Mycoplasma bovis (M.b) is one of the important pathogenic mycoplasmas. It can cause a variety of diseases if cow (Bos taurus) infected with the M.b. Pyruvate dehydrogenase complex E1 (PDHc E1) is a speed limit of enzymes that involve in biological sugar metabolic process, and widely exists in microbe, mammals and higher plants. It directly impact on pyruvate to acetyl coA. According to the gene sequences of M.b PG45 strain in GenBank, two pairs of primers were designed. PDHc E1 component subunit alpha (pdhα)(GenBank No.KU355295) and PDHc E1 component subunit beta (pdhβ) (GenBank No. KU3552956) genes of M.b Wuwei strain were amplified by PCR. After finished sequencing and point mutation, the genes were cloned into prokaryotic expression plasmid pET-28a(+), respectively. Then the recombinant plasmids pET-pdhα and pET-pdhβ were transformed into Escherichia coli BL21 (DE3), respectively. After induced expression by isopropyl β-D-1-thiogalactopyranoside (IPTG), the fusion protein recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were purified and anti-serum against rPDHA or rPDHB was prepared respectively through immunizing New Zealand rabbit (Oryctolagus cuniculus). Subsequently, subcellular location of E1α and E1β in M.b was performed using Western blot and enzyme linked immunosorbent assay (ELISA). The results showed that the recombinant proteins successfully expressed in Escherichia coli BL21 (DE3) and their molecular weights were approximately 40 and 37 kD, respectively. Western blot and ELISA analysis indicated that the E1α and E1β of M.b equally distributed in both membrane and the cytoplasm. This study provides foundational data for further investigation of biological functions of M.b.

Arabidopsis thaliana steroidogenic acute regulatory related lipid transfer (START) domain containing membrane related protein (STMP) is a function unknown protein containing transmembrane segments, it has 440 amino acid. From 84nd to 295th amino acids is START conserved domain. There is a phosphatidycholine (PtdCho) binding site in START domain. In order to clarify the role of STMP in the development and regulation of Arabidopsis thaliana, this study used the 35S promoter to construct the START domain, Agrobacterium tumefaciens was used to infect the expression vector of STMP in wild type Arabidopsis thaliana, obtained the transgenic Arabidopsis thaliana and used qRT-PCR technology to verify it; Clear the role of STMP in the process of the regulation of the branch of Arabidopsis thaliana through analysis of the developmental status of the transgenic Arabidopsis thaliana; Analysis of temporal and spatial expression of STMP by qRT-PCR technique; Preliminary analysis of the subcellular localization of STMP was performed through the establishment of STMP-GFP (green fluorescent protein) fusion expression vector and protoplast transformation. In this study, transgenic lines over-expressing STMP were obtained. The expression of STMP in transgenic plants was significantly higher than that in wild type, and the temporal and spatial expression of STMP showed that the expression of STMP in stem was the highest, Green GFP localized on cell membrane and cell cytoplasm. These results showed that STMP promotes the development of the branches in Arabidopsis thaliana, This study has important significance to improve the regulatory mechanism of Arabidopsis thaliana branching, and provide a theoretical basis for the design of directional plant type.

Trophoblast (TR) plays an important role in the process of embryo implantation and placenta development. Molecular mechanisms of TR cell differentiation and lineage formation is an important tool to study the placental development. In this study, TR cells from porcine (Sus scrofa) in vitro fertilized (IVF) blastocysts (IVF-TR) were established and had been characterized, and the differences of some gene expression between in vivo porcine TR cells and in vitro TR cells were investigated by qRT-PCR. The IVF blastocysts which were derived from trophoblast cells exhibited typical trophoblast characteristics and displayed epithelium-like morphology, and trophoblast cell marker factors expressed such as keratin 18 (KRT18), keratin 7 (KRT7) and stage specific embryonic antigen 1 (SSEA1) by immunofluorescence analyses. The result of morphologic observation showed that porcine placenta was diffuse epitheliochorial placenta with atrophy at the peripheral tips, which was easy to be stripped from the uterine wall. The cells which were derived from the placenta were elongated spindle shape, epithelial like, grew slowly, and only passaged twice in vitro. The results of qRT-PCR which performed on porcine trophoblast cells from different sources showed that the expression of certain genes were different among trophoblasts. The expression levels of caudal homeobox transcription factor 2 (CDX2), pregnancy-associated glycoprotein (PAG) and GATA binding protein 3 (GATA3) were extremely higher in placenta trophoblast than that in IVF-TR cells (P＜0.05), while heart and neural crest derivatives expressed 1(HAND1), KRT18 and cadherin 3 (CDH3) expression which were associated with TR differentiation and implantation were extremely lower in placenta trophoblast than that in IVF-TR cells (P＜0.05). Apart from TEA domain family member 4 (TEAD4), the expression levels of other trophoblast genes were lower in the placenta cells than that in placenta tissues and IVF-TR. In conclusion, the IVF-TR cells were more like early differentiated TR cells, and the gene expression was different from the mature late trophoblast cells in placenta. At the same time, the gene expression of the in vitro cultured placenta cells was also different from the placenta tissues. This study indicated the IVF-TR cells might be used as the material and tool to investigate the trophoblast differentiation and the development of porcine placenta. The results provide the preliminary assessments data for the further investigation.

