To reveal the relationship of soil bacterial community structure and its response to soil environment under continuous cropping, the Illumina platform Hiseq2500 high-throughput sequencing technique was used to sequence Luohe tobacco continuous cropping tobacco planting soil bacteria 16S rRNA V4 area with different fertilizer treatment, and find out the soil bacteria microbial community composition, diversity and interaction between the soil environment and bacteria with redundancy analysis (RDA). All 25 203 operational taxonomic units (OTUs) and 1 600 239 pieces readings in total were detected in this sequential control condition. Heatmap showed that there were significant differences among the character of bacterial communities in continuous cropping tobacco planting soil, and those differences mainly existed in different treatment or community in different growth period while diversity of species showed less. Alpha index showed that continuous cropping tobacco planting soil bacteria changed during mature period among different growth stages, and each index increased in some extent; the diversity index (Shannon and Simpson index) changed little, bacteria's abundance indexes (ACE and Chao1 index) of flue-cured tobacco raised to a high level after transplanted in 50 and 70 d with mixture of earthworm manure and microbial fertilizer(T3) and the indexes of microbial fertilizer(T2) reached to a peak which occurred after transplanted in 30 and 90 d, which indicated that the effects that different treatments posed on bacterial community showed in the abundance of bacterial community. Principal component analysis (PCA) showed that there was a strong correlation within soil environmental factors, thus based on the strong positive correlation, the original 11 factors could be divided into 4 types of soil environmental factors. Redundancy analysis (RDA) showed that the 5 environmental factors (pH, urease, available phosphorus, protease and amylase ) could represent 4 general system evolved from PCA analysis process, explain the 98.03% of the original 11 soil environment variable and highly represent the level of soil environment system. The pH not only affected the diversity of soil bacterial communities, but also the abundance of soil bacterial communities. There was a strong correlation between amylase and ACE index, the strong relationship also existed in the Chao1 index and amylase. This indicated that the rising of soil carbon level had a positive effect on the growing of soil bacterial abundance, rising soil carbon level also contribute to the activity of bacterial community; and the urease activity was negatively correlated with the abundance and diversity of soil bacterial community, which showed that the elevation in single nitrogen level actually poses a negative effect on the variation of soil bacterial community. The results of this study provide a basis for studying the formation mechanism of continuous cropping obstacles at the microbial level.

YSK2 type dehydrins (DHNs), which are the most types of DHNs, have been shown to be involved in plant response and adaptation to various abiotic stresses. In order to determine the function of YSK2 DHNs, WDHN1 from wheat (Triticum aestivum) was cloned. The CDS of WDHN1 had a length of 491 bp, contained 2 exons and 1 intron, encoding a protein of 133 amino acids, containing one Y segment, one S segment and two K segments. A phylogenetic tree analysis with the related DHNs from different plant species indicated that WDHN1 shared homology with DNA from Aegilops tauschii(EMT30992). Based on PLACE and PlantCARE database analysis, the abiotic stress-related elements of WDHN1 promoter were determined, which contained 2 ABRE (abscisic acid (ABA) response element), and 3 MBS (MYB binding site). qRT-PCR analysis indicated that transcript accumulation occurred in response to low temperature, NaCl, ABA and PEG 6000 treatments. Tissue specific expression analysis showed that the transcript levels of WDHN1 reached the highest level at 22 d in embryo. The prokaryotic expression vector of WDHN1 was successfully constructed. The expression of fusion protein was obtained with isopropyl β-D-1-thiogalactopyranoside (IPTG) and its relative molecular weight was 20 kD. We investigated the anti-aggregation effects of protein WDHN1 on Escherichia coli viability and lactic dehydrogenase (LDH) activity during multiple abiotic stress treatments. WDHN1 protein enhanced tolerance of E. coli and LDH stabilization against diverse stresses via anti-aggregation effects. The results revealed that WDHN1 might serve as a potential stress response gene for the improvement of wheat inbred lines and cultivars under stress conditions in breeding activity.

