Dibutyl phthalate(DBP) is an important plasticizer,and widely used as additives in the manufacturing of plastic, rubber and paint. But DBP is not connected with polymeric matrix by covalent bonds. Thus, DBP is very easy to diffuse into environment and pollutes environment. In order to isolate an DBP-degrading bacterium, strain S-3 was isolated from garbage-contaminated soil using selective culture medium with DBP as the sole carbon source. The strain was identified based on its morphological, physiological and biochemical tests, with reference to 16S rRNA gene sequence analysis (GenBank accession no. KF377815). Strain S-3 was finally identified as Paenibacillus sp.. With HPLC and HPLC-MS method, degrading characteristics of strain S-3 was studied under laboratory conditions and the degrading pathway of DBP was researched preliminarily. The results showed that strain S-3 could degrade more than 82.7% of 100 mg/L DBP within 5 d. The best temperature and pH for the DBP degradation by strain S-3 was 30 ℃ and 7.0, respectively.Bsed on the identified products, the degrading pathway of DBP was speculated. The product was identified as o-phthalic acid. Our research provide theoretical basis to bioremediation technology of DBP pollution.

Phenylalanine ammonia-lyase(PAL) is widespread in plant as the first key enzyme of phenylpropanoid pathway, it can be used to produce L-phenylalanine (L-Phe) through its reverse catalytic activity in biochemical industry. Nowadays, it is an important way to screen the PAL sources with high activity and stability for increasing the yield of L-Phe. In this study, a phenylalanine ammonia-lyase gene from Fagopyrum tatarcium(FtPAL) was constructed to pET-30b(+) and expressed in E.coli BL21(DE3), target protein FtPAL's reverse catalyze properties was characterized after purification. The results showed that FtPAL arrived at the highest forward catalytic activity 24.17 U/mg after induction of 5 h. Furthermore, the forward and reverse catalytic activities of purified FtPAL were 158.74 U/mg and 194.11 U/mg, respectively. Meanwhile, thin-layer chromatography(TLC) analysis also indicated FtPAL has the ability to catalyze trans-cinnamic acid into L-Phe. FtPAL was most active at 30 ℃ and pH 10, and showed the low thermal stability and moderate pH stability. FtPAL activity was activated by Mg2+ ion (P＜0.01), but was inhibited by other metal ions (Na+、Mn2+、Fe2+、Fe3+、Cu2+ and Ca2+) (P＜0.05 or P＜0.01). Particularly, Hg2+ could make FtPAL lose activity completely. Our study not only proves FtPAL has the activity to synthesize L-Phe, but also lays the foundation for improving its catalytic efficiency and stability through the genetic engineering.

As a key enzyme of the plant phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), which plays an important role in the life processes such as growth and development, and disease resistance, is the first enzyme in the anthocyanin synthesis pathway. In order to study the biological function for PAL in onion(Allium cepa L.) and its relationship with anthocyanin synthesis, the full length cDNA of PAL gene(GenBank accession no. KF421110) in onion bulb was cloned by RT-PCR and RACE technology using degenerate primers designed according to PAL genes in different plants. The sequence analysis and expression analysis using Real-time PCR were performed either. The results showed that the cloned sequence designated AcPAL1 had full length of 2 363 bp, which encoded a protein polypeptide of 708 amino acids. Blast and phylogenetic analysis indicated that the polypeptide shared a very high similarity with Allium sativum and Lycoris radiate. Expression analysis using Real-time PCR demonstrated that the transcription of AcPAL1 increase by degrees in white, yellow and red onions, and the expression was down-regulated by the continuous expansion of the bulb. In this study it is demonstrated that the expression of AcPAL1 is somewhat related to the onion anthocyanin biosynthesis and provided the basis for their relationship study between the onion PAL gene and accumulation of anthocyanin synthesis.

