In order to reduce the cost and increase the feasibility of real application in removal of heavy metals by bacteria, the discharged soybean wastewater was used as cheap culture for bacterial cultivation, and the cultured bacteria were applied to remove heavy metals from electroplating wastewater. It was found that Bacillus cereus and Ochrobactrum anthropi could be well grown in dilute soybean wastewater, means that the soybean wastewater could ensure the necessary nutrition for bacterial growth. In addition, soybean wastewater with adding sucrose residue leach solution (pH 7.5) was the optimum condition for bacterial growth. Further studies revealed that when the initial concentrations of chromium and nickel in electroplating wastewater were 214 mg/L and 367 mg/L respectively, the cultured bacteria in optimum medium had a strong capacity of Cr(Ⅵ) and Ni(Ⅱ) removal. Meanwhile, the biomass of B. cereus and O. anthropi could be greatly affected the removal efficiency, the more bacterial biomass, the better removal ability, and the removal efficiency could be higher than 80%. Moreover, microscopic investigation (such as atomic force microscopy, scanning electron microscopy and transmission electron microscopy) showed that the morphology of B. cereus was destroyed severely after reacted with chromium and nickel in electroplating wastewater, and the energy dispersive X-ray spectroscopy (EDS) analysis indicated both extracellular immobilization and intracellular accumulation involved in the bioremoval of Cr(Ⅵ) and Ni(Ⅱ). Additionally, Fourier transform infrared (FT-IR) analysis suggested that N-H and C=O functional groups from bacteria might play an important role in heavy metals immobilization process. This study provides a new idea for application of bacteria in remediation Cr(Ⅵ) and Ni(Ⅱ) containing wastewater by cheap cultivation with discharged soybean wastewater.

A fast and exact sex-specific molecular marker is important in the study of sex control and all-female breeding in half-smooth tongue sole(Cynoglossus semiliaevis). In the present study, a sex-specific co-dominace simple sequence repeat(SSR) marker which had 35 bp difference fragment between the isogeny area of Z and W chromosone was filtrated from 37 same type markers. The selected marker was cloned and sequenced, then the sequence characterized amplified region(SCAR) primer was designed to increase the proportion of the 35 bp defference fragment. Interrelated analyses concluded that 4% agarose gel, 150 V electrophoresis for 25 min were the optimum condition. The transformed SCAR marker could amplificate 134, 134 and 169, 169 bp fragment in super-female、female and male individual, respectively. The SCAR marker was applied in production practice, and 574 physiological male parent half-smooth tongue sole were tested and 100 neo-males were identified and got rid of in less harmful condition which was helpful to increase the female proportion of the offspring. The SCAR marker can be used in accurate test of female, male and super-female individual in laboratory and fast test of male parent half-smooth tongue sole to get rid of neo-male individual under simple and crude environment like fish farm.

To compare the efficiency of transposon-mediated gene trap among SB(Sleeping Beauty), PB(PiggyBac) and Tol2, a promoter trap vector based on those transposons named pT3-PST-Pro-Trap was constructed. Then the vector plasmid DNA and transposase mRNA of SB, PB and Tol2 according to 1.0∶1.5 ratio were mixed, respectively, and injected into zebrafish(Danio rerio) one-cell embryos. The gene trapping efficiency could be determined by the expression level of green fluorescent protein report gene (GFP). The results showed that promoter trap vector mediated by SB, PB, and Tol2 transposon could capture the endogenous gene promoter with high efficiency of 24.32%、30.7% and 18.87% 36 h after injected, respectively, and GFP expression were observed. PB-mediated gene trap efficiency was significantly higher than that of SB and Tol2 (P＜0.05). Three kinds of transposon capture efficiency in 60 h period were higher than that of 36 h period, SB and Tol2 group were significant differences (P＜0.05), while PB group was no significant difference (P＞0.05). The results showed that PB, SB and Tol2 transposon mediated gene capture technology can be used to vertebrate high-throughput screening functional genes, and provide effective new method for the research of functional genes.

