In order to investigate the effect of cryopreservation on spermatozoal structures in Pseudosciaena crocea, we used dimethyl sulfoxide (DMSO) or ethylene glycol (EG) as cryoprotectant, cortland solutions as extender, and two-step cooling procedure to cryopreserve P. crocea sperm in 0.25 mL straws. And we detected the DNA damage of frozen sperm by single cell gel eletrophoresis(SCGE) and membranous damage by flow cytometry(FCM). The results showed that there were no significant differences between fresh sperm and frozen-thawed sperm in vitality when 5%~20% DMSO or 5%~20% EG were used. The activation rate, moving time and life-span of frozen-thawed sperm were (87.00±2.45) %, (8.99±0.24) min and (13.11±0.65) min at 10% DMSO, and (87.50±2.52) %, (8.45±0.48) min and (12.84±0.50) min at 10% EG. A significant drop in sperm vitality was observed at 25% DMSO or 30% EG. SCGE results showed that there were no significant differences in DNA fragmentation between fresh sperm and frozen-thawed sperm diluted with 5%~20% DMSO or 5%~20% EG, but the DNA damage to frozen-thawed sperm significantly increased at 25% DMSO or 30% EG. There was a positive correlation between the DNA damage extent of frozen-thawed sperm and concentrations of DMSO and EG. FCM analysis suggested that the integrity of mitochondria and membranous structures of frozen-thawed sperm were similar to that of fresh sperm at 5%~20% DMSO or 5%~20% EG, but the proportion of frozen-thawed sperm maintained the integrity of mitochondria and membranous structures was significantly declined at 25% or 30% DMSO or EG. So we conclud that the weakening of sperm vitality and the enhanced damage of DNA, mitochondria and membranous structures in frozen-thawed sperm are mainly induced by the high concentration of cryoprotectant. 10% DMSO or 10% EG can be considered as the best cryoprotectant to ensure the quality of frozen sperm.

To investigate the transcriptional regulation of pig melanocortortin-4 receptor(MC4R) gene, 5' promoter fragment (spanned from －1 264 bp to －31 bp) was amplified with PCR method and the potential transcriptional start sites were predicted with bioinformatics method; Eleven promoter segments with different length were obtained by promoter deletion analysis and cloned into PGL3-Basic vector; then the promoter activities were determined in transiently transfected pig PK15 cell by the dual-luciferase assay system. The results indicated that there was a potential transcription start site in the region from intiation codon to －682 bp. Further analysis demonstrated that the region from －514 bp to －486 bp was important for the basic transcriptional activity and bioinformatics analysis showed that there was a cis-acting element possessing a functional binding site(HSF). HSF maybe play an important role in the regulation of MC4R gene expression.

PopW from Ralstonia solanacearum is a member of the harpin protein family, eliciting hypersensitive cell death in non-host plants, inducing disease resistance in plants and enhancing plant growth. To achieve secretory expression of PopW protein in Bacillus subtilis, we constructed recombinant expression vector pHTPOW, which carried the p43 promoter, a signal peptide element of nprB, and the full length PopW gene. The expression of PopW in the genetically modified strains was confirmed by SDS-PAGE and Western bloting analysis. In vitro test showed that on the 24 and 48 h after treated with the genetically modified strain BsW, the J2 mortality rates of Meloidogyne incognita were 53.4% and 79.6%, respectively. In the green house trials, the number of eggs and galls reduced significantly 42-days after tomato (Lycopersicon esculentum) treatment with the transgenic strain BsW and the biocontrol efficacy reached 60.0%. The genetically-modified bacteria strain which enhances plant growth and inhibits nematodes activity offers a new promising perspective on the reasearch and development of new microbial pesticides.

