full-length DNA of tfchi1 was 2 561 bp, containing six introns with the length of 129, 78, 68, 65, 53 and 59 bp, respectively, ORF was 1 194 bp and it encoded 397 amino acids. Based on the predicted amino acid sequence of tfchi1 and the bioinformatic analysis of protein structure, two prevalent conserved catalytic domains with the sequences of SXGGW and DGXDXDWE were identified, Tfchi1 belonged to family 18 glycoside hydrolase, and shared 96% homology with the sequence of chitinase (XP-002480365) from Talaromyces stipitatus ATCC 10550. Its predicted molecular weight and isoelectric point were 43.47 kD and 4.97, respectively. Tfchi1 had no signal peptide sequence but 15 potential N-glycosylation sites. Random coils and α-helices were the major structural elements in secondary structure of Tfchi1, while(α/β) 8 rounded buckets were evident in tertiary structure. A recombinant protein with chitinase activity was obtained after tfchi1 was transformed into Pichia pastoris GS115 and its molecular weight was also consistent to the theoretical value. The results show that a chitinase gene, belonging to family 18 glycoside hydrolase, is correctly cloned from T. flavus in this study.

The epidermal growth factor receptor have been identified as a key players in female reproductive functions, the objectives of the present study were to identify the polyorphism of epidermal growth factor receptor gene(EGFR) and find the association between reproductive traits and the gene in goats. According to the Bos taurus EGFR gene sequence provided by The National Center for Biotechnology Information, we designed fourteen pairs of primers to detect single nucleotide polymorphisms of EGFR gene in Matou, Boer, Gulin Ma, Chuandong White, Dazu Black, Guizhou White, Jintang Black, Banjiao, Chengdu Ma, Nanjiang Brown of goats(Capra hircus) by PCR-single strand conformation polymorphism(SSCP). The result showed that the DNA fragments amplified by primer P3 and primer P13 had polymorphisms among primers P1~P14. For the fragments amplified by primer P3, three genotypes (AA, BB and AB) were detected in different goat breeds. The sequencing results revealed that the genotype BB had one point mutation (115837C→T) compared to AA genotype. The litter size with genotype AA and genotype AB had no significant difference in three breeds of goats (P>0.05), and goats with BB genotype rarely existed. For primer P13, three genotypes (GG, GH and HH) were detected in different goat breeds. The sequencing results revealed that the genotype HH had one point mutation (173015G→A) compared to GG genotype. The goats with genotype GH had significant higher litter size than those with genotype GG in Guizhou White and Chuandong White goat(P<0.05), but Gulin Ma didn't have polymorphisms of primer P13, and goats with HH genotype rarely existed. The results initially indicate that the EGFR gene may be either a major gene that influences the prolificacy in goats or a molecular marker linked with such a gene closely.

The current study was aimed to analyze the association of promoterⅢ in Acetyl-CoA carboxylases-α gene(ACACA) with milk traits in Laoshan dairy goat(Capra hircus). Denaturing high-performance liquid chromatography (PCR-DHPLC) and direct sequencing were applied to identify SNPs in promoter Ⅲ of ACACA gene in Laoshan dairy goat population containing 174 samples, and least square method was performed to evaluate the association of polymorphism in promoter Ⅲ with milk traits. The SNPs have been identified as followed, B/D at 1206 bp, A/C at 1255 bp, M/N at 1322 bp. The distribution of alleles and genotypes in both Group F and Group P was not balanced, but still under Hardy-Weinberg equilibrium. The variation between two genotypes was not significant in the whole population, only the difference between different herbs existed. Further, the difference between different genotypes were analyzed in Group F and Group P, respectively. The 1206 site in F group, allele B had a positive effect on protein , lactose and non-fat rate, and the difference between genotypes BB and BD was statistically significant for protein rate and fat rate (P<0.01), for non-fat and milk density (P<0.05). The substitution effects for protein, lactose and non-fat rate were significant (P<0.05). While in P group, the difference between genotypes BD and BB for all milk traits was not significant (P>0.05). Allele N was the dominant allele for lactose rate，while this was the negative allele for milk yield at 1322 site in F group, and the difference between genotypes MN and MM was significant for milk yield (P< 0.01) and for lactose rate(P<0.05). The substitution effects for lactose rate and milk yield were 0.12% and －52.22 kg (P<0.01), respectively. In P group, allele M was the dominant allele for protein rate, lactose rate, non-fat and milk density, the difference between genotypes MM and MN was significant for protein rate, lactose rate, non-fat and milk density (P<0.05). The substitution effects for lactose rate and non-fat were －0.25%, －0.44%(P<0.05), respectively. This study shows that the same alleles of promoter PⅢ in ACACA gene vary in their effects in different breeding groups, for milk protein rate, lactose rate, non-fat material rate, milk density and 300 d milk yield.