The selection of a suitable reference gene is a critical condition for improving the precision of gene expression analyzing by quantitative Real-time PCR (qRT-PCR). In this study, switchgrass(Panicum virgatum L.) root tissues were used as materials. Under different abiotic treatment conditions, mRNA expression stability of ten candidate reference genes, including UBQ1(ubiquitin conjugating enzyme1), ACT2(actin), UBQ2 (ubiquitin conjugating enzyme2), TUB(tubulin), GAPDH(glyceraldehyde-3-phosphate dehydrogenase), CBP20(cap binding protein 20), UCE2(E2 ubiquitin-conjugating enzyme), 18S rRNA1(18S ribsomal RNA protein), NAC(NAC domain protein) and CYP2(cyclophilin) was detected by qRT-PCR technology. Delta-Ct method, Genorm(ver. 3.5), Bestkeeper(ver. 1.0) and NormFinder (ver. 0.953) were used to analyze the all data. The results showed that the most candidate reference genes of switchgrass had some differences in the various abiotic stresses. In the switchgrass root tissues the ranking order of ten reference genes from better to average was as follows: ACT2, TUB, UCE2, CBP20, CYP2, UBQ2, 18S rRNA1, NAC, UBQ1 and GAPDH. Among them, ACT2 gene had the highest expression stability under the drought stress in the root tissue. The 18S rRNA1 gene was the best gene under salt and heat stresses. Under cold and waterlogging stresses, ACT2 was the most comparatively suitable genes. The results of software comprehensive analysis indicated that ACT2, TUB and UCE2 genes were the most stable reference genes which were suitable for switchgrass abiotic stress studies. UBQ1 and GAPDH were not suitable reference genes. The research results can be used for further gene expression study under various abiotic stresses in switchgrass.

This experiment was designed to study the effects of polymorphism of growth differentiation factor 9 gene (GDF9) on egg production traits in female line of Shaobo chicken(Gallus domesticus), and 216 female line of Shaobo chicken breeding in Poultry Institute, Chinese Academy of Agricultural Sciences were used in the present study. The genetic polymorphism of GDF9 was investigated by PCR-RELP, and analyze the association of polymorphisms sites with egg production traits in female line of Shaobo chicken, which included age at first egg (AFE), body weight at first egg, weight at first egg (WFE), average egg weight at 300 days (AWEN) and the total number of eggs at the age of 300 days (EN). The results indicated that three SNPs site were detected in exon2 of GDF9 gene. Three genotypes(AA, AG and GG) were found at G2051A mutation locus，two genotypes(CT, TT) were found at T2273C mutation locus, three genotype(CC, CT, TT) were found at C2336T mutation locus. The fitness test showed that all Shaobo chicken at three mutation locus were in Hardy-Weinberg equilibrium. The relationship between GDF9 gene and reproduction traits in female line of Shaobo chicken was analyzed by least squares linear model. The individuals with GG genotype had significant lighter weight at first egg (P<0.05), GG genotype birds had also significant more the total number of eggs at the age of 300 d (P<0.05). Least squares means of reproduction traits for TT was not significantly lower than that for CT (P>0.05) at T2273C mutation locus. CT genotype birds had significant younger age at first egg (P<0.05), the individuals with CT genotype had also significant more the total number of eggs at the age of 300 d (P>0.05). The combine genotypes also had significant genetic effects on age at first egg (AFE), weight at first egg (WFE), body weight at first egg (BWFE) and the total number of eggs with 300 days (EN). However, it seemed that the effect of GDF9 gene with combine genotype on egg production traits was more significant. The results indicate that polymorphisms of GDF9 gene are significantly correlated with egg production traits in female line of Shaobo chicken, and individuals with GGCTCT combine genotype can be used as the molecular genetic marker to select female line of Shaobo chicken for reproduction traits.