The mechanism of high concentrate diet which induces milk fat depression in lactating dairy cows (Bos taurus) has not been well explained. The purpose of this study is to investigate the changes of metabolism of milk fat precursors in liver by high concentrate diet fed, and to reveal the mechanism of milk fat depression (MFD) in lactating dairy cows. Ten Holstein cows (25±5 days in milk) were divided into two groups and assigned to 2 dietary treatments: Control group (forage-to-concentrate ratio: 4∶6) or a high concentrate group (forage-to-concentrate ratio: 6∶4). The liver vascular cannula was performed after 20 weeks feeding followed by convalescence for 4 weeks. The following experiments were carried out: 1) Milk production was recorded, and milk fat content was tested once a week; 2) Fasting blood from portal and hepatic vein was collected to test the concentration of triglyceride (TAG) and free fatty acid (FFA); 3) Live biopsy samples were collected to detect mRNA expression of fatty acid metabolism related genes. Results showed that milk yield of high concentrate group was increased during 6~18 week, while milk fat content was significantly decreased after 18 weeks (P＜0.05) compared with control group. Net content outputs of TAG and FFA in high concentrate group were significantly (P＜0.05) or extremely significantly (P＜0.01) decreased compared with control group. The mRNA expression of sterol regulatingelement bindingprotein-1c (SREBP-1c), acetyl-coacarboxylase (ACC), fattyacidsynthetase (FAS) in liver of high concentrate group were significantly (P＜0.05) or extremely significantly (P＜0.01) decreased, while the mRNA expressions of peroxisome proliferators-activated receptor α (PPARα) and carnitinepalmitoyl transterase-1 (CPT-1) were significantly increased (P＜0.05) compared with control group. High concentrate diets decreased fatty acid synthesis but enhanced fatty acid catabolism, which led to the redistribution of milk fat precursors in liver, and decreased its output, and affected milk fat synthesis. This study revealed the mechanism of high concentrate diet fed inducing milk fat depression in lactating dairy cows, and provides a theoretical basis for improving milk fat content.

Hot shock protein 90 (Hsp90) regulates multitudes of biological processes, such as cell metabolism, normal development and environmental stress tolerance. Herein the full-length cDNA and the corresponding gene of the heat shock protein 90, named Ab-hsp90, were isolated and characterized in the plant parasitic nematode Aphelnchoides besseyi, the agent of the rice white-tip disease. The full-length Ab-hsp90 cDNA contained a 5' UTR of 69, an ORF of 2 139 bp encoding a polypeptide of 712 amino acids and a 3' UTR of 137 bp. The deduced amino acid sequence of Ab-hsp90 showed that high similarity with other known Hsp90s, and the Maximum Likehood tree indicates Ab-hsp90 was located in the clade of plant nematodes. Five conserved amino acid signature indicated that Ab-hsp90 was a cytosolic member of the Hsp90 family. The gene consisted of 9 exons and 8 introns. Expression levels of Ab-hsp90 were determined by quantitative real-time PCR. Ab-hsp90 mRNA was constitutively expressed in all developmental stages of A. besseyi, but the highest transcript abundance existed in egg and the adult nematodes. Exposing the nematode to desiccation condition, relatively low osmotic stress and short-term abamectin solution promote Ab-hsp90 transcription. Ab-hsp90 was also induced in the nematodes under the low temperature (4 ℃) at 1.5 h but continuously expressed in high temperature (40 ℃) in 18 h. Interestingly, differential expression of Ab-hsp90 was detected in A. besseyi infecting the resistant and rice seedlings in 72 h. A burst of expression of Ab-hsp90 was present in the inoculated nematodes on the resistant rice seedlings, while its expression was upregulated in 48 h and then dropped at 72 h in the susceptible rice. Moreover, although the differential expression along with the developmental stages of A. besseyi, the mRNA level of Ab-hsp90 was also largely induced in the nematodes feeding on the callus of Daucus carota and hyphae of Botrytis cinerea in 9 d. Our data suggested that Ab-hsp90 can assist A. besseyi to cope with environmental stresses, infect plants and feeding. The results may provide a new perspective to dissecting the functions of hsp90 proteins in plant parasitic nematodes, which thus is conducive to the further study of nematode and plant interactions.