Molt inhibiting hormone (MIH) is an essential hormone in regulating molting and growth of crustacean, which is produced in eyestalk of crustacean. However, it is very difficult to obtain MIH content in single individual because separation and purification of this protein require large numbers of samples, which limits the physiology and biochemical study of MIH gene. Thus, an alternative way is to use prokaryotic expression for conducting large amount of recombinant proteins. By now, the influence of the exogenous recombinant MIH protein on the endogenous MIH gene expression of Chinese mitten crab (Eriocheir sienesis) has been remained unclear. In this study, a series of experiments were conducted to identify the optimal conditions for prokaryotic expression of MIH gene. Its exogenous recombinant protein was firstly used via injecting into the eyestalk of Chinese mitten crab for observing the expression profiles of endogenous MIH gene. The experiments conditions included different concentration of IPTG (isopropy-β-D-thiogalactopyranoside)(0.5, 1.0, 2 and 4 mmoL/L), different induction time (0, 2, 4 and 6 h) , and different induction temperature (27 and 37 ℃). The results indicated that the highest amount of content of recombinant MIH protein were obtained under the conditions of IPTG with concentration of 1.0 mmol/L for 6 h at 27 ℃. The MIH recombinant protein concentration was 2.3 mg/mL under this condition. Then the recombinant protein from the induced bacterial expression (pET-28a-MIH) were injected into living crabs. It was found that the expression level of MIH gene in eyestalk was significantly higher after 4 h injection than those of PBS control group (P＜0.05), while no significant difference was found among 8 h, 24 h injection and PBS control groups (P＞0.05). Furthermore, the expression level of endogenous MIH gene in eyestalk was significant higher after 4 h than that after 8 and 24 h (P＜0.05), while there were no significant difference among the 4, 8 and 24 h with injection of PBS (P＞0.05). Our results indicated exogenous MIH recombination protein could shortly promoted the expression of endogenous MIH protein, and then partly restrain the molting in crab. This result provided insight into the molting mechanism of crab and made a foundation for antibody preparation in future.