This experiment is conducted to study the 3-hydroxy-3-methylglutaryl coenzyme A synthase gene (HMGCS1), to obtain the cDNA sequence， and to quest the correlation between HMGCS1 gene expression and cholesterol biosynthesis in the overfeeding goose liver. We selected landes goose(Anser anser) as experiment animal, which were needed for overfeeding 28 d. The experiments were designed to clone the whole cDNA of HMGCS1 gene by RT-PCR and RACE techniques and the expression of HMGCS1 in different tissues were analyzed by fluorescence quantitative RT-PCR. We choosed a cholesterol kit to detect the cholesterol level in goose liver. Finally we got nucleotide sequence of a full-length 2 947 bp cDNA for the HMG-CoA synthase, encoding a protein of 522 amino acids(GenBank accession No.KF425515). The results of RT-PCR showed that the gene expression in liver was extremely higher than that of other tissues(P＜0.05). After overfeeding for 28 d, the cholesterol in the liver decreased in the experiment group, but the decrease was not significant(P＞0.05). Nevertheless, there was a significant decrease in the level of HMGCS1 mRNA expression in experiment group(P＜0.05). The results of amino acid sequence analysis indicated that the gene maintained a high conservatism in evolution with ducks(Anas platyrhynchos), chickens(Gallus gallus) and pigeon(Columba livia). The gene was mainly expressed in the liver. There was a positive correlation between the cholesterol levels and the activity of the HMGCS1 in the cholesterol biosynthetic pathway. These results may provide the basic data for further research on the structure and functions of HMGCS1 gene.

Sugarcane (Saccharum spp.) is a commercial important sugar crop in South China. In order to achieve the breeding aim of increasing sugar yield, it is essential to focus on the principal rate limiting steps in sugarcane sucrose accumulation processes. Sucrose phosphate synthase (SPS) is considered a key rate-limiting enzyme in the pathway of sucrose synthetization and carbon partitioning processes. In this study, two different genomic DNA framents(GenBank accession No. EU278617 and EU278618) which encoding Sugarcane SPSⅢ protein were cloned by long?distance?PCR(LD-PCR). DNA Exon-intron?structure analysis suggested that both sequences contained twelve introns, thirteen exons, and the open reading frame encoded a protein of 964 amino acids. Subsequently, the 5'-flanking sequence (GenBank accession No. KC422670) of SPSⅢ was aslo isolated from sugarcane genome DNA using Adaptor-ligation PCR method. This novel sequence contained the important DNA putative cis-acting elements as well as TATA box. To identify promoter activtity of SPSⅢ, we generated five different vectors which contained different length of the 5'-flanking fragment fused to the the β-glucuronidase (GUS) reporter gene . Transient expression experimen was carried out by the method of particle bombardment. Histochemical activity of b-glucuronidase was observed in callus. The gene expression results confirmed its promoter activtity. This stuty will provide theory basis for further study on the gene structure and biological function of SPSⅢ via gene clone and promoter analysis.

For starry flounder (Platichthys stellatus), the male is less than the female and the volume of sperm is few, which limits the development for artificial reproduction and crossbreeding. In present study, the series of selecting experiments for sperm diluents, the osmotic pressure of diluents, the suitable temperature of seawater and the suitable dilution degree of frozen sperm were selected using the mature starry flounder. In order to check the application effect of frozen sperm, the experiments of fertilization and hatchability with starry flounder eggs were made. and made comparison with control group (fresh sperm). The results were analyzed by One-way ANOVA and Student-newman-keuls from SPSS 11.5. The results indicated that the frozen sperm motility was the highest in Ts-2 (37.65 g/L sucrose+10.01 g/L KHCO3+1.21 g/L Tris alkali) and SFs-4 (60 g/L glucose+10.01 g/L KHCO3+1.21 g/L Tris alkali) with (56.67±5.78) % and (66.67±5.77) %, respectively. It was also used computer aided sperm analyse(CASA) to analyze and test for motion parameters, (curvilinear velocity(VCL), straight line velocity(VSL), beat cross frequency(BCF), linearity(LIN), straightness(STR)) from frozen sperm sample in Ts-2 and SFs-4. The results indicated the motion parameters of frozen sperm with SFs-4 significantly higher than the ones in Ts-2. Using sucrose, KHCO3, and Tris alkali to prepare different osmotic pressure of sperm diluents, the frozen sperm motility was the highest (61.67±5.00)% in 319.94 Pa and it was no significant difference compared with fresh sperm (P＞0.05). In the experiment of selecting of the suitable temperature of seawater, the frozen sperm were activated with 3~18 ℃ seawater, theit showed the sperm motility was the highest at 12 ℃ and there was no significant difference compared with fresh sperm (P＞0.05), but there was significant difference between others and fresh sperm (P＜0.05). For the dilution degree of the post-thaw sperm was diluted for 10~40 time, and fertilized with eggs from starry flounder, it indicated that the frozen sperm rate of fertilization was the highest in the 20 times diluted, then it was stated the different dilution degree of frozen sperm had certain effect on fertilization ratio. In present study, the sperm from starry flounder could be effective cryopreserved with Ts-2 +20%DMSO and SFs-4+20% DMSO. This research result firstly provide important the theory and technology basis.

