bro (baculovirus repeated ORF)-d gene of Bombyx mori nucleopolyhedrovirus (BmNPV) is a kind of repeated open reading frame sequence in baculovirus and is widespread in a variety polyhedrosis virus genome which specially infects Lepidopteran insect. In order to understand the role of bro-d gene in viral infection and the relationship with host cell apoptosis, the bro-d gene was knocked out by Red recombination system to construct the bro-d-deficiency type virus (bro-d-ko-Bacmid), and was encapsulated by liposome and then was transfected into BmN cells, and wild type bacmid (Wt-Bacmid) was used as the control. The effects of bro-d gene on the replication and transcription level of virus genome and inhibition of apoptosis protein 2 (iap2) were detected by qRT-PCR, and the results showed that bro-d gene deletion would lead to a significant decrease on the replication level of viral genome and the transcription levels of viral early gene lef-3, late gene vp39 and very late gene p10 would be also significant decrease (P＜0.05). These results indicated that bro-d gene was a nonessential gene for BmNPV replication and it was involved in the regulatory process of viral gene in different transcription phase. In order to further explore the effects of bro-d gene deletion on virus-induced apoptosis of host cells, the bro-d-ko-Bacmid was co-transfected into BmN cells with the transient expression plasmid pIEx-1-bro-d which contained the IE promoter which only triggered in eukaryon. The results showed that the transcription level of iap2 was decreased significantly (P＜0.05) in the cells transfected with bro-d-ko-Bacmid and was increased after cotransfected with pIEx-1-bro-d. Furthermore, in spite of the viral replication and transcription levels of bro-d-ko-Bacmid were significantly lower than that of Wt-Bacmid (P＜0.05), and bro-d-ko-Bacmid would cause a significant decrease in cell viability compared with Wt-Bacmid (P＜0.05). These results implied that bro-d deficiency would strengthen the virus pathogenicity. Moreover, the B-cell lymphoma-2 (Bcl-2) protein and Bax (Bcl-2 assaciated X protein) expression levels in post-transfected cells and the key regulatory proteins in mitochondrial signaling death pathway were detected by Western blot. The results further confirmed that bro-d gene deletion would lead to significant decrease in the level of Bcl-2 protein (P＜0.05) and significant increase in the level of Bax protein (P＜0.05) compared with wild type virus, which resulted in a increased apoptotic level of host cells. These results indicated that bro-d gene could not only up-regulate the gene expression levels of different phages, but also could down-regulate the apoptotic level of host cells through mitochondrial pathway by regulating the expression of iap2. These results provide a new basis for understanding the function of bro-d gene in virus infection cycle, and a new way to transform the baculovirus expression system by prolonging the host cell life-span, and improve the expression amount of target proteins.

Major histocompatibility complex (MHC), one of the vital functional gene families in vertebrates, encodes MHC molecule which has the function and ability to maintain the organism adapt to environmental changes from generation to generation, and is the best genetic marker for investigating adaptive evolution in animals. Tibetan sheep (Ovis aries), the main livestock resources in Qinghai-Tibetan plateau, was chosen as research object, and the genetic characteristics of DQA genes in 5 ecological Tibetan sheep populations from Gansu (Oula, Qiaoke and Ganjia type) and Qinghai (Oula and plateau type) were investigated using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) techniques in this study, which aimed to reveal its adaptive evolutionary mechanism in plateau Tibetan sheep. The results showed that 33 alleles and 125 SNPs were discovered in 2 genes of Tibetan sheep DQA locus. This indicated that the DQA genes in 5 ecological Tibetan sheep populations exhibited abundant polymorphism. Subsequent analysis demonstrated that balancing selection was one of the mechanisms to maintain the polymorphism of DQA genes in Tibetan sheep. The changes of DQA genes among groups in 5 ecological Tibetan sheep from Gansu and Qinghai were less than those among population with groups, suggesting that the DQA genes in Tibetan sheep generates positive selection and adaptive evolution. This research results will provide basic data for genetic improvement and germplasm innovation of Tibetan sheep.