In order to investigate the molecular mechanism of salt tolerance, the proteome in roots of 30 d seedling age southern type Alfalfa (Medicago sativa 'Millenium') under control condition were compared with those under 72 h treatment at 250 mmol/L NaCl. Proteins were abstracted from root under control and NaCl- treated conditions, reduction and enzymolysis of proteins were performed. Isobaric tags for relative and absolute quantitation (iTRAQ) technique and 2-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS /MS) were used to identify the differentially expressed proteins in southern type alfalfa root upon salt stress, biological analysis on differentially expressed proteins was conducted to some of the potential target proteins to salt stress. Quantitative Real-time PCR (qRT-PCR) technique was used to validate the expression data of five randomly selected differentially expressed proteins. The results indicated that 3 857 quantitative proteins were identified 534 differentially expressed proteins(fold change ratio≥1.2, P＜0.05) were identified, in which 281 proteins were up-regulated and 253 proteins were down-regulated. Gene ontology (GO) molecular functional analysis showed that the differentially expressed proteins were mainly involved in transporter activity, catalytic activity and enzyme regulator activity. Significantly enriched KEGG pathway were related to metabolic pathways, biosynthesis of secondary metabolites, phenylalanine metabolism and ribosome and so on (P＜0.05, False discovery rate (FDR)＜0.05). The identified differentially expressed proteins were involved in signal transmission (8.99%), oxidation resistance (7.68%), defense(5.61%), protein synthesis, processing and degradation (14.79%), energy and transport (5.81%), metabolism (26.97%), membrane and intracellular transport (5.62%) and cell structure, division and cytoskeleton (2.06%) and so on. Functional analysis of the differentially expressed protein species showed an generally increased abundance of proteins related to signal transmission, oxidation resistance and defense and a decreased generally abundance of proteins related to metabolism and energy and transport. The expression levels of differentially expressed proteins were verified by real-time PCR and the results were not consistent with those of the quantitative mass spectrometry (P＜0.05).The results also showed that histidine phosphotransfer protein, mitogenactivated protein kinase, h-type Thioredoxin, plastidial methionine sulfoxide reductase, guanosine diphosphate (GDP) dissociation inhibitor, Lipid transfer protein, β-1, 2-xylosyltransferase and core histone H2A/H2B/H3/H4 and so on were potential target proteins in alfalfa in response to salt stress. Differentially expressed proteins in southern type alfalfa root to salt stress can be effectively screened by iTRAQ technique combined with 2D-LC-MS /MS in order to lay a solid basis for a holistic understanding of the molecular regulatory mechanism in southern type alfalfa to salt stress.

Chemical-induced male sterility (CIMS) system, which depends on some chemical hybridization agents (CHAs) such as tribenuron-methyl (TBM) herbicide, is one of the important ways for heterosis utilization in rapeseed (Brassica napus). Selective breeding TMB herbicide resistant rapeseed as male parent in CIMS system can simplify the protocol and cut the cost of hybrid seed production for better yield and quality of rapeseed. Acetohydroxyacid synthase (ALS) is a key enzyme in the biosynthetic pathway of branched-chain amino acids, including leucine, isoleucine and valine. Several herbicidal ALS-inhibitors can bind to ALS and inhibit the activity of ALS, resulting in plant death by blocking the synthesis of branched-chain amino acids. Sulfonylurea (SU) is a kind of herbicidal ALS-inhibitors such as TBM. In this study, we cloned the mutated acetolactate synthase gene (GenBank No. EU520490) from a TBM resistant native flixweed (Descurainia sophia) mutant (#108), and the mutated gene was named as DsALS-108. The results from amino acid sequence alignments revealed a substitution of Pro with Thr at position 197 according to Arabidopsis acetolactate synthase AtALS protein sequence (named P197T). In order to verify whether the P197T mutation in DsALS-108 gene was responsible for the TBM resistance, expression vector PBI121-DsALS-108 was constructed and Agrobacterium tumefaciens carrying the constructs was used for transformation of Arabidopsis. We obtained twelve positive transgenic Arabidopsis lines. When treated with ≥10-4 g/L of TBM, the positive transgenic plants grew normally. In contrast, wild-type plants could not survive. These results suggested that the expression of mutated gene DsALS-108 resulted in the TBM resistance. In Agrobacterium-mediated transformation of rapeseed, hypocotyls were cut as explants. Agrobacterium tumefaciens harboring pCAMBIA3301-DsALS-108 constructs was used to generate transgenic rapeseed plant. Twenty-three positive transgenic lines were obtained. The positive transgenic rapeseed lines expressing DsALS-108 were found to grow well with the treatment of 7.5×10-3 g/L of TBM, which was three times higher than lethal concentration of wild-type plants. Moreover, male fertility of the DsALS-108-expressing rapeseed plants was not affected when treated with by 0.05 mg/L of TBM, which was the concentration used for induction of male sterility, showing the potential application of DsALS-108-expressing rapeseed plants as male parents for hybrid rapeseed production using CIMS. This study can supply new herbicide resistant breeding material for utilization of heterosis in rapeseed using CIMS.