Elongase of very long chain fatty acids 6 (ELOVL6) is the rate-limiting enzyme in the elongation cycle in mammals. In this study, ELOVL6 coding sequences (CDS) was cloned by RT-PCR, tissue expession was analyzed by Real-time quantitative PCR (qRT-PCR) and constructed a recombinant Adenovirus overexpression vector for ELOVL6 of dairy goat (Capra hircus) to investigate its effect of related genes on fatty acid metabolism after infecting recombinant Adenovirus in dairy goat mammary epithelial cells(GMEC). The results showed that the CDS of ELOVL6 gene was 795 bp (GenBank No. KF667508), encoding 264 amino acids. Compared with cattle(Bos taurus), rat(Rattus norvegicus) and human (Homo sapiens), the nucleic acid sequence homology of dairy goat ELOVL6 was 97%, 90% and 93%, and the amino acid sequence homology was 99%, 92% and 95%, respectively. The protein structure analysis using TMpred showed that 5 transmembrane helixes were speculated in ELOVL6 protein, and there was a conserved histidine box (HWYHH) between the second and the third transmembrane helix. The ELOVL6 protein had strong hydrophobic properties analysed by ProtScale. Tissue expression analysis showed that ELOVL6 gene was themost abundant in adipose tissue(P<0.01), followed by small intestine(P<0.01), and minimum in heart. The titer of recombinant Adenovirus containing ELOVL6 gene was 108 U/mL. Compared with Ad-GFP group, ELOVL6 mRNA level increased by about 200-fold in in GMEC after incubated with the virus for 48 h. Sterol regulatory element binding protein-1 (SREBP-1) was decreased significantly (P<0.05), and adipose differentiation related protein (ADRP) was increased significantly (P<0.05). There was no significant effect on the expression of peroxisome proliferator activated receptor γ (PPARγ) and fatty acid translocase (CD36). These results indicated that ELOVL6 can influence the expression of genes related to fatty acid metabolism, and may have certain function on fatty acid metabolism in dairy goat mammary epithelial cells. This study provides the theoretical foundation for the study of fatty acid metabolism in the process of lactation.

To research the quickly construction of universal plant expression vector with different objectives, one expression cassette was constructed with partial sequence of multiple cloning site cloned from pUC19, CaMV 35S promoter and NOS terminator cloned from plasmid pEGAD. The whole expression cassette was inserted into the multiple cloning site of vector pCAMBIA1300 to generate the universal plant expression vector pCamE (GenBank accession No. JX841315). Plasmid pCamE offerred 8 restriction endonuclease sites at the multiple cloning site, 4 upstream of the promoter and 3 downstream of the terminator. These restriction endonuclease sites were helpful to insert foreign gene into vector pCamE, and modify or replace promoter/terminator by any other interested elements. With the report gene of enhanced green fluorescent protein (GFP), 3 expression vectors, pCam::GFP, pCam::SalGFP and pCam::DAHPS-GFP, were constructed and transformed into onion (Allium cepa L.) epidermal cells and Arabidopsis thaliana, respectively. The proteins of eGFP and DAHPS-GFP were expressed normally in onion epidermal cells. In GFP transformed Arabidopsis, eGFP fluorescence was clearly observed in root, root hair, spire base, epidermal hair and anther. The results showed that this universal plant expression vector pCamE could integrate into the plant genome and normally express with different genes, the length of the inserted gene sequence and the site that target gene inserted into did not affect the transcription or expression of these target genes. All these demonstrated that vector pCamE was an economic and wide versatility vector, by which the target gene could be modified efficiently and inherit stablly in transgenic plant with different objectives. Thereby, pCamE provides a powerful tool for large-scale genetic transformation and analysis of gene function.