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB), a disease affecting watermelon, melon and other cucurbits. A transposon (Tn5) mutant library was constructed with strain xjL12. The mutant △xj48 was selected for its loss of pathogenicity on hami melon (Cucumis melo var. saccharinus) seedlings and its ability to elicit hypersensitive reaction (HR) on tobacco. The gene disrupted by Tn5 was identified as a homolog of the hpaP gene, a conserved gene of type Ⅲ secretion system (T3SS). Further characterization of the mutant using genetic recombination revealed that swimming motility, biofilm formation and the expression of the hrcV gene requires a functional hpaP gene. Pathogenicity was partially recovered in a hpaP complemented strain. The genetic evidence in this report demonstrates that the hpaP gene is critical for the pathogenicity of Acidovorax citrulli, and plays an important role in infection.

Starch, protein and oil are main nutritional contents in normal maize. In this study, the F2 population derived from the maize(Zea mays L.) cross Huang C × 178 was used to construct genetic linkage map by simple sequence repeat (SSR) markers. Based on the linkage map and nutritional quality detected in F2, F2:3 and F2:4, a total of 16 QTLs were identified using MapQTL5.0, of which, 6 QTLs were associated with oil content, explaining phenotypic variation for 6.2%~17.8%, 5 QTLs with protein content, explaining phenotypic variation for 6.3%~12.0% and 5 QTLs with starch content, explaining phenotypic variation for 6.3%~10.0%. About all of QTLs detected appeared to be of over dominant and partially dominant. We conclude that 16 QTLs for nutritional quality traits will provide useful information for molecular assistant selection(MAS) in maize genetic improvement.

Sucrose phosphate synthase (hereinafter as SPS) is the key enzyme that controls the sucrose biosynthesis in plants. By searching NCBI GenBank, 77 SPS genes identified to date from plants were obtained, in which 43 were in full length sequences. By phylogenetic analysis of the 43 full length sequences, the SPS in plants were classed into four families, named A, B, C and D family, respectively, and there were several SPS genes in the same plant belonging to different families. By alignment of the four genes, SofSPSII from sugarcane(Saccharum officinarum), OsSPS4 from rice(Oryza sativa), LpSPS from ryegrass (Lolium perenne) and BoSPS from bamboo(Bambusa oldhamii) had very close phylogenetic relation. A pair of primers were designed in conserved sequence region to amplify partial sequence of SofSPSA. With the primers, a 2 553 bp fragment was amplified and sequenced. According to this partial sequence of SofSPSA, a 3 906 bp full length cDNA sequence was obtained by 5'-RACE and 3'-RACE. The full length cDNA contained a 3 180 bp open reading frame (ORF) encoding a protein of 1 060 amino acids. The theoretical molecular weight and isoelectric point (PI) of the protein were 118.4 kD and 6.09, respectively. The start codon (ATG) lies 359 bp after the transcription start site, and a 364 bp non-coding sequence with a typical polyA tail lies after stop codon (TAA) on the full length cDNA sequence (GenBank accession No. HM854011). The amino acid sequense of SofSPSA showed 99.3%, 91.8%, 91.6% and 91.9% similarity, and 99.2%, 87.9%, 87.5% and 87.8% identify to that of SofSPSII, OsSPS4, LpSPS and BoSPS, respectively. Finally, sense and antisense SofSPSA gene ORFs were amplified and connected to pBI121 to construct the plant expression vectors, pBI-SofSPSA and pBI-anti-SofSPSA, respectively. This work will lay a foundation for further study on the biological function of SPS from sugarcane and variety impovement by transfer of sugarcane SPS gene.