The purpose of this research was to screen high efficient decomposing fungi for practical applications to accelerate the decay of moso bamboo (Phyllostachys edulis) stump, and analyze the changes of lignocellulose enzyme activities and gene expression during the decomposing of bamboo sawdust. Lignocellulose enzyme activity were detected primary on the plates containing congo red, aniline blue, and guaiacol for the 61 fungi strains isolated from soil of bamboo plantation (P. violascens). The results showed that 5 fungi strains (P17, P26, P68, P69 and P73) represented significantly higher lignocellulose enzyme activity. Three saprophytic fungi strains of them (P17, P26 and P69), which hold the higher efficiency to degrade the bamboo sawdust in terms of mass loss rate, were tested for their enzyme activities of lignin (laccase (Lac), lignin peroxidase (Lip)) and cellulose (endoglucanase(EG), cellobiohydrolase(CBH)), and gene expression of lignocellulose (cbhⅠ, laccase-like multicopper oxidase gene (lcc)). Based on morphological characteristics and sequence alignment analysis of rDNA-ITS region, the 3 fungi strains were Trichoderma virens, T. asperellum and Mucor racemosus for stains P69, P26, and P17, respectively. The mass loss rate of bamboo sawdust caused by T. virens, T. asperellum and M. racemosus were 20.56%, 17.66% and 13.11% after 20 days decomposition, respectively. The activities of CBH and Lip reached its peak on the 12th day during the decomposition, while Lac activity reached its peak on the 9th day for all the three fungi. The highest of the activities for EG, CBH, Lac and Lip were detected always in T. virens when the activities of 4 enzymes reached their peaks of the three fungi. The analysis of fluorescence quantitative PCR (qPCR) showed that the cbhⅠ gene expression number in T. virens (1.93×105~8.76×106 per microgram of RNA) was significantly higher than those in T. asperellum (1.29×104~4.64×105 per microgram of RNA) and M. racemosus (2.42×104~7.8×105 per microgram of RNA) at all points of observation. The expression number of lcc gene in T. asperellum (9.23×105~4.89×106 per microgram of RNA) was also significantly higher than that in T. asperellum (3.74×105~9.36×105 per microgram of RNA) and M. racemosus (8.91×104~8.16×105 per microgram of RNA) at all points of observation. The results of Pearson's correlation coefficient (r) analysis showed that Lac activity secreting by the 3 fungi was positively correlated with the mass loss rate of bamboo sawdust during decomposition. At the same time, there was a certain synergy between EG and CBH of the 3 fungi stains. However, EG activities secreting by M. racemosus was positively correlated with the mass loss rate of bamboo sawdust, which be differ from the other 2 stains, suggesting there are differences in species characteristics of fungi during decomposing bamboo sawdust. Moreover, the expression of cbhⅠ gene showed a positive correlation with CBH activity of the three fungi during decomposing bamboo sawdust. These results indicated the capacity of decomposing bamboo sawdust for the three saprophytic fungi from higher to lower was as follows: T. virens, T. asperellum and M. racemosus, and Lac played an important role during the decomposing process.

In order to construct a Hemagglutinin-stem based universal vaccine and investigate its immunogenicity, the hemagglutinin (HA) gene of A/Puerto Rico/8/1934(H1N1) virus was used as template in this study to amplify the stem region, namely HA2(main protein of HA stem region) and HA-stem (whole stem region). The amplicons were inserted into pCAGGS vector, and the constructed recombinant plasmids were transiently transfected into HEK-293T cells. Then, the proteins expression was identified by RT-PCR and indirect immunofluorescence (IFA) assay. 6~8 weeks female BALB/c mice (Mus musculus) were divided into 2 recombinant plasmids group, pCAGGS group, and PBS group. The mice were intramuscularly inoculated twice at 0 and 30 d . Orbital blood was sampled at 0 and 60 d post immunization, and subjected to HI antibody titration, neutralizing antibody titration and INF-γ detection. The results showed that recombinant plasmids pCAGGS-HA2 and pCAGGS-HA-stem were successfully constructed, and could normally express target proteins in HEK-293T cells. pCAGGS-HA2 and pCAGGS-HA-stem induced antibody against A/Puerto Rico/8/1934(H1N1)in mice with HI titre 5 log2, and neutralizing antibody 1∶27 and 1∶24, respectively. While HI titre and neutralizing antibody against A/Chicken/Hebei/4/2008(H9N2) was 3 log2 and 6 log2, 1∶24 and 1∶22. The INF-γ level in pCAGGS-HA2 and pCAGGS-HA-stem group was (2362.40±98.24)ng/Land (2497.06±120.44) ng/L.The antibody titre and INF-γ level induced by pCAGGS-HA2 and pCAGGS-HA-stem was significantly higher than those induced by control group(P＜0.05). It indicates that pCAGGS-HA2 and pCAGGS-HA-stem can induce cross reaction with heterologous influenza virus, and may be a hopeful candidate universal influenza virus.

The goal of this study was to determine the differences in morphology and microbiota among 5 intestinal sections (duodenum, jejunum, ileum, cecum and rectum) of Shan partridge ducks (Anas platyrhynchos). Ducks were randomly divided into control and treatment groups, fed with basal diet and basal diet with 2% fish oil for 28 days, respectively. Histological staining and 16S rDNA V3–V4 region sequencing were applied to examine the morphology and microbiota of intestines. The highest ratio of villus height to crypt depth (VH/CD) was found in the ileum, followed by the rectum, whereas goblet cell count (GCC) was the highest in the rectum, with no differences detected between the other sections. Dietary supplementation with fish oil significantly reduced the VH/CD in the ileum, but had no significant effects on the other intestinal sections. Operational taxonomic units (OTUs) were the lowest in the duodenum and the highest in the ileum, but supplementing with fish oil reduced the number of OTUs overall. Alpha diversity analysis indicated that the lowest microbiotic diversity occurred in the duodenum, and that fish oil significantly reduced the diversity of the rectal microbiota. Taxa of Lactobacillus, Clostridia, Megamonas and Campylobacterales were chosen for further analysis on the results of linear discriminant analysis coupled with effect size (LEfSe). The results indicated that of the intestinal sections, the ileum is the primary site for digestion and absorption in Shan partridge ducks, as this section exhibited optimal morphology and contained the largest number of microorganisms that participate in these processes. Dietary supplementation with 2% fish oil compromised intestinal morphology and reduced the richness and diversity of the microbiota as a whole. This study provided reliable evidences for comprehensively evaluating the intestinal morphology and microbiota of Shan partridge duck, and the intestinal research foundation of Shan partridge duck supplemented with fish oil as well.