The excision of selectable marker gene is an important foundation for transgenic crop to release into the environment or commercialized planting. In order to establish a low-temperature induced site-specific marker gene deletion system in wheat (Triticum aestivum), low-temperature induced promoter wcs120 was cloned from wheat Xinong 928 genome DNA. The sequencing result showed that the whole wcs120 promoter sequence was successfully cloned , which was the same as AY493570.1 and a insertion of 21 bp with AF031235. Using the plasmid pXL5513 which contained the CinH/RS2 system as basal vector, the final vector pXL5513-fwcs-RDA was constructed, in which the CinH recombinase gene was under the control of cold-induced promoter wcs120 and the interest gene pyrabactin-resistance like gene 5 (PYL5) was inserted outside the RS sites near the left border. All of CinH recombinase gene, selectable gene bar and reporter gene green fluorescent protein (GFP) were flanked by 2 recombination sites RS2, which would be expected to be excised after the expression of CinH recombinase. pXL5513-fwcs-RDA was transferred into wheat immature embryo calli of Mianyang 19 (M19) by particle bombardment. After selection and generation, 9 transformed plantlets were gained from 1 745 calli and 6 positive transgenic seedlings were confirmed by PCR of PYL5 gene. Induced by low temperature, all of these positive transgenic seedlings showed selectable gene deletion, which were initially confirmed by PCR. Because of the features of particle bombardment, the patterns of gene insertion were complex, the subsequent experiments were planned to identify the deletion pattern. This investigation will lay a foundation for the application of low-temperature induced site-specific recombination system in marker-free transformation on wheat.

S-adenosyl-L-homocysteine hydrolase (SAHH) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (SAH) into homocysteine and adenosine. SAH is the inhibitor of most transmethylation reactions which is often compared with S-adenosyl-L-methionine (SAM, one of methyl donor) to evaluate the overall methylation level in plant. In order to identify the function of SAHH in Kalanchoe daigremontiana (KdSAHH), primers were designed according to the full length of KdSAHH cDNA in GenBank (Accession No. KF953475). The KdSAHH gene expression pattern under drought stress (light drought stress(20 d); middle drought stress (35 d); high drought stress (50 d)), subcellular location in cell and ectopic transformation in tobacco (Nicotiana tobacum) were done to unveil the basic biological function. Drought stress was simulated with 20% PEG6000 in the seedling stage between transgenic and wild type tobacco and drought-related physiological indexes like leaf relative water content (RWC), chlorophyll (Chl) content, malondialdehyde (MDA) content, peroxidase (POD) content, superoxide dismutase (SOD) content were measured at 3 different time points (0, 12 and 24 h). The semiquantitative RT-PCR analysis showed that the highest expression level of KdSAHH gene was meatured in high drought stress (50 d), followed by middle drought stress (35 d) and light drought stress (20 d). The result of subcellular location indicated that KdSAHH gene was located both in nucleus and cytoplasm. Nine positive tobacco transgenic line were obtained by PCR. Physiological indicators of transgenic and wild type tobacco under 3 time periods (0, 12 and 24 h) of 20% PEG6000 treatment showed that both of them showed obvious water loss symptoms at 24 h, but the transgenic tobacco of KdSAHH gene had lower wilting degree compared with wild type tobaccos, and some leaves at the upper end of the transgenic plant were still standing, while all leaves of wild type tobaccos showed obvious water loss symptoms. With the increasing of drought stress, both leaf RWC and Chl content of transgenic and wild type tobaccos tended to decreased but the decline extent of transgenic tobacco was relatively slight. Leaf RWC was significantly different between transgenic and wild type tobaccos at 24 h (P＜0.05) and Chl content were significantly different between the 2 types at 12 and 24 h (P＜0.05), which showed that under drought stress, the transgenic tobacco was still able to maintain a stable metabolism. MDA content was increased in transgenic and wild type tobaccos but more MDA content was in the wild type tobacco than that in the transgenic tobacco, and it was significantly different between the 2 types at 24 h (P＜0.05), which showed that transgenic tobacco had a lower degree of membrane lipid peroxidation and suffered lighter injury. The changing trend of SOD and POD content in transgenic and wild type tobaccos were almost the same, however, the wild type tobacco produced more SOD and POD content than the transgenic tobacco with the increasing drought stress. POD content was significantly different between the 2 types at 12 and 24 h (P＜0.05), while there was no significant difference of SOD content. The reason might be analyzed as follows transgenic tobacco had a stronger resistance to drought stress and could not generate too much reactive oxygen. By comparing different physiological indexes between the transgenic and wild type tobaccos under drought stress, we could conclude that transgenic tobacco had a higher resistance than that of wild type tobacco. The result of this study has a great significance that it identified KdSAHH gene was tightly involved in Kalanchoe daigremontiana drought resistance. SAHH widely spreads in various organisms, which is a key enzyme of methyl cycle in organism. The study enriches the function of KdSAHH gene and provides a reference for follow-up study aiming at the function of this gene.