In order to obtain the full-length sequence of polyphenol oxidase gene (PPO) and its expression in different tissues, it was cloned from lotus rhizome (Nelumbo nucifera Gaertn. ssp. nucifera) bud by using RACE technique. The full length cDNA of PPO was 2 074 bp with open reading frame 1 503 bp, encoding a protein of 501 amino acids, 5'UTRs of 160 bp and 3'UTRs of 411 bp (GenBank No. HQ380894). Bioinformatics analysis showed that the molecular weight of PPO gene was 56.8 kD with pI value 8.60, which had tryosinase family domain, hydrophilic characteristics and trans-membrane properties but no signal peptide. Both α-helix and β-turn were found spreading in the whole polypeptide chain. The expression of PPO gene in five lotus tissues (rhizome bud, young leaf, petal, fresh rhizome cut and stem) was also investigated and the variance analysis results showed that the relative expression level of PPO in rhizome bud was only slightly higher than that in young leaf, but significantly higher than those in the other three tissues. The expression level in young leaf was no difference with that in fresh rhizome cut, but significantly higher than both in petal and stem. The expression levels in petal and stem were lowest and no significance between them. These results indicate that PPO gene produced in rhizome bud (an important meristem) may have no activity or be unstable, which is distributed to different tissues after production accompanying with organ development and responsible for corresponding functions under specific spatial and temporal conditions.

Dehydrins is one of the responsive proteins under drought stress. The aim of this study was to explore distribution of dehydrins in cells and the relationship with degree of drought tolerance under water stress. Two different drought resistance winter wheat(Triticum aestivum) as experimental materials，with the colloidal gold immune electron microscopy technique and Western blot, ithe distribution of subcellular structures of dehydrated protein expression in wheat was nvestigated under water stress, and also analyzed its relationship with plant drought resistance was also analyzed. The results showed that: at the initial stage of the stress(4~8 h), the golden particles were mainly distributed in the cytoplasm, and also with a small amount in the nucleus. During the interim of the treatment(12~24 h), golden particles were mainly located in the cytoplasm and the nucleus, and also with a small amount in the organelle, at the end of the treatment(36~48 h), most golden particles were detected nearby the plasmalemma, the golden particles in the cytoplasm and the nucleus were relatively more after 24 h of the rehydration. Using the normal leaf tissue without any treatment and dehydrated protein antibody as contrast, however, none was detected. With the increase of stress time, the membrane permeability increased at beginning, the soluble protein content was also increased first; Western blotting analysis showed that ,there was a specific protein of 37 kD under water stress and its expression was positively correlated with wheat drought tolerance. After rewatering, relative permeability of cell membrane and soluble protein content were all decreased, and dehydrin could exist in plant for some time. In conclusion, these data provides a direct evidence for studying the expression and distribution of the dehydrated protein in wheat mesophyll cell.

To identify Insertion-deletion (Indel) markers tightly linked to the fruit bitterness (Bt) gene in cucumber (cucumis sativus L.), a F2 population including 184 individuals derived from two cucumber inbred lines 931(btbt) and 46GBt (BtBt) was used in this study. Results showed that the inheritance of fruit bitterness in cucumber was controlled by one dominant gene Bt. Based on primary mapping of Bt gene and re-sequence of two parental lines, Bioinformatics was employed to analyze the sequence difference between the two parental lines in the primary mapping region of Bt gene. Then seven Indel markers were developed. The InDel marker Bt-InDel-1 tightly linked to Bt gene with 0.8 cM was identified after analysis on F2 population with 184 individuals using the seven Indel markers. The veracity of Bt-InDel-1 was tested using another F2 population with 58 individuals, and the accuracy rate for this marker was 94.8%. The results indicated that the Bt-InDel-1 marker can be used in non-bitter MAS cucumber breeding and fine-mapping of Bt gene.