Fibroblast growth factor 10 (FGF10) is a critical growth factor, which can promote the differentiation of Mus musculus and Homo sapiens preadipocytes. In order to clarify the expression profile of FGF10 gene of Capra hircus, elucidate its expression changes during intramuscular preadipocyte differentiation, and analyze the correlation between gene expression and intramuscular fat deposition, Jianzhou Big-Eared goat (C. hircus) was used as laboratory animal in this study and the C. hircus intramuscular preadipocytes were obtained usingⅡcollagenase. Reverse transcription PCR (RT-PCR) was used to clone FGF10 gene and Real-time quantitative PCR (qPCR) was used to detect the expression level of genes. The expression level of FGF10 was detected both in different tissues and in various stages during C. hircus muscle development, including stages of kid, youth and adult. Besides, the temporal expression profile of FGF10 during intramuscular preadipocyte differentiation was also constructed. Following the determination of intramuscular fat (IMF) content which was performed by soxhlet extraction method, a correlation between FGF10 mRNA expression and IMF content was built in longissimus dorsi. The full-length sequence of the Jianzhou Big-Eared goat FGF10 gene was 1 252 bp including a complete 642 bp ORF flanked by 166 bp 5' untranslated region (UTR) and 444 bp 3' UTR, encoding 213 amino acids (Accession no. KT899958). The amino acid identity of FGF10 protein between the C. hircus and Ovis aries, Bos Taurus, Rattus norvegicus and Homo sapiens was 100%, 100%, 97% and 94%. The secondary structure prediction of C. hircus FGF10 protein showed that 12.68% α-helix, 27.70% extended strand and 59.62% random coil might be formed. The FGF10 protein of C. hircus possessed a transmembrane region and a FGF region, as well as a signal peptide structure whose cleavage site was between Ala38 and Leu39. Phylogenetic tree analysis showed that FGF10 was conserved among different species. FGF10 widely expressed in all examined tissues of Jianzhou Big-Eared goat, including heart, liver, spleen, lung, kidney, adipose tissue, longissimus dorsi, biceps femoris and triceps brachii. The highest expression level of FGF10 was found in lung which was significantly higher than that in other tissues (P＜0.05). And it also highly expressed in spleen and adipose tissue while less expressed in heart, longissimus dorsi and triceps brachii. The qPCR results also showed that FGF10 continuously expressed during the growth of longissimus dorsi. And it's expression level was increased with the muscle development and reached the highest in adult goat, significantly higher than in kid and youth (P＜0.05). Correlation analysis result showed that the expression level of FGF10 in longissimus dorsi of C. hircus was significantly positively correlated with IMF content (P＜0.05). The mRNA expression of FGF10 in the intramuscular preadipocytes was the lowest and reached the highest at 2th day after the induction of differentiation. Then the mRNA level decreased significantly in 4 d (P＜0.05), but gradually increased again in the following days. These results provide theoretical basis for further elucidating the molecular mechanism of FGF10 gene in regulating C. hircus IMF deposition.