Soybean cyst nematode (SCN)(Heterodera glycines) is the most devastating pathogen of soybean (Glycine max) in the world. To complete the life cycle, SCN enters into host roots and causes dramatic morphological and physiological changes in host cells. At the same time, the gene's expression changed in host roots. In this paper, Illumina HiSeqTM 2500 technique was used to sequence the genes expression of the soybean, which was resistance to race 4 of SCN, after SCN infection 9 and 17 d. These time points were chosen to contrast with plant gene expression without inoculated cyst nematode. The previous time point was identified because it was the development transition period of second-stage juvenile (J2) to third-stage juvenile (J3). Similarly, the later time point was the development period of third-stage juvenile (J3) to the fourth-stage juvenile (J4). Finally, a total of 1.96×1010 bp bases were obtained. There were 2 180 and 4 210 genes with altered expression in roots at 9 day and 17 day after SCN infection, respectively, and a total of 840 common genes were expressed between 2 periods. Gene ontology (GO) significant enrichment analysis displayed 1 898 and 3 627 differentially expressed genes (DEG) which could be divided into cellular component, molecular function and biological processes of these 3 ontology. And there was relatively more DEG classified to extracellular region, antioxidant activity, nutrient reservoir activity and cell killing. Differentially expressed genes were classified into 25 categories using Cluster of Orthologous Groups of proteins (COG) annotation, and a total of 880 and 1 751 DEG annotated to this database at those 2 periods respectively. The function of COG was more comprehensive, the general function prediction only had the largest proportion, and the second is the transcription. In addition, gene expression patterns were integrated with biochemical pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG). Part of DEG may be involved 83 and 105 different metabolic pathway based on KEGG database at 9 day and 17 day after SCN infection, respectively. Genes encoding enzymes involved in phenylalanine metabolism, energy metabolism and plant hormone signal transduction were altered. This result will be useful to clarify the mechanism of disease resistance. In a word, these data provide some support for screening the key resistant gene and vivificating the function.

The completion of Chinese cabbage whole genome and the reduced cost of re-sequencing provide possibility for high-throughput development of InDel markers. In the present study, two Chinese cabbage inbred lines He102 and 06-247 with significant differences in phenotype such as bolting tolerance, virus resistance and tip-burn tolerance were re-sequenced for genome-wide InDels identification. The sequencing depth of these two lines was 10×and 8×respectively. A total of 330 218 polymorphic InDels loci were predicted based on the sequence data. The frequency of InDels occurrence was 1.2/kb, among which 11 238 loci were located in deduced coding region and represented for 5184 genes. We randomly selected 933 predicted polymorphic InDels loci that distributed on the 10 chromosomes of He102 and 06-247 respectively for PCR validation. Results showed that the positive rate varied greatly with sequencing depth. When the choosed loci from a higher sequencing depth material He102, the false positive rate was higher either, and vise versa. In the present study, the average false positive rate was 51.9% and 22.5% for the selected loci from He102 and 06-247, respectively. In other words, higher false positive rate based on higher-depth material reflected that the false positive loci were missed in lower-depth material; contrarily, lower false positive rate based on lower-depth material reflected that the positive loci were detected in higher-depth material. A total of 593 verified loci including 375 loci located in coding sequence (CDS), were identified. More than 90% of the polymorphism loci were co-dominant while about 5% of them were dominant. The rest couldn't produce clear band or multiple bands were generated. The detected polymorphic loci were not evenly distributed on ten chromosomes. Chromosome A01 contained the most number (86) of loci, while A08 contains only 29 loci. The co-dominant polymorphic loci were used to detect the residual heterozygous line of F7 which was generated by single seed descend from F2 of He102×06-247. The results showed that in F7 population, more than 90% of the alleles were homogenous while less than 10% of the them were remain heterogenous. The hemogenous/heterogenous state and the physical position of each locus in all 217 lines of F7 were confirmed. The residual heterozygous lines and the identified polymorphic markers provided useful information for elaborate decompose and fine mapping of QTLs/genes related to bolting, virus resistance and tip-burn tolerance in Chinese cabbage.