Ralstonia solanacearum causes a lethal wilting disease on many important crops in tropical, subtropical and warm regions in the world. The research of genetic diversity of R. solanacearum plays an important part in knowing the appearance and prevalence of bacterial wilt diseases. The special primer was used to identify R. solanacearum strains, in which 84 strains of R. solanacearum from different host plants in the Fujian Province were identified with a specific band in the location of 504 bp. Meanwhile, the genetic diversity of these R. solanacearum strains was assessed by Box element polymerase chain reaction (BOX-PCR) and Repetitive extragenic palindromic polymerase chain reaction (REP-PCR) method. Based on their genomic fingerprints, 19 specific bands were amplified in the BOX-PCR and 20 specific bands were amplified in the REP-PCR. The cluster analysis showed that the genetic diversity of R. solanacearum was concerned with geographic origin and host plants, respectively. The different host plants played a leading role in the genetic difference of R. solanacearum, while the difference of regions was mainly provided by the BOX-PCR, and the difference of host plants was mainly provided by REP-PCR. At the same time, there were some special fragments of R. Solanacearum from different regions in BOX-PCR patterns. Moreover, there were also some special fragments of R. Solanacearum from different host in REP-PCR patterns. Therefore, R. Solanacearum from different regions and hosts could be identified by these special fragments. All of results indicate that REP-PCR and BOX-PCR can provide an alternative way for the study of genetic diversity of R. solanacearum.

To know the genetic diversity and constitution of population of Ustilaginoidea virens in Fujian Province, the genetic diversity of 102 isolates on rice(Oryza sativa) collected from different rice growing areas in Fujian Province were assessed with RAPD(random amplified polymorphic DNA). A total of 157 bands were amplified by 10 primers selected from 100 random primers, with the percentage of polymorphic bands of 82.17% and genetic distance between 0.02~0.67. The results showed that there were significant genetic diversity in the population of Fujian, the highest genetic diversity of population was observed at rice growing areas of Mingxi(PPB=76.43， H=0.2212， I=0.3383)in comparison to the population of the other areas, and the genetic diversity of population from late season (PPB=91.08，H=0.2402， I=0.3655) were more diverse than that of early season (PPB=63.06, H=0.1892, I=0.2870). The result of cluster analysis revealed that 102 isolates tested could be classified into 7 genetic groups(R1 to R7) at 0.349 genetic distance level, and R1 was the predominant group contained 80 isolates in which existed some subgroups. And these groups had no obvious correlation with the geographic origin and the rice varieties origin of isolates. However, 10 isolates from Ninghua could be classified into 2 genetic groups, early season and late season, at 0.330 genetic distance level. The preliminary analysis show that geographic origin of isolates, rice varieties and their growing season may be the main factors for the genetic diversity of U. virens in Fujian, and play an important role in genetic variation of the pathogen, and occurrence and prevalence of the rice false smut.

Tomato mosaic virus (TAV) is an important pathogen in agriculture, infecting more than 100 species of 24 dicotyledoneae and 3 monocotyledoneae, such as Chenopodiaceae and Solanaceae. In order to analyze the role of genomic RNAs, full length sequence of TAV isolated from Beijing (TAV-BJ) was obtained by RT-PCR and infectious cDNA clones was constructed. Using total RNA from Nicotiana glutinosa infected by TAV-BJ as template, full length of RNA2 and RNA3 were amplified by RT-PCR, and RNA1 was obtained from cDNA of TAV-BJ double stand RNA1. PCR products of RNA1, RNA2 and RNA3 were cloned into pUC118 vector and sequenced. TAV-BJ RNA1 contained 3 409 nucleotides (nts), encoding 1a protein of 994 amino acids, RNA2 was 3 032 nts, encoding 2a protein of 829 amino acids and 2b protein of 78 amino acids and RNA3 consisted of 2 218 nts and encoded 3a protein of 247 amino acids and coat protein (CP) of 219 amino acids (GenBank accession No of TAV-BJ genomic RNA1, 2 and 3 was HQ424163, HQ424164 and HQ424165, respectilvely). cDNA clones of TAV-BJ genomic RNAs were transcribed into RNA and the mixture of transcripts RNA was mechanically inoculated onto N. glutinosa. At days post-inoculation (dpi), T1T2T3 induced host plants to express mosaic symptom, which was in accordance with that appearing on N. glutinosa infected by TAV-BJ viral RNAs. Seedlings of N. glutinosa infected by CMV-Fny lack of 2b gene (CMV-FnyΔ2b) was symptomless. Co-inoculation of T1, T2, T2Δ2b or T3 respectively with CMV-FnyΔ2b showed that the pseudorecombinant virus with T2 or T3 could resue the pathogenicity of CMV-FnyΔ2b in host plant, thus RNA3 of TAV-BJ might paly some function of 2b protein of CMV-FnyΔ2b. TAV-BJ RNA3 chimera infectious clones that replaced with 3a or CP gene fragment of CMV-Fny were constructed by Overlapping PCR. Biological assays of F1F2Δ2bRNA3T3aFcp and F1F2Δ2bRNA3 F3aTcp suggested that TAV 3a protein could compensate some function of 2b protein.In this study, infectious cDNA clones of TAV-BJ are successfully constructed and results of pseudorecombinant virus between CMV-Fny and TAV-BJ confirm that 3a gene of TAV-BJ have some role of CMV-Fny 2b gene.