Single sequence repeat (SSR) has the advantages of high polymorphism and comparability, great reproducibility and co-dominance, and has been widely used in genetics and breeding of vegetable crops. In this study, the SSR loci information of spinach (Spinacia oleracea) was analyzed, the SSR molecular markers were developed and genetic diversity of different spinach materials were analyzed. The study used the high generation inbred lines 1012 as the test material, the spinach tissues of flowers, roots, stems and leaves were collected at the flowering stage, and the Illumina HiSeqTM 2500 technique was used to sequence the genes expression. A total of 44 796 unigenes were screened by using MISA software from Spinacia oleracea transcriptome, 2 045 SSR loci occurred in 1 940 unigenes, 1 835 unigenes contained a single SSR loci, 105 unigenes contained 2 or more than 2 SSR loci, and the frequency of these SSRs was 4.33%, and the mean distance was 19.81 kb. Among all SSR loci of Spinacia oleracea, mononucleotide, trinucleotide and tetranucleotide repeat were major types, accounting for 19%, 65% and 7% of the total SSR, respectively, and the repeat types of dinucleotide, pentanucleotide and hexanucleotide were lesser, and the distribution frequency of these types were 0.18%, 0.10% and 0.17%, accounting for 4%, 2% and 3% of the total SSR, respectively. The average repeat length was 21 bp, and the main repeat length was 18~24 bp, accounting for 93.55% of the total SSR, the secondly repeat length was 25~30 bp, accounting for 4.11% of the total SSR，and number of SSR repeat length at 31~39 bp and ≥40 bp were fewer. The main number of repeats was 6 and 7, accounting for 31.42% and 31.05%, the secondly number of repeats were ＞12 and 5, accounting for 19.17% and 8.41%, respectively, the number of repeats was 8~12. The mononucleotide repeat motif of A/T was the predominant repeat types, dinucleotide repeat motifs of GA/TC and AG/CT were the predominant repeat types, trinucleotide repeat motifs of CAA/TTG, GTT/AAC, GAA/TTC, TGT/ACA and GAT/ATC were the predominant repeat types. The repeat motifs of tetranucleotide, pentanucleotide and hexanucleotide were dispersed, each repeat motifs were relatively lower. All 218 primers of 2 199 SSR primers were selected randomly for PCR amplification using 9 Spinacia oleracea materials with different sources, including 38 SSR markers which performed polymorphical bands, accounting for 17.43% of the total test primers. The 9 materials were divided into 3 major groups by UPGMA, and the first major group included 4 materials, which were 11231, 1408, 1439 and 13190, the second major group included 2 materials, 1012 and 09248, and the third major group included 3 materials, 0928, 8301 and 1723. The SSR information and obtaining abundant polymorphisms SSR markers were studyed, which can be used for the genetic diversity, genetic mapping construction and molecular marker-assisted breeding in Spinacia oleracea.

When plants of annual herb are being invaded by a pathogen, the gene of salicylic acid methyltransferase (SAMT), which converts salicylic acid (SA) produced at the inoculation site into methyl salicylate (MeSA), plays an important role in plant systemic acquired resistance (SAR) and signal transduction of SA. However, the function of SAMT in perennial woody plants needs to be further verified. In this study, a pair of primers was designed according to the complete CDS of Populus trichocarpa SAMT and was used to amplify the SAMT gene of 84K popular by polymerase chain reaction. Under Gateway technology, we performed a BP recombination reaction between the attB-flanked SAMT fragment and an attP-containing donor vector pDONRTM222 with the action of BP clonase enzyme to convert the gene of interest into entry vector. Then the vector were transformed competent Escherichia coli DH5α. Plasmids, which were extracted and digested by MLU Ⅰ enzyme, and over-expression vector pMDC32 were performed an LR recombination reaction with the action of LR clonase enzyme to transform the SAMT into expression vector. Successfully over-expression vector of SAMT gene were constructed, and SAMT-overexpressing transgenic 84K popular plants were obtained by Agrobacterium-mediated genetic transformation using leaf-disk transformation method. Then transgenic lines were tested and analyzed by RT-PCR and qPCR methods. From the results, leaf-disk transformation is appropriate transformation method, with high conversion efficiency. Eccept one line of the SAMT -overexpressing genetically modified lines is lower, 57 times more than CK. Compared with control, other lines of transgenic 84K popular plants are more than 3 729 times. These results laid the foundation for the future research of the gene function and SAR in poplar and other roles, as well as provided the reference about the function of the gene in other perennial woody plants.