A full-length cDNA sequence of glycine-rich protein(GRP) gene, Sc-GRP, was obtained from sugarcane(Saccharum officinarum L.) leaf full-length cDNA library based on EST(expressed sequence tag) sequencing and bioinformatics analysis. Bioinformatics analysis revealed that this gene was 518 bp long including a 273 bp complete open reading frame(ORF), a 74 bp 5' untranslated region(UTR), and a 311 bp 3' UTR. Typical AATAA motif and poly(A) tail at the 3' UTR were also found. This gene corresponded to a 90 amino acid residues protein. The theoretical molecular mass and the theoretical isoelectric point of Sc-GRP was 10.12 kD and 5.86 respectively. Besides, glycine residue was found as the most abundant amino acids in Sc-GRP and reached 13%. Sc-GRP contained GGGX, GGX and GX motifs. No signal sequence or RNA binding region were found in Sc-GRP. Real-time PCR analysis showed that the expression of Sc-GRP in sugarcane mature leaves was much higher than that in young leaves, and increased with the mature degree of internodes in cane. However, Sc-GRP was hardly expressed in mature roots. Under the challenge of Al3+, the expression level of Sc-GRP gene went up firstly, and decreased subsequently in sugarcane primary roots. The result showed that the Sc-GRP gene is assumed to locate in cytoplasm and participate in the osmotic regulation of cell in sugarcane. This study aimed at understanding the functions of the Sc-GRP and providing basal information for its potential application in sugarcane molecular breeding.

The sexual polyploidization through 2n gametes, which could result in intergenomic recombination to produce new genetic variations, is paid more and more attention by plant breeders. In this study, the young flowers of colored Zantedeschia diploid cultivars were treated by the chemical method (colchicines) to induce 2n gamete, and the cytological characteristics of 2n male gamete (2n pollen) formation were observed. The result showed that 2n pollen of coloured Zantedeschia was induced successfully by colchicine and 2n pollen grain was larger than that of normal 1n pollen. The best concentration of colchicines was 0.15%. There are obvious differences on cytological matters during 2n pollen formation. The abnormal phenomena like parallel spindles and fused spindles at metaphase, dyads and triads at the tetrad stage, multinuclear cell with abnormal meiosis, nuclei fusion in pollen mother cell, etc. were observed. The seeds of the treated pollen crossing with normal 1n female gametes were harvested. The chromosome identification showed that the triploid (2n=3X=48) progeny was obtained successfully. The results implied that 2n pollen induced artificially can be a powerful method to create the sexual polyploids of colored Zantedeschia.

The tonoplast Na+/H+ antiporter gene (AtNHX1) of Arabidopsis thaliana playes important roles on saline tolerance of the plant. In order to identify of (AtNHX1) gene function and create new germplasm of tobacco, the AtNHX1 gene under the control of stress-induced rd29A promoter was transferred into tobacco (Nicotiana tabacum L.) tissues by Agrobacterium-mediated transformation. A total of 72 regenerated plants resisted to Kanamycin were obtained, of which 17 grew well in MS medium supplemented with 85.5 mmol/L NaCl. Subsequent PCR, Southern and Northern hybridizations validated the genomic insertion and normal expression of the AtNHX1 gene in the transgenic tobacco plants. The up-regulated expression of AtNHX1 gene was induced by salt challenge. The growth and root development of the transgenic plants were significantly better than that of the controls in the mediums supplemented with 171, 256 and 342 mmol/L of NaCl while the concentration of malonyldialdehyde in the leaves of transgenic plants was significantly lower than that in the controls. The results indicated that the insertion and expression of AtNHX1 gene controlled by rd29A promoter could significantly enhance the salt tolerance of transgenic tobacco plants. These results revealed that the AtNHX1 gene regulated by rd29A promoter could be normally expressed in heterologous plant and its expression could improve the saline tolerance of the receipt plant. Therefore, genetic transformation of the AtNHX1 gene can be one of gene-engineering strategies for breeding saline tolerant crop.

To probe the infrageneric phylogenetic relationship of Lilium, 19 sequence-related amplified polymorphism (SRAP) primer combinations were chosen to analyze genetic diversity of 23 wild Lilium species, and NTSYS-pc2.1 and POPGENE1.32 were used for data analysis. Result showed that a total of 236 clear DNA bands were amplified, 233 of which were polymorphic, the proportion was 98.72% with average 12.3 polymorphic loci per SRAP primer combination. The genetic similarities ranged from 0.3750 to 0.7679, and the genetic distance ranged from 0.2642 to 0.9808. Twenty three species were classified into four clustering groups by UPGMA. L. regale and L. leucanthum were the mostly closely related systems, whereas L. brownii and L. amabile were the mostly distantly related systems. Materials with similar morphology and from the same source mostly clustered together, and gene exchanges existed between Sect. Lophophorum and Sect. Sinomartagon. The clustering results were basically consistent with morphology classification. The results indicated that SRAP markers are suited to study genetic diversity and phylogenetic relationship of Lilium.