Marbled flounder (Pseudopleuronectes yokohamae) is an important natural fishing and aquaculture object in the flounder fish. The number of germplasm resources has been decreased, due to factors such as changed ecological environment, artificial overfishing and so on. So research the sperm cryopreservation technology which is of great significance for germplasm resources. In present study, the series of experiments for sperm diluents, cryoprotectant, the suitable salinity of seawater and frozen semen artificial insemination were selected using the mature male marbled flounder. The experiment results were analyzed by One-way ANOVA and Student-Newman-Keuls from SPSS 19.0. Figures made with Origin 8.0 software. The results indicated that the antifreeze effect of sperm cryopreservation was good, using MFs-3 (8 g/L NaCl+0.65 g/L KCl+15 g/L glucose) with 20% PG and EG respectively, which the sperm motility, fast moving time and life time was (95.26+0.39)%, (46.00+1.00) s, (124.33+4.04) s and (95.15+0.41)%, (45.67+0.58) s, (124.00+3.00) s, respectively. It indicated the diluent MFs-3 suits for marbled flounder sperm cryopreservation, and the best cryoprotectants were PG and EG. In the experiment of selecting of the suitable salinity of seawater, the frozen sperm were activated with 10‰~50‰ seawater, showed that the sperm motility was the highest at 30‰ and there was no significant difference compared with fresh sperm but there was significant difference between others and fresh sperm (P＜0.05). And sperm motility has been gradually increased from (86.13+1.44)% to (95.07+0.69)% when the salinity is 16.7‰~30‰; Sperm vitality has been gradually decreased from (95.07+ 0.69)% to (84.09+2.61) % with 30‰~45‰ sea water. Lower than 16.7‰ and higher than 45‰ of sea water, sperm was almost no movement. The thawed sperm with PG and EG cryopreservation and marbled flounder eggs made fertilization experiments, fertilization rate and hatching rate was (80.08+0.68)%, (77.44+1.76)% and (80.17+0.45)%, (77.92+1.33)%, respectively, and there was no significant difference compared with fresh sperm. It was also used computer aided sperm analysis (CASA) to analyze and test for motion parameters from frozen sperm sample in MSs+20% PG and MSs+20% EG including curvilinear velocity (VCL), straight line velocity (VSL), beat cross frequency (BCF), linearity (LIN) and straightness (STR) and so on, showed that there was no significant difference between them. In present study, the sperm from marble flounder could be effective cryopreserved with MSs-3+20% PG and MSs-3+20% EG. This study has been successfully preserved marbled flounder sperm with 20% MFs-3+PG and MFs-3+20% EG at -196 ℃. And the frozen sperm motility, fast moving time and life time was no significance with the fresh sperm. The technology had preserved the marbled flounder sperm cryopreservation, which has set a solid foundation for artificial cross breeding and genetic resources center. The research result firstly provides important the theory and technology basis.

As a key enzyme of plant eighteen carbonate metabolic pathway, lipoxygenase (LOX), which is closely related with seed aging. Onion (Allium cepa) seeds are a short-lived seeds. In order to study the mechanism of LOX gene in the process of onion seeds aging, the full length cDNA of LOX gene (GenBank accession No. KU363822) in onion seeds was cloned by RT-PCR and rapid-amplification of cDNA ends (RACE) technology. The bioinformatics methods were used to analyze the sequence characteristics of this gene and the combination of RT-PCR and qRT- PCR methods were used to investigate the temporal and spatial expression pattern of AcLOX1 gene. The cloned full-length cDNA of AcLOX1 was 2 847 bp in length, containing a 2 616 bp ORF which encodes 872 amino acids. Amino acid sequence analysis showed that AcLOX1 contained a PLAT_LH2(polycystin-1, lipoxygenase, alpha-toxin_lipoxygenase homology) domain and a Lipoxygenase domain. Protein sequence identity search showed that AcLOX1 was similar to LOX gene of Eucalyptus grandis, Prunus dulcis, Vitis vinifera with similarities of 66%~73%. Phylogenetic tree reconstructed by neighbor joining method clearly showed that AcLOX1 protein was classified into 9-LOX subgroup. RT-PCR and qRT-PCR analysis showed that AcLOX1 were expressed in onion tissues and there were differences in expression. The expression was increasing from 5 day to 25 day after pollination, but there was a small amount of sustained expression in the seeds from 25 days after pollination to maturation and seeds stored a year. In this study it is demonstrated that is somewhat related to the onion seeds aging and can promote the development of onion seeds, which lay the foundation for elucidating the mechanism of seed aging and provide theoretical basis for improving the storage of seed.