Myogenin (MyoG) plays a central control role in the process of muscle differentiation. The aim of this study is to research the activity efficiency and tissue specificity of bovine(Bos) myogenin (MyoG) gene promoter. In order to construct a eukaryotic expression vector pDsRed-BosMyoG, promoter fragment of bovine MyoG was obtained by PCR, and pDsRed2 was used as an exogenous vector in which red fluorescence was as a target gene. Then pDsRed-BosMyoG was transfected into a sheep(Ovis aries) skeletal muscle satellite cells and fibroblasts using lipofectamine. Red fluorescence was detected by RT-PCR and Western blot to study activity efficiency and tissue specificity of bovine MyoG-promoter. The results showed that red fluorescence could be observed in transgenic skeletal muscle satellite cells under microscope but not in transgenic fibroblasts. In mRNA level, red fluorescence mRNA expression value in transgenic muscle cells was significant higher than that in the transgenic fibroblasts(P＜0.01). Similarly, Western blot result showed that red fluorescence protein of transgenic skeletal muscle satellite cells was high, but there was not red fluorescence protein of transgenic fibroblasts. The conclusion was that bovine MyoG-promoter can specifically promote the expression of exogenous genes in skeletal muscle cells, and was an effective muscle cells-specific promoter. Cloning bovine MyoG-promoter and exploring the activity of promoter region, was good to reveal a key expression sites of MyoG-promoter and the underlying regulation mechanism of muscle development, and to provide experimental evidences of improvement of meat quality in livestock.

The mutations of β3-adrenergic receptor gene(ADRB3) have effect on function of heat production in animal. To study the ADRB3 polymorphisms were associated with sheep(Ovis aries) production in Gansu Alpine Merino. The genetic polymorphisms of part of intron of ADRB3 gene in Gansu Alpine Merino were detected by PCR-SSCP. Meanwhile, effects of different PCR-SSCP genotypes and alleles of ADRB3 gene with related growth traits were analyzed. The results showed that 6 SNPs(T/C, C/T, A/G/C, T/C, A/C and T/C) corresponding to 5 alleles(A,B,C,D and E) and performing 7 kinds of genotypes(AA, AD, BD, BE, CC, CD and DD) were detected in the intron of ADRB3 gene in Gansu Alpine Merino. The allele D with the frequency of 0.4159 and genotype CD with the frequency of 0.2540 were the highest. Through the analysis of polymorphism with growth rate, the results indecated that the different genotypes had different effect on growth rate, which the difference of genotypes had significant differences with weight of 2 months(P<0.05) and had no significant differences with other stages(P>0.05). The samples with the allele A and D or the genotype AD and BD had better weight than samples with other alleles and genotypes, and the growth rate of the sample with genotype BE was the lowest. The results of this study showed that the intron of ADRB3 gene have abundant polymorphism in Gansu Alpine Merino, and the allele A and D can be used as an effective genetic markers to improve the group production performance of Gansu Alpine Merino. And the genotype AD an BD can be used as the effective markers to improve the meat production performance in hybridization and guide the improvement of meat performance in sheep.