Natural resistance-associated macrophage protein 1(NRAMP1) can defense the invasion of the intracellular bacteria such as Mycobacterium and Brucella to improve the ability of resisting disease. In this study we cloned a NRAMP1 isoform from the spleen of Qinchuan Bos Taurus. Sequence analysis showed that NRAMP1 isoform had a third intron in comparison with NRAMP1, which resulted in the protein only containing 200 amino acids because the translation stopped in the third intron. In order to get further study on the expression of NRAMP1-ISO, we constructed the prokaryotic expression vector pET-41-NRAMP1-ISO and the eukaryotic expression vector pEGFP-NRAMP1-ISO. The prokaryotic expression vector pET-41-NRAMP1-ISO could efficiently express in the BL21 Escherichia coli under different concentrations of the IPTG. The eukaryotic expression vector pEGFP-NRAMP1-ISO could both express in the nucleus and the cytoplasm of the bovine fibroblast while NRAMP1 was normally expressed around the lysosome membrane in the cytoplasm. Semi-quantitative RT-PCR showed that NRAMP1-ISO gene was highly expressed in the heart, spleen and lung while lowly expressed in the brain, pancreas and the genital ridge. The results provided the useful imformation for further anzlyzing the function of NRAMP1-ISO gene.

The expression characterization of MC4R gene before and after overfeeding plays an important role in fat metabolism of Lades geese. SYBR GreenⅠReal-time quantitative PCR was developed to analyze the expression characterization of MC4R gene in 13 tissues. PCR results showed that both MC4R and β-actin were expressed in all 13 tissues before and after overfeeding of Landes Goose(Casuarius casuarius). SYBR GreenⅠReal-time quantitative PCR results showed that the relative expression of MC4R in kidney, intestine and heart after overfeeding was lower than those before overfeeding, their 2-ΔΔCt values were 0.41±1.80, 0.65±5.75 and 0.72±1.22, respectively. But the relative expressions of MC4R in other tissues were all higher after overfeeding, their 2-ΔΔCT values from high to low were 22.78±1.00 (brain), 9.08±2.80 (liver), 5.28±1.83 (lung), 3.78±3.01 (pancreas), 3.07±3.64(leg muscle), 2.90±0.97 (stomach), 2.34±1.66 (subcutaneous adipose), 2.18±3.01 (abdominal adipose), 2.07±0.37 (chest muscle) and 2.01±1.75 (pleen). We conclude that the results are consistented with the mechanism of MC4R and can provide indirect basis for researches on animal production and human obesity.

50 ICR(Institute of Cancer Researcch, USA) rats (Mus musculus) with 18~22 g body weight were randomly divided into 5 groups(control, compound Chinese herbal medicine, Pulsatilla, Phellodendron and Pulsatilla ∶ Phellodendron (1∶1) group) to investigate the effects of Chinese herb additives on diarrhea inhibition and intestinal bacteria. Rats were given extract by intragastric administration according to 4 g crude drug/kg body weight at the same time everyday, the controls were given distilled water with the same dose. Each rat was given Escherichia coli bacteria according to 0.02 mL/g with 108 cfu /mL by intraperitoneal injection at the 10th day to establish the model of diarrhea, and observe the effect of extract to diarrhea. The dejecta was collected at the 13th day for DGGE analysis. Results showed that, the changes in body weight of the controls were the most obvious at the 10th day, diarrhea was the most serious and significantly different with the other groups(P<0.05); The diarrhea frequency of four treatment groups were reduced 51%, 26%, 33% and 35%, respectively compared with the control group. The diarrhea inhibition of compound Chinese herbal medicine group was significantly(P<0.05) higher than that of other treatments; The faecal DGGE profiles showed that, for compound Chinese herbal medicine group, the number of DGGE bands and diversity index of compound group were more than that of the other treatment groups with significant difference(P<0.05). In conclusion, Chinese herb medicine feed additives can significantly reduce diarrhea frequency, optimize the structure of intestinal microbial community in mice, and the best effects were showed in group of compound Chinese herbal medicine.