Aurora-B is a serine/threonine protein kinase which plays important regulatory role during mitotic cell cycle progression, but its expression, localization and functionality during mammalian oocyte meiosis are seldom reported, and no related reports in pigs (Sus scrofa). In the present experiment, Aurora-B expression, subcellular localization, and its possible function during porcine oocyte meiotic maturation were investigated via confocal microscopic analysis and highly selective Aurora-B inhibitor treatment. Confocal microscopic analysis revealed that Aurora-B distributed uniformly in the cytoplasm at germinal vesicle(GV) stage. After germinal vesicle breakdown, Aurora-B expression was mainly associated with chromosomal. During MⅠ period, with spindle assembly and sister chromatids were arranged in the equatorial plate, Aurora-B overlaped with chromatin. In AITI , chromosomes began to migrate to the poles under the spindle traction, then Aurora-B located in the centre of chromosomes, but not overlaped with sister chromatids. Oocytes discharged the first polar body(pbI) after 44 hours when Aurora-B enriched on the oocyte nucleus and pbI. After using inhibitors AZD-1152 treatment, the results showed that more and more oocytes arrested in GV stage with the increasing concentrations of AZD-1152, GVBD incidence suppressed; compared with control group, the oocytes arrested in Pro MⅠ-AITI were significantly increased (P<0.05), arrested in MⅡ were significantly reduced(P<0.05). At the same time, MⅡ oocyte chromosome misaligned, some failed to align; abnormal spindle morphology, even disappeared; actin filaments had a certain degree of fracture and dispersion. In addition, porcine parthenogenetic embryos development severely affected after suppressing the expression of Aurora-B by inhibitor on oocytes. Cleavage rate, blastocyst rate and the total number of blastocyst cells are significantly lower with the increasing concentrations of AZD-1152 in each treatment group, and no blastocyst when the AZD-1152 concentration reached 50 nmol/L. Our study showed that Aurora-B may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly, chromosome segregation during porcine oocyte meiotic maturation, and also plays a role in the subsequent development of oocytes.

Cysteine proteinases (CP), an essential component of proteolysis are widely implicated in physiological processes in plants, e.g. plant growth and development, biotic and abiotic stresses. The full-length cDNAs of 2 CPs were cloned from the latex of para rubber tree(Hevea brasiliensis), named HbCP2 (1 245 bp) (GenBank accession No. KF771829) and HbCP3 (1 268 bp) (GenBank accession No. KF771830), and predicted to encode proteins of 41 and 40 kD, respectively. The genomic DNA sequences of HbCP2 (1 919 bp) and HbCP3 (1 634 bp) shared the same genomic structure of 3 introns and 4 exons. The 2 Hevea CPs were classified into the papain C1 family based on the existence of a typical papain C1 domain and phylogeny analysis. Homology analysis showed that HbCP2 was highly homologous to a CP homolog of Ricinus communis (86.86%), whereas HbCP3 homologous to a CP homolog of Jatropha curcas (84.74%). Real-time PCR analysis revealed that HbCP2 and HbCP3 showed different tissue expression patterns as assayed among Hevea tissues (leaf, bud, female flower, male flower, seed, young bark, mature bark and latex). HbCP2 had the highest expression in male flowers, followed in mature and young barks, and the least in buds. In comparison, HbCP3 showed predominant expression in latex, followed in leaves, and much less expression in the 6 other tissues with the least in buds and mature barks. Expression of HbCP2 and HbCP3 in the latex were regulated to different levels by the treatments of tapping, wounding, ethrel, GA and JA, not affected by the treatments of 2, 4-D, ABA and SA. In virgin rubber trees, with the process of tapping cuts, the expressions of HbCP2 and HbCP3 showed a similar manner of first down-regulation and then gradual recovery. HbCP3 expression decreased with the time of wounding treatment, whereas HbCP2 expression showed a fluctuation with increased wounding treatment. Hormones treatment showed overall low regulation on HbCP2 and HbCP3 expressions, only GA treatment led to a more than 2-fold expression change in gene HbCP2 of the 6 hormones examined. In addition, the expression of HbCP3 in Hevea leaves was apparently regulated by Corynespora inocualtion. In comparison, HbCP2 expressed stably at 3 d after Corynespora infection, increased significantly at 5 d, which most possibly resulted from leaf senescence. These results suggested that HbCP2 might play a role in stress responses and tissue senescence regulation, whereas HbCP3 might be involved in the regulation of latex regeneration and disease response in Hevea tree. The study provides useful information for research on cysteine protease gene family which regulates latex regeneration and stress response of rubber tree.