Lignin is a complex, noncrystalline polymer, which is one of the important components of the pear stone cells. Stone cells are a type of brachysclereid, contain abundant lignin and cellulose, which formed by the secondary deposition of lignin on the primary walls of parenchyma cells. The synthesis, transfer and deposition of lignin is closely related to the development of stone cells. Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) catalyzes the hydroxylation of trans-cinnamic acid to 4-hydroxycinnamate and is the second key enzyme of common phenylpropanoid pathway. C4H gene belongs to CYP73A subfamily of cytochrome P450-dependent monooxygenase superfamily, and specifically converts the trans-cinnamic acid into p-coumaric acid. In this study, the C4H (designated as PbC4H) from Pyrus bretschneideri cv. Dangshan Su fruit was cloned. The full-length PbC4H cDNA (GenBank No. KF663548) was 1 764 bp, 5'-UTR was 85 bp, 3'-UTR was 164 bp and contained 26 bp PolyA tail, which also with an ORF of 1 515 bp encoding a putative protein with 504 amino acid residues. Functional structural analysis showed that PbC4H had a transmembrane domain, and T binding groove motif A306AIETT311, E363-R366-R420 triplet, the necessary proline hinge structure P34PGPIPVP41 for the enzyme to correct orientation. In addition, PbC4H had 5 characteristic substrate binding site motifs (substrate recognition sites and SRSS): SRS1(S100RTRNVVFDIFTGEGQDMVFTVY),SRS2(L216AQSFDYNY), SRS4(I299VENINVAAIETTLWS), SRS5 (R368MAIPLLVP) and SRS6(K484GGQFSLHI). Homology comparison indicated that the amino acid sequence of PbC4H was highly homologous to the sequences of amino acids encoded by other C4H, indicating that C4H gene conservation during evolution. In order to further clarify the phylogenetic relations between PbC4H and other species C4H, a phylogenetic dendrogram was constructed using MEGA5.1 software and found that Rosaceae plant Pyrus bretschneideri, Malus and Prunus C4H clustered as a class. qRT-PCR analysis showed that PbC4H expression increased first and then decreased during the fruit development. The expression levels of 23~63 days after flowering was higher than that in other stages, expression peak of PbC4H was observed at 55 days after flowering. The change trends were consistent with the changes of lignin and stone cell content during fruit development, which suggested that there was a significant relationship between them. PbC4H was constructed into prokaryotic expression vector of pET-32a, and the recombinant PbC4H expressed in Escherichia coli strain BL21. The induced protein, which molecular weight is consistent with the predicted molecular weight. We constructed eukaryotic expression vector to identify its subcellular localization. PbC4H was observed to be localized on membrane by subcellular localization analysis. This study provides a theoretical basis for the regulation of the synthesis of lignin and the development of stone cells in pear by using molecular biology technology.

The cut-and-paste piggyBac (PB) transposon system has been shown to be a useful tool for genetic modification in a number of organisms including mammals, insects, and yeast. However, little is known whether it is active in the green microalga Chlamydomonas reinhardtii. In this study, we constructed stable PB element-containing C. reinhardtii strains bearing either a single copy or multiple copies of PB element. Subsequently, we transiently expressed TPase or codon-optimized crTPase in the PB element-containing strains to assay for transposition of PB element using restriction fragment length polymorphism (RFLP) methodology. Our analyses show that initial RFLP patterns of PB element are altered in cells one week and two weeks after plasimds containing TPase or crTPase expression cassette. The size of PB element-containing fragment remained unchanged in TR1 cells after transformation of the pJR38 plasmid containing no TPase or crTPase sequences. This result indicated that PB element was stable without TRpase or crTPase activity in cells. On the contrary, RFLP patterns of the PB element were altered 1 week and 2 weeks after transient expression of TPase or crTPase in TR1 or TR3 cells. For example, a fragment of 1.5 kb containing PB element was present in TR1 prior to transformation of TPase-containing plasmid. One week after transformation, one initial fragment of 1.5 kb and 3 new fragments of 2.8, 4.2 and 6.5 kb disappeared and appeared, respectively, suggesting transposition of the PB element occurred in TR1 cells. Two weeks after transformation, the size of all 3 fragments changed again, implying that transposition persists. In TR3, 1 week after the transformation, 3 fragments of 1.5, 3 and 4 kb and 1 fragment of 6.5 kb remained unchanged and disappeared, respectively. Meanwhile, a new fragment of 2.8 kb appeared. The RFLP pattern was hardly altered again 2 week after the transformation. These results indicated that the sizes of PB element-containing fragments changed, implying the PB element transposed after introducing the TPase and crTPase activities in C. reinhardtii TR1 and TR3 strains. Furthermore, to test whether PB element had been transposed in these cells, we examined the presence of a new joint generated by excision of the PB element in the genome. To this end, primers flanking to the new joint were applied for PCR analysis using the genomic DNA derived from the colonies. Clearly, excision event of the PB element was detected. This result supports the results that PB transposon system is active in C. reinhardtii. To find out the reason that the cells could survive the transposition of PB element, PCR fragments containing the PB element excision joint were subjected to nucleotide sequence analysis. Alignment of the sequences indicated that cells from the colonies suffered from illegitimate excision of PB element. The study shows that illegitimate excision of PB element occasionally occurs at the short repeat sequences 5'-TTT-3' and 5'-ACGCAG-3', but the standard excision site 5'-TTAA-3'. This study shows that the TPase and crTPase are active in transposition of PB element in C. reinhardtii. Hence, we propose that PB transposon systems can be adopted for genome modification in C. reinhardtii.