The diacylglycerpl acyltransferase(DGAT) plays an important central role in the metabolism of cellular diacylglycerol. This study was conducted to investigate the DGAT genes (DGAT1 and DGAT2) acyl CoA: diacylglycerol acyltransferase(DGAT) gene expression characteristics during 60, 90 and 120 days in 3 different tissues, liver(L), subcutaneous fat(SF) and rib eye muscle(RE), by Real-time PCR. The association between tissue expression profile and backfat of shoulder, backfat of 6~7 ribs, backfat of the last rib and backfat of gluteus medius was analyzed. The results showed there were no any difference (P＞0.05) expression of DGAT1 in different days, whereas significant differences (P＜0.05) in DGAT2 mRNA expression between 60 and 90 days in the different tissues of the Large Whites. The analysis of DGAT1 and DGAT2 expression in different tissues indicated that the highest levels of DGAT1 was expressed in the L, followed in SF, the highest levels of DGAT2 was expressed in the SF, followed in L ,and the least in ovary of the RE. The result of association analysis showed there were the significant positive correlation between expression of DGAT1 and BF of 6~7 ribs(r = 0.71, P＜0.01) and backfat of the last rib (r = 0.62, P＜0.01) in the L, and the significant negative correlation(r = -0.46, P＜0.05) between expression of DGAT2 and BF of shoulder, and the significant positive correlation(r = 0.49, P＜0.05) between expression of DGAT2 and BF of 6~7 ribs in SF. The results suggested that the expression of DGAT2 gene was different levels in two days, and DGAT1 and DGAT2 were expressed in three tissues with different levels. The expression of DGAT1 and DGAT2 genes were significant correlation with part BF. DGAT1 and DGAT2 can be considered as candidate genes of BF in Large Whites' breeding.

Green fluorescent protein (GFP) is an ideal marker for cell sorting. Integrating green fluorescent Protein (GFP) into bovine Y chromosome is the key for sorting X and Y sperm by GFP. In this study, one loxp site and one Lox2272 site were inserted into the vector pEGPP-N3 through special designed primers amplified by high-fidelity enzyme and the vector pEGFP-loxp-Lox2272 was constructed. The bovine fetal fibroblasts were transfected with linearized pEGFP-loxp-Lox2272 by LipofectamineTM (Lip) 2000. The results revealed that the best transfected effect was mediaed by 3 μL Lip 2000 and the best concentration of G418 for screening transgenic cell clones was 600 μg/mL. This study provided a useful method for constructing vectors and important reference for screening bovine gene-targeted cell clones.

Transcription factor nanog and oct-4 are the fundamental features of pluripotent Stem Cell, which are the key factors to keep their pluripotency and self-renew ability. We transplanted 10 fibroblast cells(BEF422) into bovine oocytes at GⅤ and MⅡstages respectively, and cultured the oocytes for 24 h in vitro, than we used the RT-PCR and immunofluorescence technique to check the expression and location of nanog and oct-4 in the injected oocytes. The results showed that nanog and oct-4 were all expressed in the injected oocytes and located on transplanted cells nuclear. However, they weren't detected in fibroblast cells and oocytes control group; This result demonstrated that GⅤ and MⅡ stage oocytes both can activate the transcription factor nanog and oct-4 expression to reprogram the fibroblast cells. This study will lay a foundation for further verifying the reprogramme mechanism and related material on donor cells and oocytes.