Rotylenchulus reniformis is a semi-endoparasitic nematode, which widely distributes in tropical and subtropical area of the world, and is pathogenic nematodes of many vegetables and tropical fruit trees. Our research was focus on identification and phylogenetic relationship among different geographical populations of reniform nematode. To characterize the genetic diversity of intra-species populations of Rotylenchulus reniformis, sequence variations of COII- LrRNA mitochondrial DNA (mtDNA) between individuals and 3 intra-species populations of reniform nematode from Zhejiang (ZJ), Fujian (FJ) and Chongqing (CQ) were analyzed based on DNA sequence analysis technology. The result showed that the length of COII-LrRNA gene region ranged from 557 to 563 bp using primer 1108 and C2F3-R. The average content of A, T, C and G were 32.6%, 52.9%, 4.5% and 10.0%, respectively. The content of A+T was 85.5%, and it was much higher than that of G+C content. Obvious genetic diversity was found from the amplified region. All 389 conserved sites, 176 variation sites, 51 parsimony-informative sites and 125 singleton sites were identified. There were 40 haplotypes among the 64 sequences, which suggested a high level of heteroplasmy in R. reniformis, and 17 haplotypes were found in ZJ population, 13 haplotypes in CQ population and 12 haplotypes in FJ population. The number haplotype diversity (Hd), and nucleotide diversity (π) of 3 population were 0.946 and 0.157 4, respectively. The analysis of molecular variance (AMOVA) showed that the Fst (F-statistics) of coefficient of genetic differentiation was 0.058 15 (P＜0.01), which placed these isolates into a moderate range of genetic differentiation and no obvious geographic isolation. The variation within populations was 94.18% and only 5.82% of the variation occurred among the 3 intra-species populations. In general, the variation among COII-LrRNA gene sequence of reniform nematodes in China was obvious, which indicated that reniform nematodes in China had abundant genetic diversity, and a strong ability to adapt to environmental change. This study enriches the phylogenetic information of R. reniformis, and provides the basic evidence for the inherent genetic factors of damage from reniform nematode.

Aurora A is one of the serine/threonine protein kinase who participates in a series of biological events such as centrosome replication, spindle assembly and cytokinesis. The purpose of this study was to explore the dynamic expression and function of Aurora A during oocyte-to-embryo transition in porcine (Sus scrofa). As Aurora A has an extremely close relationship with microtubules, our examination began with the expression and distribution of α-tubulin during the oocyte-to-embryo transition. Meanwhile, The dynamic expression and subcellular localization of Aurora A during oocyte-to-embryo transition were detected using indirect immunofluorescence method and laser confocal microscopy analysis. Then, the specific inhibition test was used to analyze the regulatory function of Aurora A in this particular transition stage. The results showed that Aurora A expressed in each period in the oocyte-to-embryo transition and had the similar subcellular localization with α-tubulin. At the metaphaseⅠ (MⅠ) and MⅡ stage, α-tubulin assembled a typical spindle in the cortex of oocyte, and a similar distribution presented in the same place where Aurora A was. During the prophase of mitosis, α-tubulin gradually gathered and reticulated, and Aurora A also had a net-like distribution. Following, α-tubulin was translated from the symmetric and barrel shaped spindle in the central of embryo to migration towards poles of embryo, at the same time Aurora A had a similar trend from mitosis metaphase to anaphase. When oocytes were treated with a highly selective inhibitor (MLN8054) of Aurora A, the extrusion rate of first polar body was significantly reduced with the increasing concentrations of MLN8054 (P＜0.05), and the percentage of abnormal spindle morphology, mainly including monopolar spindles and multipolar spindles, was significantly increased (P＜0.05). What's more, the high frequencies of abnormal spindle and chromosome alignment varied with the different exposure times. When oocytes at MⅡ stage which were treated with MLN8054 were parthenogenetic activation by electric pulse, the rate of embryo cleavage was decreased, but the difference was not significant (P＞0.05). The results showed that the expression of Aurora A was closely related to the α-tubulin distribution, with obvious stage characteristics during the transition of oocyte-to-embryo, and Aurora A might be involved in the regulation of oocyte maturation and embryo development via regulating the assembling and stability of spindle. The inhibition of Aurora A which were treated with MLN8054 had no significant effect on the transition of porcine MⅡ oocyte-to-embryo development, and the perhaps reasons might be the activity of Aurora A which was suppressed restored in the subsequent embryonic development and early embryo mitosis went through smoothly, when the competitive inhibition of MLN8054 was removed. This experiment mainly studied the expression of Aurora A and its effect on the structure of spindle during porcine oocyte-to-embryo transition, which provides an important experimental basis for porcine oocyte maturation and early embryo development as well as related signaling pathway in this process.