α-amylase is not only involved in plant sugar metabolism and biological energy transfer, but also associates with the stress resistance of plants. The aim of this study was to clone the Bn-α-amylase gene, analyze the gene sequence and the gene expression pattern. The full-length coding sequence of Bn-α-amylase gene was cloned by RT-PCR methods, based on the sequence of Unigene4746 in the transcriptome library of ramie(Boehmeria nivea). Then the sequence was analyzed using bioinformatics methods. And the expression patterns of Bn-α-amylase in various ramie tissues or under different stress conditions were analyzed by Real-time PCR. The full-length coding sequence of Bn-α-amylase was 2 295 bp(GenBank accession no.KF860891). The encoded protein contained 765 amino acids, whose predicted pI was 5.685 and molecular weight was 86.11 kD. Bioinformatics analysis demonstrated that two conserved domain located in the carboxyl terminal, Subcellular localization prediction showed that the protein was located in the cytoplasm, there was no signal peptide and Transmembrane domain, the protein sequence was highly identical to α-amylase of other species. Bioinformatics analysis demonstrated that the sequence was highly identical to α-amylase of other species. It shared a 80% nucleotide identity to Malus × domestic, 76% to Arabidopsis thaliana and to Actinidia chinensis, 73% to Ricinus communis, and 67% to Glycine max. And it also shared the high protein similarity to these species with the valure of 68%, 65%, 65%, 69% and 51%, respectively. Phylogenetic tree analysis showed that the α-amylase of Glycine max, Vitis vinifera, Actinidia chinensis and Ricinus communis was closer to Bn-α-amylase than the α-amylase of Arabidopsis thaliana, Oryza sativa and Sorghum vulgare, then the α-amylase of Selaginella tamariscina was less closer to to Bn-α-amylase than the α-amylase of Arabidopsis thaliana, Oryza sativa, Sorghum vulgare. The results of Real-time PCR analysis suggested that Bn-α-amylase expressed in root, bark, stem xylem, stem apex and leaves, with a highest level in leaves, while a lowest level in root. Interestingly, the expression of Bn-α-amylase could be strongly induced by high salt or drought stress, while down regulated by ABA treatment. These results indicate that the Bn-α-amylase may play a role in ramie stress torlerance.

In current, cucumber fruit color is one of the most important commodity characters. When breeding towarded diversity and specialty, it has attracted more and more attention in the process. To study the inheritance of tender cucumber fruit peel color traits, this research used three inbred lines of cucumber(Cucumis sativus L.) which were distinct different in fruit color as the parents material, maked two hybrid combinations, respectively which were Q16(dark green of fruit color)×Q8(yellowish white of fruit color)and Q16×Q24(yellow white of fruit color)and maked six genetic populations (P1, P2, F1, B1, B2 and F2), respectively. And this research adopted the combination of visual and chromatic meter to observe and class for the fruit peel color traits of tender cucumber on each individual plant in six genetics populations. The data of fruit colors were analyzed by joint analysis of multiple generations of the major gene plus poly-genes model of plant quantitative traits. The results showed the generation of segregation population of fruit color traits of cucumber presented unimodal or bimodal skewness distribution in these two hybridized combinations Q16×Q8 and Q16×Q24. This phenomenon indicated that this traits was quantitative traits and part of this traits was controlled by major genes. By the genetic analysis, the results showed that the inheritance of fruit color in these two hybridized combinations (Q16×Q8 and Q16×Q24) were appropriate for the model of two major genes with additive-dominance-epitasis (model B-1). This indicated that fruit color traits of cucumber was controlled by two major genes and the heritability of major genes in two hybrid combinations was highest in F2 population which reached 87.4% and 93.1%, respectively. In addition, the additive effect of two major genes in two hybrid combinations approximately equal which were 1.368, -0.132 and 1.451, -0.049, respectively. And the dominance effect of two major genes respectively were 0.625, 1.625 and 0.529, 1.530 in two hybrid combinations. It could thus be seen that in these two pair major genes, the additive effect of the first pair gene was obvious, and dominant effect of the second for the major gene was notable. In these two crosses, the heritability of major genes was high, this showed that fruit color of cucumber was suited to breeding in earlier generation. This result lay a foundation for the gene location in fruit color traits of cucumber and creating new germplasm of cucumber.