In order to efficiently select some advanced fragrant sterile lines by means of molecular breeding technology, the functional molecular marker 1F/1R of fragrance(fgr) gene was used to detect the polymorphisms of 13 maintainers of indica rice. Yixiang B was identified to haveⅠ- type band (fgr/fgr) (strong fragrance) by PCR strategy, and others had Ⅱ- type bands (Fgr/Fgr). 1F/1R was used to screen offsprings derived from a cross Ⅱ-32B / Yixiang B. The result suggested that 1F/1R showed codominance and was controlled by a single gene. By marker assisted selection(MAS) in the F2 generation, the homozygote rate of the marker 1F/1R in selected plants in F3 reached as high as 96.0%. Selecting the plants with fgr gene, together with the selection of traits in field and laboratory, such as plant type, grain type, transparency, chalk degree, gelatinization temperature, amylase content and so on, we obtained some plants interested among them and pairing mated with Ⅱ-32A. Through selection and pairing mating for 5 generations, with each generation into scale of about 50~60 pairs, we obtained some advanced fragrant sterile lines with stable traits, named Zhenongxiang A(B). Four representative sterile lines were selected to evaluate their comprehensive characteristics. These lines hadⅠ- type band at 1F/1R locus and they were considered as ideal advanced fragrant sterile lines with obvious fragrance, good plant types, slender grain type, high degree oftransparency, low degree of chalkiness, medium gelatinization temperature and amylase content. In conclusion, it is more effective to select the sterile lines of aromatic rice by functional marker fgr-1F/1R.

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawning (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embyos (P＜0.05) compared with FD and CT group，while no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24 , 48 and 60 h), the results showed that the blastocyst rate in the 60 h group was the highest (36.11±1.78%) compared with the other groups (P＜0.05). While the reconstructed embryos derived from donor cells treated with TSA for 24h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39±1.78%) in the 60 h group was significantly higher than that of 24 h (25.48±1.34%) group (P＜0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural estrus recipients, no significant difference in terms of pregnancy rate among groups was found, however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can development to term.

Glucose-6-phosphatase(G6Pase, EC3.1.3.9) catalyzes the hydrolysis of glucose-6-phosphate into glucose, the final step in the gluconeogenic and glycogenolytic pathways. A full-length cDNA of glucose-6-phosphatase catalytic subunit(G6PC) from Lateolabrax japonicus was amplified by SMART RACE method. The cDNA was 1 651 bp in size, with a 75 bp 5'-UTR, 496 bp 3'-UTR and 1 080 bp ORF, encoding a protein of 359 amino acids with a molecular weight of 40.45 kD and pI 9.30(GenBank accession No. HQ317736.1). The deduced amino acid sequence of L. japonicus G6PC shared high identity with other eleven species, which was from 58% to 85%, and the highest was 85% with Osmerus mordax. L. japonicus G6PC contained three conserved domains. RT-PCR was used to test the expression profile of G6PC in ten tissues, including muscle, eye, spleen, kidney, liver, fat, heart, gill, intestine and brain. The results showed that G6PC was mainly expressed in liver, intestine and kidney, and weakly in brain and gill. A 1 243 bp 5'-flanking region sequence upstream of the translational start of G6PC gene was cloned using genomic walking technique. It contained highly conserved 2 TATA boxes, 2 IRS(insulin response sequence), C/EBPb(CCAAT-enhancer-binding protein b), CRE-BP(cAMP response element binding proteins) and HNF-3b(hepatocyte nuclear factor 3 beta, FoxA2) potential transcriptional factor binding sites. We conclude that L. japonicus G6PC are abundant in liver, intestine and kidney, and the 5'- flanking region of G6PC contains multiple binding sites for transcriptional factor. These results will be helpful foundation for understanding the regulation mechanism of G6PC.