Abstract In order to investigate the molecular mechanism of salt tolerance, the proteome in leaves of 30 days seedling age southern type alfalfa (Medicago sativa 'Millenium') under control condition were compared with those under 72 h treatment at 250 mmol/L NaCl. Isobaric tags for relative and absolute quantitation(iTRAQ) technique with and 2-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS /MS) were used to identify the differentially expressed proteins in southern type alfalfa leaf under salt stress, biological analysis on differentially expressed proteins was conducted to some of the potential target proteins to salt stress. The results indicated that 3 712 quantitative proteins were identified, 417 differentially expressed proteins (fold change ratio≥1.2, P≤0.05) were identified, in which 291 proteins were up-regulated and 126 proteins were down-regulated. The identified proteins were then categorized based on their sub-cellular localizations and biological functions using Gene Ontology (GO). Significantly enriched the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway were related to metabolic pathways, biosynthesis of secondary metabolites, pagosome, fatty acid metabolism, photosynthesis and so on (P＜0.05, false discovery rate(FDR)＜0.05). The identified differentially expressed proteins were involved in photosynthesis(7%), signal transmission(3%),oxidation resistance(6%), defense (2%), protein synthesis, processing and degradation(18%), energy and transport(7%), carbohydrate metabolism(11%), amino acid metabolism(7%), lipid metabolism(5%), other metabolism(7%), membrane and intracellular transport(7%) and cell structure, division and cytoskeleton(3%) and function unknown protein(16%). Functional analysis of the differentially expressed protein species showed an generally increased abundance of proteins related to oxidation resistance, energy and transport, defens and signal transmission, metabolism and a decreased generally abundance of proteins related to protein synthesis, processing and degradation, metabolism and photosynthesis. The results also showed that cytochrome P450, oxygen-evolving enhancer protein, phosphatidylinositol-specific phospholipase C, fructose-1,6-bisphosphate aldolase, trehalose-6-phosphate synthase, aspartate aminotransferase, E3 ubiquitin ligase, vacuolar H+-ATPase subunit C and so on were potential target proteins in alfalfa in response to salt stress. Our results suggested that differentially expressed proteins in southern type alfalfa leaf to salt stress can be effectively screened by iTRAQ technique combined with 2D-LC-MS/MS, may play roles in alfalfa adaptation to salt stress. Further study of these proteins will provide theory evidences into the molecular mechanisms of salt stress in alfalfa.

MicroRNAs are a class of tiny, single-stranded noncoding RNAs that can target mRNA via base pairing and lead to translational repression or mRNA degradation at the level of gene posttranscription, which are involved in organ development, cell differentiation, proliferation, apoptosis and other biological processes, closely related with many diseases. Basic leucine zipper and W2 domains 2 (BZW2) functions in protein synthesis metabolism. The purpose of this study was to validate whether miR-122 could inhibit BZW2 gene expression in chicken (Gallus gallus). The bioinformatics method was used to investigate whether miR-122 could pair chicken BZW2 3'-UTR, furthermore dual-luciferase report system and mutant assay were used to validate that miR-122 repressed BZW2 expression. qRT-PCR was used to detect the expression level of BZW2 in leghorn hepatocellar (LHM) cells transfected with miRNA-122mimic and primary chicken hepatocytes transfected with LNA-antimiR-122. Bioinformatics prediction and dual luciferase report system proved that BZW2 was target gene of miR-122, target site mutation assay showed the complementary site of 3'UTR with miR-122 seeds region were target site of miR-122. After overexpression of miR-122 in leghorn hepatocellar (LMH) cells, BZW2 mRNA expression level decreased obviously. Using LNA-antimiR-122 to knockdown the miR-122 expression in primary chicken hepatocytes, the target gene BZW2 mRNA expression level increased significantly. These data suggested that BZW2 gene was target gene of miR-122 and BZW2 mRNA was negatively regulated by miR-122 in chicken hepatocytes. It will provide scientific basis on revealing a wide range of functions in the liver of miR-122 in chicken.

Start codon targeted polymorphism (SCoT) is a single primer gene targeted marker technique, and the primers are designed according to conservative areas from the flanking of translation initiation site (ATG). After the first pioneer successfully applied this technique to analyze the gene differential expression in sugarcane (Saccharum officinarum), cDNA-SCoT has been applied in many kinds of plants, such as peanuts (Arachis hypogaea), corn (Zea mays), Dendrobium officinale, mango (Mangifera indica), watermelon (Citrullus lanatus), etc. In this article, the principle, methods of designed primers and the characteristics of cDNA-SCoT were introduced. Then, the screening and optimization of cDNA-SCoT PCR system, as well as its application in disease resistance, cold resistance, drought resistance, genetic evolution, etc, were emphatically summarized. This article provides a new technology of gene differential expression, which can be used as an effective complement to other technologies, and a reference for researchers to choose different technologies when they analyze gene differential expression.