Glyphosate is the dominant herbicide worldwide. Herbicide tolerance is the most important agronomic traits of biotech crops. This article briefly stated the mechanism of action of glyphosate and its tolerant gene, and introduced the current status of excavation the glyphosate-tolerant gene and the relative patents in China. Until the end of 2012, there are 15 patent applications of domestic units and individuals in glyphosate-resistant have been patented in China, and another 22 applications are under review. It is clearly that the domestic exploring new glyphosate-tolerant gene and getting the independent intellectual property made great strides since 2007. By the authentication glyphosate resistance in transgenic plants, some resistance genes have appeared the potential value of commercial applications.

With the increase of human population density in coastal areas, the anthropogenic input of nutrients and organic matter into coastal waters has resulted in hypoxic events of increasing magnitude, frequency, and duration. Adaptation to hypoxia in cells and tissues leads to the transcriptional induction of a series of genes that participate in erythropoiesis/iron metabolism, angiogenesis, glucose metabolism, cell proliferation/survival and apoptosis. The primary factor mediating this response is the hypoxia-inducible factor-1 (HIF-1): an oxygen-sensitive transcriptional activator. HIF-1 is a dimeric transcriptional complex comprising a HIF-α subunit and a HIF-β (also called the aryl hydrocarbon receptor nuclear translocator, or ARNT) subunit, both of which are members of the basic helix–loop–helix/Per-Arnt-Sim (bHLH/PAS) proteins that are characterized for containing bHLH and PAS conserved domains. They have been recognized primarily for its role in the maintenance of oxygen and energy homoeostasis. The HIF-1α acts as the important regulator of the expression of some genes in response to hypoxia. At the same time, the HIF-1α subunit is O2 labile and is degraded by the proteasome following prolyl-hydroxylation and ubiquitination in normoxic cells. Stability of HIF-1α protein is primarily regulated via an oxygen dependent degradation domain (ODDD) while its transcriptional activity is facilitated via two transactivation domains (N-TAD and C-TAD). These TADs, besides being essential for interaction with transcriptional co-activators such as CBP/p300, are targets for regulation via post-translational modifications such as phosphorylation, acetylation and redox modifications. Under hypoxic conditions, HIF-1α accumulates and forms a heterodimeric DNA-binding complex with HIF-1β, and binds to the hypoxia response element (HRE): 5'-RCGTG-3' on the promoter region of target genes. However, HIF-1β subunit is constitutively expressed in cells. HIF-1β is an obligate dimerization partner for some bHLH-PAS proteins, including AhR (aromatic hydrocarbon receptor): HIF-1α and 2α, and the Sim proteins. The bHLH domain of HIF-1β is mainly responsible for identifying and binding the DNA specific sequence. For PAS domain, it is necessary to form a functional DNA binding complex of HIF-1β and bHLH-PAS family proteins. Although some information is available about the gene expression in response to hypoxia, little is known about the molecular mechanism of the regulation of their expression in aquatic animals. The present review summarized structure and function of HIF-1, O2/PHDs/pVHL degradation pathway of HIF-1, expression regulation, target genes of HIF-1 and it' s research advance in aquatic animals. This article can provide the reference for research on HIF-1 signaling pathways of aquatic animals in the future.