Most of the Trichoderma strains are deficient in the β-glucosidase production, results in end product inhibition, and the cellulase from Trichoderma shows relatively low growth rates, Thus, cellulase from Penicillium has attracted renewed attention. The complete gene of exo-β-1,4-D-glucanases (cbh1) was cloned and expressed in Pichia pastoris to characterize the recombinant enzyme. The gene of cbh1 (GenBank Accession No.HQ843504), cloned from M strain of Penicillium oxalicum by the modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was 1 641 bp without intron and encoding 547 amino acids. Because its Glu236, Asp238 and Glu241 were the classical catalytic residues, we predicted cbh1 belongs to glycoside hydrolase family 7. The recombinant plasmid pPIC9-cbh1 was constructed by cloning the gene into vector pPIC9 and then was transformed into Pichia pastoris strain GS115 by electroporation. The sequencing and activity analysis of the positive recombinant showed pPIC9-cbh1 could successfully express cbh1 in P. pastoris. To our knowledge, it was the first report that exo-β-1,4-D-glucanases from Penicillium oxalicum successfully expressed in P. pastoris. The enzyme activity of the recombinant reached up to 56 IU/mg. The optimum pH and temperature of the recombinant enzyme were 6.0 and 55℃ respectively, and the stability of pH and temperature were both good. The results suggest that cloning of the cbh1 enriches the gene resources of cellulases, and its successful expression in P. pastoris provides the fundament for the construction of gene engineering strains.

Low enzymatic activity and high cost are the two main problems that limit the industrial applications of cellulose. In order to enhance the enzymatic activity of neutral endoglucanase activity, error-prone PCR was conducted to engineer the Bacillus subtilis C-36 endoglucanase gene. Two optimum mutants, b-15 and b-28 were obtained, with an endoglucanase activity 2.1 folds and 3.6 folds increased, respectively. The sequence of b-15 endoglucanase gene showed six nucleotide substitutions leading to four mutated amino acids; and b-28 endoglucanase gene showed one nucleotide substitution leading to one mutated amino acid. According to the 3D structure of endoglucanase mimicked by SWISS-MODEL, the four mutated amino acids of b-15 were either located at the corner between the fourth and fifth α-helix in the catalytic domain or at the fifth β-fold or the corner between the ninth and tenth β-fold in the carbohydrate-binding domain. And the mutation of b-28 was located at the fourth β-fold in the carbohydrate-binding domain. Following the orthogonal experiment, the mutant b-15 and b-28 could reach to an endoglucanase activity of 4.542 U/mL and 5.136 U/mL through fermentation, respectively, both of which were much higher than the wild-type control. These results have provided a base for further research of endoglucanase.

To obtain the optimism co-incubation time of boar sperm with DNA and set up the technique platform of swine sperm-mediated gene transfer, the Real time quantitative PCR, fluorescence microscope imaging and regular semen quality analysis were applied to detect the influence of incubation time of sperm with DNA on viability, motility, transfect efficiency, and the amount of adsorbed DNA and internalized DNA of sperm. The result revealed that, after 15, 30, 60, and 90 min co-incubation of the parceled and labeled DNA with sperm, the sperm motility and viability decreased extremely significantly with the increase of incubation time (P<0.01), while the transfect efficiency increased extremely significantly (P<0.01) or significantly (P<0.05), as well as the amount of adsorbed DNA and internalized DNA increased, However, there was no difference among incubation times (P>0.05) after 30 min incubation. In addition, compared with the increase of transfect efficiency of sperm, the decrease of motility and viability of sperm relatively was more obvious with the increase of co-incubation time. Overall, considering the parameters of motility, viability, transfect efficiency and the amount of adsorbed DNA and internalized DNA of sperm, the best co-incubation time of swine sperm with foreign DNA is 30 min.