Processing bodies (P-bodies) are distinct foci within the cytoplasm of the eukaryotic cell consisting of many enzymes involved in mRNA turnover. P-bodies have been observed in somatic cells originating from vertebrates and invertebrates, plants and yeast. To date, P-bodies have been demonstrated to play fundamental roles in general mRNA decay, nonsense-mediated mRNA decay, adenylate-uridylate-rich element mediated mRNA decay, and microRNA induced mRNA silencing. Not all mRNAs which enter P-bodies are degraded, as it has been demonstrated that some mRNAs can exit P-bodies and re-initiate translation mRNAs are temporarily stored in stress granules. Stress granule is a similar granule of P-body. Stress granules are dense aggregations in the cytosol composed of proteins & RNAs that appear when the cell is under stress. The purpose of stress granules might be to protect RNAs from harmful conditions. Both P-bodies and stress granules play an important role in Post-transcriptional gene regulation. Fully grown mouse (Mus musculus) oocytes in Graafian follicles are transcriptionally silent during the period before the resumption of meiosis until after fertilization when most of the transcriptional reactivation occurs at the 2-cell stage. Products of the oocyte genome support oocyte growth and development before silencing and are also stored for use during the silent period to support oocyte maturation and the early stages of preimplantation embryo development. However, the maturation of oocytes needs a lot of proteins which are mainly generated from the translation of maternally stored mRNA. During the maturation of oocytes, different gene transcriptions expressed at specific time and precise regulation are vital. Oscillatory degradation and resynthesis of cyclins drive the somatic cell through its cycle, and the oocyte utilizes these same processes to control levels of cyclin B1 throughout the period of prophase I arrest. In the fully grown GV oocyte that contains high cyclin B1 levels, ubiquitin-mediated proteasomal degradation of cyclin B1 becomes key to regulating CDK1 activity and is essential during the subsequent stages of meiosis I and II. Known for its role as the driver of anaphase progression in mitosis, the anaphase-promoting complex (APC) is a multimeric E3 ubiquitin ligase, responsible for the ubiquitylation of cyclin B1 and other substrates, which thereby targets them for degradation by the 26S proteasome. The activity and substrate specificity of the APC are regulated by its binding to one of two coactivator proteins that function in a reciprocal manner to coordinate the different phases of the cell cycle. In somatic cells, there are two ways to regulate one gene expression, namely transcriptional regulation and post-transcriptional regulation. The cooperation of both two regulations ensure an orderly life activities in cells. On the contrary, transcriptional regulation does not exist during the maturation of oocytes. Post-transcription regulation is the only way to regulate one gene. Here, we review P-bodies and their function, discuss the function of maternal P-bodies in mouse oocyte maturation, come up with new models about how mRNAs are stored in maternal P-bodies and how mRNAs released from these granules, put forward that oocytes are novel model for the research of post-transcriptional regulation.

Excess accumulation of lipids in the adipose tissue leads to obesity, which is associated with cardiovascular diseases, type Ⅱ diabetes and other pathologies. Adipogenesis is the process by which precursor stem cells differentiate into lipid laden adipocytes. Adipogenesis is regulated by a complex and highly orchestrated gene expression program. In order to discovery key regulatory genes and pathways in adipogenesis and explore the molecular regulation mechanism of adipogenesis, in the previous study, we constructed a Adipogenesis Regulation Network (ARN) Database (http://210.27.80.93/arn/) by mining over 9 000 papers related to adipogenesis, which contains more than 30 000 adipogenesis related data and information. In order to fully explore the potential value of ARN database to promote the study of adipogenesis, in this study, we designed an online analysis tool "ARN-analysis" to analyze the experimental data or to construct the scientific hypothesis about adipogenesis through two hypothesis construction processes: "open" and "closed". Furthermore, by count the number of the relation records, the number of the expression records and the number of the prediction records for each node (gene and micro RNA), the impact factor (IF) value which reflects the importance of each node was calculated. Finally, by analyzing and mapping the number of the relation records of nodes, the topology of the adipogenesis regulation network was explored. The results showed that the analysis tool of ARN database can effectively analyze 3 kinds of data: "node" and "expression" and "user input", it would be useful for researchers to analyze test data or build scientific hypotheses. Understanding of the regulation network topology can deepen our understanding of the mechanism of the formation of fat. In this study the analysis and prediction functions of ARN database was explored, ARN provided a new way for professional researchers to analyze data and construct hypotheses, explored the possibility of using a large number of scientific research data accumulated in the past to promote future research and practice.