Chalcone synthase (CHS) is a pivot for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. To clone CHS gene from Conyza blinii Lévl. and analyze its expression in different tissues during florescence, the full- length DNA cloned by RACE was 2 098 bp, including 2 introns (486 and 415 bp); the coding region of cDNA consisted of 1 692 nucleotides, and predicted to code a protein of 399 amino acids which included all the active sites of CHS, named CbCHS (GenBank accession No. KJ155749). Bioinformatic analysis result showed that CbCHS amino acid sequence had a high homology with other opened CHS genes in plants (the highest value of 95%). The RT-PCR result showed that CbCHS was highly expressed in stems (87.03%) and leaves (98.33%) , showed no significant difference between them (P＞0.05), while roots had the lowest expression (9.43%), and showed a significant difference with other tissues (P＜0.05). The anthocyanin content was the highest in stems and leaves, and the lowest in roots, which indicated that there was some relevance between the CbCHS expression and anthocyanins accumulation. The DNA and cDNA sequence of CbCHS gene were firstly obtained from Conyza blinii Lévl. with the research of relationship between the anthocyanin content and gene espression in different tissues of Conyza blinii Lévl.. The results of this study provide some technology basis for the key enzyme expression and regulation mechanism analysis in effective components biosynthesis pathway of Conyza blinii Lévl..

Programmed cell death (PCD) is essential for normal development and reproduction which is also induced by various biotic or abiotic stress. Aluminum (Al) stress is a major factor limiting plant growth and yield of crops in acid soils. One of Al toxicity on cells is induced PCD. Whether Al stress causes soybean cell apoptosis is still poorly understood. In this study, 4',6-diamidino-2-phenylindole (DAPI) staining and content assays of mitochondrial cytochrome c (Cyt c) were performed in Tamba black soybean (Glycine max) root tip cells under Al stresses. DAPI staining results showed that there was a nucleus aggregation in the cell edge, and nuclear chromatin condensation displayed a crescent-shaped distribution around the nucleus with dense stain. The content of mitochondrial malondialdehyde (MDA) of soybean root tip cells increased with the increased Al concentration and time of Al treatment, which suggested that the degree of mitochondrial membrane oxidation increased and membrane damage became more serious. Cyt c/a can reflect the content change of Cyt c within the inner mitochondrial membrane. Mitochondrial permeability transition pore (MPTP) of soybean root tip cells opened unceasingly, mitochondrial membrane potential (ΔΨm) decreased, mitochondrial membrane integrity was destroyed under Al stress promoting Cyt c off from the inner mitochondrial membrane. A reduced value of mitochondrial Cyt c/a and content of Cyt c under Al stress suggested that Cyt c might be released from mitochondria to the cytoplasm through the mitochondrial membrane. These findings, from cellular and physiological level, revealed the roles of mitochondria and Cyt c in Al-induced PCD of Tamba black soybean root tip cells, will furtherly know about PCD process induced by Al tress and toxic mechanisms of Al to plants.

To explore the biological functions of SPMI (sperm motility inhibitor) and Spermadhesins genes in swine(Sus scrofa) sperms, the high and low motility sperms were prepared by swim-up method. Sperm total RNA was isolated by Trizol method. Somatic cells marker genes were detected to determine the RNA purity. Then SPMI and Spermadhesins mRNA level in high motility sperms was compared with low motility sperms by semiquantitative RT-PCR. The results showed that AQN1, AQN3, AWN (Spermadhesins), PSPⅠ(porcine seminal protein Ⅰ), PSPⅡ (porcine seminal proteinⅡ) and SPMI mRNA level in high motility sperms were significantly lower than that in low motility sperms. The result of one-way ANOVA showed that the ralative expression level of AQN1, AQN3, AWN, PSPⅠ, PSPⅡ and SPMI mRNA was significantly higher in low motility sperm than in high motility sperm. This result suggested that SPMI and Spermadhesins mRNA level in sperms could be as a molecular marker for sperm motility, and provide a reference for molecular breeding pig.