Long non-coding RNA (lncRNA) is a group of RNAs with broad biogenesis, which are longer than 200 nt and highly conserved in their secondary and tertiary structures. lncRNA that broadly participates in varied physiological processes in organisms has abundant biological function and can regulate expression of target genes at transcriptional, post-transcriptional and epigenetic levels. Furthermore, its sophisticated molecular regulatory mechanisms have already been widely revealed in biological processes such as chromosome dosage compensation, genomic imprinting, target mimicry, functional protein trafficking, and so on. As with the biological function of lncRNA being continuously revealed, researchers find that the highly conserved structure of lncRNA is crucially important to exert its biological function. Therefore, the advanced structure prediction of lncRNA becomes much more significant during the process of studying its biological function. At present time, researchers mainly use computational and mathematic methods to predict lncRNA functional domains, which is expected to illustrate its biological mechanisms at structural levels. In terms of the lncRNA related research methods, they also get rapid development. Among them, some comparatively mature approaches such as chromatin isolation by RNA purification (ChIRP) and RNA-binding protein immunoprecipitation (RIP) have already become widely used in the field of investigating the interaction between lncRNA and chromosome, and the interaction between lncRNA and proteins, respectively. This review focused on the origin, standard classification, advanced structure prediction, biological function and related approaches of lncRNA, which was aimed at providing researchers better understanding of lncRNA biological function in organisms.

Polymerase chain reaction-single strand conformation ploymorphism (PCR-SSCP), as an important technique in detecting minor differences between genomic DNA bases, has the characteristics of simplicity, rapidity and sensitivity. So far, a large number of genome sequences provide lots of DNA sequences for the development of novel genetic markers through comparative genomics and the mapping of target genes. Hence, the possibility was investigated on the application of PCR-SSCP technique combined with comparative genomics to develop novel molecular markers in cotton (Gossypium spp.) genetic mapping in the study, taking Ligon lintless-1 (Li1) mutant gene mapping as an example and the published sequences of Arabidopsis and cotton genome D as references. The optimization of SSCP conditions for samples of large fragment PCR products (＞400 bp) were carried out in the experiment. The results showed that the optimal experiment conditions were as follows: voltage of 150 V, gel concentration of 10%, electrophoresis temperature of 4 ℃, and 5∶1 of loading dye to PCR product. According to the linear segments of Arabidopsis and cotton, we selected 102 cotton EST sequences to develop the SSCP primers. The optimized PCR-SSCP system was utilized to construct a genetic map consisting of 28 markers and spanning 149.6 cM . This genetic map contained 4 genes with functions closely related to Li1 loci. Additionally, using genome D sequences, 185 SSCP primers were developed and there were 86 primers showing polymorphism between the two parents (Li1 and H7124). In this experiment, Li1 gene was firstly mapped in a genetic map consisting of 17 SSCP markers and the Li1 loci, which spanned 40.3 cM. The Li1 gene was flanked by W058 and P095, which were the nearest markers, and the distance from Li1 loci was 0.6 and 0.3 cM respectively. Using the published Li1 genetic map to analyze the relationship between D genome sequences and two genetic maps constructed in this experiment, the study suggested that there were good homologous between these genetic maps. The results presented here could offer a useful foundation for future research work on fine mapping and cloning of Li1 gene. In addition, with the development of the third generation genome sequencing technology, constantly enrichment of the GenBank and the publishment of cDNA sequences, the optimization of SSCP technology plays an important role in cotton genetic linkage map construction, map-based cloning, molecular marker-assisted breeding and other aspects.