Animal embryonic development is regulated by many genes. In order to explore the molecular mechanism of duck early embryonic development, we constructed the subtractive library of duck early embryonic development related genes to screen the related candidate genes of duck early embryonic development. Two different periods (6 and 20 h) duck (Anas platyrhynchos) embryo were used respectively as the driver and tester, and a designated duplex-specific nuclease(DSN) mediated subtractive hybridization method was employed to construct the subtractive library of duck early embryonic development related genes, and the genes were sequenced and analyzed by bioinformatics. Results showed that 54 genes were obtained by DNA sequencing from 216 positive clones picked randomly. Their average length was 1 084 bp. Nucleotide BLAST homological analysis showed that 44 genes had similarities to known genes. Gene ontology showed that these genes were related with a variety of biological pathways，such as metabolism, establishment of cellular localization, biological regulation，etc. And these genes played very important roles during early embryonic development of duck in many different aspects (such as nervous system development, immune regulation, transcriptional regulation, etc.). We conclude that isolation and identification of these differentially expressed genes will contribute to the future related research of poultry early embryonic development.

At present, the researches on crop genetic engineering are mainly for the sake of the improvement of plant resistance, while yield is a complex agronomic trait, controlled by various genes and environment. Photosynthesis, nitrogen assimilation, distribution of carbon source and plant architecture are widely considered as the base of yield formation. With the advance of Plant Genome Project, a lot of genes regulating the above physiological process have been identified and genetically modified, which resulted in the improvement of crop yield. Isolation of the genes as well as validation of gene function will provide the novel mind and a set of new methods for breeding high-yielding varieties, although the molecular mechanism that the genes regulate the traits is still unknown. With the implementation of the Important National Project of Transgenic New Variety Cultivation, identification of functional genes will be paid more and more attention. The researches on the important genes related to plant yield are summarized in the article on the basis of source, translocation, sink, plant architecture and nitrogen utilization contributing to yield formation in crops, which are expected to contribute to cultivating transgenic crops of high yield.

A bacterial blotch disease, caused by Pseudomonas syringae pv. averrhoi is one of the most damaging disease of carambola(Averrhoa carambola). A polymerase chain reaction(PCR) protocol was developed to specifically detect Ps. syringae pv. averrhoi based on internally transcribed spacer(ITS) region of 16S~23S ribosomal. PCR products from the 16S~23S rDNA of Ps. syringae pv. averrhoi were cloned and sequenced. Based on a multiple sequences alignment among these obtained sequences, a pair of specific PCR primers PsaveF(5'-CTTATCGACGACTCAGCTGCG-3')/PsaveR(5'-TCATGCGTTGATCGTCAGGATC-3') were designed. The primer pairs amplified a single 373 bp product from all isolates of Ps. syringae pv. averrhoi that was not amplified from any other isolates tested. The sensitivity of detection with primers PsaveF / PsaveR was 10 pg genomic DNA. It could amplified a specific single product from natural infected carambola that was not amplified from healthy tissue. The results showed that the PCR protocol provides a rapid, sensitive and reliable tool routine detection and identification of Ps. syringae pv. averrhoi. In addition, the classification implication of ITS sequence homology was found by comparing sequences from Ps. syringae pv. spp. This study is beneficial to control the bacterial blotch disease of carambolan.

The aim of this assay is to investigate on the TaqMan Real-time PCR method for phytoplasma general detection which has the superiorities in saving time and preventing contamination. One developed TaqMan Real-time PCR assay for general detection of phytoplasma was evaluated in comparison to conventional nested-PCR method with the aim to assess its potential for research and routine applications. In this study, specificity and sensitivity of the Real-time PCR was evaluated using 12 phytoplasma infected and 9 healthy plants DNA. All the phytoplasma got high fluorescence signals, healthy plant DNA and blank control got negative results. The TaqMan Real-time PCR procedure had high test specificity and higher sensitivity 10 times than that of the nested-PCR. The TaqMan Real-time PCR had the simplest and fastest testing process, especially decreased the risk of PCR contamination. Results inclicate that has a high potential for automation, and seems to represent the currently most suitable method for large-scale phytoplasma general detection.