Keratin relates to the output of cashmere wool and the quality of fluff fiber. The purpose of this study is to research the change rule of differentially expressed protein of hair follicle in Inner Mongolia cashmere goat(Capra cashmere). Two- dimensional gel electrophoresis(2-DE) was used to separate total protein of hair follicle in 12 months and protein map for hair follicle cycle was established. Differentially expressed protein spots were identified and the expression trends of partial protein spots were analyzed by mass spectrometry. The results indicated that 255 protein spots were distributed at pI 4~7 and their molecular weight were 30~85 kD in each 2-DE maps of 12 months. 20 differentially expressed protein spots were found with Image Master 2-D platinum software. Finally, 12 protein spots were successfully identified by mass spectrometry, and they were mainly keratins (K) which participated in cell biological process and played important biological function. The 2-DE maps of 12 months were divided as follows: Four months of November, December, January and February were classified as hair follicle catagen; March and April were classified as hair follicle telogen, when proteins expression significantlly reduced; six months of May, June, July, August, September and October were classified as flourishing period with the highest expression. Preliminary analysis results showed that the expression level of K1, K5, K71 and K25 were the highest in flourishing period, which might promote the growth of hair follicle. At the same time, the expression level of K83 and K10 were the highest in the period of telogen, which would have the relationship with hair follicle telogen. In conclusion, excellent protein expression profiles for hair follicle were established and differentially expressed protein spots were successfully analyzed in this study, which will provide basic data for research on hair follicle cycle based on proteomics for Inner Mongolia Cashmere goat and searching marker protein.

This study aimed to compare the in vitro effects of laminin, fibronectin, collagen, and coculture with sertoli cells on the efficiency of chicken (Gallus gallus) spermatogonial stem cells (SSCs) proliferation. Chicken SSCs were isolated by two-step enzyme digestion and purified by gelatin differential adherent. Three passaged SSCs were inoculated on laminin, fibronectin and collagen coated culture dishes and co-cultured with sertoli cells. The efficiency of cell proliferation under each culture condition was detected by alkaline phosphatase activity(AKP), EDU(5-ethynyl-2'-deoxyuridine) staining and quantitative polymerase chain reaction (qPCR). Results showed that the number of SSCs AKP positive clones was highest in sertoli cells group with (32±3) clones each vision, while that of laminin group, fibronectin group and collagen group were (26±3), (24±2) and (23±2), respectively, and the later three groups showed no significant difference (P>0.05); EDU staining showed cell proliferation rate in sertoli cell group reached (92.82±2.15)%, which was significantly higher than that of other groups (P<0.01); qRT-PCR results showed that the expression of proliferation marker genes such as c-myc(myconcogene), Klf4(kruppel-like factor 4) and PCNA(proliferating cell nuclear antigen) was highest in sertoli cells, followed by laminin group, the expression of c-myc and Klf4 in fibronectin group were higher than that of collagen group, while PCNA was on the contrary. The results indicated that both matrix proteins and sertoli cells could promote the proliferation of chicken SSCs in vitro, and sertoli cells was identified as the most appropriate factor for in vitro cell proliferation, followed by laminin, fibronectin and collagen showed no significant difference (P>0.05). The results provide theoretical and experimental references for the further optimization of chicken SSCs culture system and understanding of SSCs proliferation mechanism in vitro.