The purpose of this investigation was to improve the fertilization efficiency of bovine sexed sperm. In order to search an appropriate incubation time, we co-cultured bovine oocytes with sexed sperm for 8 and 22 h, respectively. Meanwhile, we compared effect of two mediums on developmental capacity of sexed zygotes, including G-1/G-2 sequential medium and SOFaa single medium. The results showed that the cleavage rates of sexed zygotes were (50.88±4.44)% and(55.9±4.52)%(P>0.05) for 8 and 22 h incubation time, respectively. Furthermore, the blastocyst rates were (41.16±5.12)% and (35.05±5.02)%(P>0.05), respectively. However, the hatched blastocyst rate of sexed zygotes in 8 h incubation time was (52.56±6.95)%, obviously higher than that of 22 h(39.81±6.38)%(P<0.05). On other hand, in terms of the cleavage rate, the blastocyst rate and the hatched blastocyst rate, we found that there were no significantly difference between G-1/G-2 sequential medium and SOFaa single medium. Whereas, compared to the SOFaa medium, the sexed blastocysts cultured in G-1/G-2 medium had a larger cell number of blastocyst(113.9±9.46 vs 102.0±9.97)(P<0.05). These results suggest that reducing oocytes and sexed sperm incubation time can improve the hatched blastocyst rate of bovine sexed zygotes. According to cell number of blastocyst, G-1/G-2 sequential medium can be more suitable for in vitro culture of bovine sexed zygotes.

Using plasmid vector to build standard curve for absolute quantification PCR (AQ-PCR) might induce error because of the molecular difference between super-helix double-strand plasmid DNA and linear single-strand cDNA sample. To increase the exactitude of the AQ-PCR, we improved the method by adding two steps during the quantity assay procession of target gene mRNA copy in unknown samples. 1) The plasmid was digested to be linear by restriction enzymes for building standard curve; 2) The unknown cDNA samples were amplified to make a sense strand, followed the fluorescence quantitative PCR cycle with standard plasmid together. The results indicated that 1) The same copy of circular and linear plasmid started AQ-PCR test at the same time,the Ct value of circular plasmid was larger than that of linear plasmid (P<0.05); 2) The measured starting copy numbers of cDNA samples amplified once were larger than that of cDNA samples unamplified. The measured results of starting copy number between amplified and unamplified showed that there have 3 of 5 samples were significant differences (P<0.05).Using these improvements, the standard curve of chicken GATA5 gene was improved and reflected accurately the amplification of purpose products. Our improved method of using plasmid to build standard curve for AQ-PCR was more feasible, and the measure was more accuracy.

In this study, we have constructed and injected short hairpin RNA(shRNA) eukaryotic expression vectors into fertilized eggs. Upon incubation, the transferred plasmids were analyzed for their metabolism in the embryo gonads during gonadal differentiation, and their interference on the expression of polypeptide 1(CYP19A1) gene (belonging to cytochrome P450, subfamily A, family 19) was assayed subsequently. The feasibility of the method was then further discussed for gene-specific interference in the chick embryo. In the experiment, four specific shRNA expression vectors and a non-specific expression vector were constructed. For each vector, 45 fresh eggs were injected through the subgerminal cavity. Twenty non-treated eggs served as controls. Embryos were sacrificed after incubation for 12 days (E12, stage 38) and their livers were collected to detect the metabolism of the injected plasmids. Specifically, expression of each vector was investigated in the left gonads by quantitative RT-PCR and the co-produced enhanced green fluorescence protein (EGFP) was detected in all the injected embryos. The quantitative RT-RCR results showed that the specific expression vector cyp-580, cyp-1083 and cyp-1295 significantly suppressed CYP19A1 mRNA levels with a silencing efficiency of 73%、52% and 85%, respectively. However, the vector cyp-1403 could not suppress the expression of the CYP19A1 gene. The present study has provided a method to produce the sex-reversed chicken and has established a platform for gene-specific interference in vivo.