Phytophthora cactorum-fragaria (PcF)proteins, distributing in Phytophthora only, are considered to be important virulence factors. There are 20 PcF/SCR(small cysteine-rich)-like genes which have been found by genomics research in Phytophthora infestans, and no necrosis-inducing function confirmation for plants yet. In this study, one of them was selected to testify its necrosis-inducing function, which was predicted to have 4 disulfide bonds in the mature protein, hereby named PcF/SCR.1 (GenBank: XM_002902885). The full-length cDNA of PcF/SCR.1 was obtained by RT-PCR with the length of 336 nt and then inserted into binary expression vector pBI121 to produce recombinant plasmid pBI121-PcF/SCR.1. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 and analyzed necrosis-inducing function by heterologous expression in Nicotiana benthamiana. The results showed that PcF/SCR.1 caused leaf yellow at 5 days post induction (dpi) and shrinkage and necrotic symptoms at 7 dpi, indicating that the gene was able to induce plant necrosis. Furthermore, sequence alignments among PcF/SCR.1 and PcF protein showed that there were 3 disulfide bonds (between C89 and C99, C68 and C100, C73 and C104), similar to that of PcF proteins, and 1 additional potential disulfide bonds (between C88 and C110) existed only in PcF/SCR.1. All those disulfide bonds were predicted to important for maintaining protein structures and function. In order to furtherly understand the necrosis-inducing function of PcF/SCR.1, site directed mutagenesis that replaced 4 cysteines with alanine at amino acid sites at 99, 100, 104 and 110 of PcF/SCR.1 was used to destroy each disulfide bridge. The resulted plasmids then heterologously expressed in N. benthamiana. The agro-infiltration assay results showed that a cysteine point mutation at aa 110, or double mutations at aa 99 and aa 110 had no effects on the necrosis-inducing function of PcF/SCR.1. However, 3 cysteine mutations (C99/100/110) or 4 (C99/100/104/110) of PcF/SCR.1 only caused slightly yellow symptoms in leaves in contrast to that of PcF/SCR.1, suggesting that the necrosis-inducing function was compromised. For better understanding of the gene expression patterns during P. infestans infection potato(Solanum tuberosum), Real-time RT-PCR was applied to analyze gene expression levels at different infection stages. The results showed that gene PcF/SCR.1 was up-regulated after P. infestans inoculation in potato leaves and at 48 h highly expressed about 80 times compared with control. The expression pattern indicated that gene PcF/SCR.1 played a role in an infection process, probably through suppressing host defenses and facilitating colonization of the pathogen. The study provides useful data to understand the role of the gene during P. infestans pathogenesis and valuable references for researching gene functions of other members in PcF/SCR-like gene family.

Bacteria's twin-arginine translocation system(Tat) can affect its pathogenicity seriously. In order to explore the relationship between Tat and pathogenicity of Acidovorax citrulli, a tatB disruption mutantstrain ΔtatB and its complementary strain CtatB were constructed by homologous recombination, and 6 factors (virulence, motility, growth ability, extracellular polysaccharide and cellulase producing ability and biofilm formation) were tested to illustrate Tat system's influence on Acidovorax citrulli. Comparing the expression of TrbC, hrcN, Aave_1810, Aave_0034 and hrpE genes by Real-time fluorescent quantitative PCR, to explore the relationship between tatB gene and type Ⅲ secretion system, Tat system related genes and cell toxicity related genes. ΔtatB could trigger the hypersensitive response in non-host plant tobacco(Nicotiana tabacum). The deficiency of tatB weakened Acidovorax citrulli's virulence, motility and growth ability, while had no effect on the production of extracellular polysaccharide (EPS)，extracellular cellulase and biofilm formation. Real-time fluorescent quantitative PCR analysis showed that the deficiency of tatB made the expressions of TrbC and hrcN genes in mutant strain significantly increased, while the expressions of other 3 genes notably decreased. Consequently, the deficiency of functional gene tatB in Tat system affected some biological characteristics of Acidovorax citrulli, and resulted in the decrease of pathogenic ability. These results supply basic information for pathogenicity and protein secretion research apart from secretion systemⅢ.

Italian ryegrass (Lolium multiflorum) is widely grown in southern China from December to May of the next year. It is also one type of palatable digestible and leaf annual forage as hay and silage during the entire season. However, due to the high annual precipitation in this area and the humid climate, Italian ryegrass supplied unbalancedly as hay. Silage is found to be an ideal way of producing forage in southern China. It is very positive to explore Italian ryegrass silage for developing grassland animal husbandry in this area. Therefore, this was a review about silage techniques development of Italian ryegrass, the usages and effects of silage additives were also mainly discussed in southern China. By summarizing, wilting silage technique is one important silage technique for Italian ryegrass, but it is not suitable in high humid season. Besides, Italian ryegrass silage with additives will become the major way in future. The silage additives include acid, lactic acid bacteria, cellulase, water soluble carbohydrate and mixing additives. Formic acid, propionic acid and sorbic acid are frequently used as acid additive which is the fermentation inhibitor. Lactic acid bacteria, cellulase and water soluble carbohydrate are used as the fermentation accelerant. Mixing additives are referred to mixing lactic acid bacteria, enzyme, sugar, and other minerals to be one additive, but its effect on forage silage was different based on different researches. In addition, the mixing silage could solve the problem of high moisture content in Italian ryegrass, and it would be a properly extension and application perspective in the productive practices.