The tripartite motif-containing protein (TRIM) family belongs to a E3 ubiquitin protein ligase with conserved structure and rapid evolution. They are involved in a variety of biological processes, such as cell regulation, cell differentiation, metabolism and innate immunity. TRIM36 is one of members of TRIM family. In this study, trim36 gene (GenBank No. KU244470) was RACE cloned in the half-smooth tongue sole (Cynoglossus semilaevis) based on the entire genome and transcriptome sequence. trim36 gene contained 2 899 bp, and the ORF was 2 214 bp, which encoded 737 amino acids, 289 bp of 5'-untranslated regions (UTR) and 396 bp of 3'-UTR. The bioinformatics analysis showed that the amino acid sequences contained a RING finger, 2 B-Box-type zinc finger (BBOX), a coiled-coil motif, a fibronectin type Ⅲ(FN3) and spla kinase and ryanodine receptor (SPRY) domains. In addition, trim36 and its adjacent genes had conservative genomic synteny in different vertebrates. The upstream of the trim36 gene appeared geranylgeranyl transferase type-1 subunit beta (pggt1b), coiled-coil domain-containing protein 112 (ccdc112) and Fem-1 homolog C (fem1c) in sequence. According to the alignment result of TRIM 36 amino acid sequence of different species, the TRIM36 protein sequence of Cynoglossus semilaevis presented the highest consistency with that of Larimichthys crocea, and the homology was 89%, followed by Stegastes partitus and Oryzias latipes (88%). Moreover, the consistency between Gorilla gorilla and Macaca mulatta was the lowest of 63%. The results of RT-PCR and qRT- PCR showed that trim36 mainly expressed in ovaries and brains, which was extremely higher than that of other tissues (P＜0.05). The results of gonad in different periods by RT-PCR and qRT-PCR showed that trim36 gene began to rise at 120 d, which was consistent with the time of ovarian cavity formation, and the ovary expression was significantly higher than that in testis (P＜0.05). The in situ hybridization results demonstrated that trim36 expressed in cytoplasm at stage Ⅰ and Ⅱ of oocyte. This specific expression pattern suggested that trim36 might play a role in ovarian cavity development and stageⅠand Ⅱ of oocyte formation in half-smooth tongue sole. This study provides clues and references to support further research on trim36 gene function and TRIM family function.

Photosynthesis-associated nuclear genes (PhANGs) are able to respond to multiple environmental signals which include light, drought, sugar and abscisic acid (ABA). In previous studies, several cis-elements related to light regulation have been identified in promoters of PhANGs. However, systematic research on cis-elements of PhANGs which are response to abiotic stress, such as drought and ABA, has not been conducted yet. Here, we performed a series of investigation that mainly aimed at the cis-element that located on ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene (RBCS) promoter and roles in abiotic stress response. Firstly, a 1 839 bp upstream sequence of the TaRBCS11 gene was isolated from the genomic DNA of wheat (Triticum aestivum). Sequence structure analysis showed that the transcriptional start site was located at 59 bp upstream of initiation codon (ATG). Then, the sequence was submitted to the PlantCARE database and the analysis result revealed that the sequence contained large numbers of light-related cis-elements such as BoxⅠ, G-Box, I-box and GATA-motif, and dehydration, salt and ABA response-related cis-elements such as MYB binding site (MBS), CCAAT-box, ethylene-responsive element (ERE) and gibberellin-responsive element (GARE-motif). Based on these results, the promoter regions of TaRBCS11, TaRBCS13, TaRBCS10 and TaRBCS14 that from the same subfamily had been isolated from wheat genome. The alignment indicated that these 4 promoter sequences shared highly identity ranged from 50.9% to 74.17%. Many light-responsive motifs such as MNF1, GATA-motif, I-box, G-Box and Sp1 were also observed at the relatively same position in the proximal region (-200 bp~ATG) of these promoters. In order to obtain the promoter deletion fragment, five forward primers (F1, F2, F3, F4 and F5) and one reverse primers (R1) were designed according to the distribution of the structure and expression element of the TaRBCS11 promoter sequence. PCR amplification was conducted and 5 deleted fragments in length of 1 656, 1 258, 866, 533 and 277 bp were obtained, respectively. Subsequently, the 5 different length of deletion fragments of TaRBCS11 promoter were fused with the β-glucuronidase gene (GUS) and Agrobacterium tumefaciens-mediated transient expression assay were conducted. The histochemical staining of GUS showed that the leaves with 35S and TaRBCS promoter were stained blue which indicated that all promoters possessed activity to drive GUS. Meanwhile, quantitative analysis of GUS activity showed that GUS activities decreased gradually as the length of TaRBCS promoters decreased from P1656 to P277 in the leaves. The result also indicated that light induction could enhance GUS activities. Furthermore, the deletion of light responsive element such as ATCT-motif, Box I, Box 4, G-Box, and chs-CMA2b could reduce GUS activities. Assay of tobacco (Nicotiana tabacum) leaves which harboured the complete promoter in the fusion construct indicated that P1656 was negatively regulated by salt, ABA, dark and especially drought. The 5' deletion analysis of TaRBCS promoter confirmed that a region between -804 and -474 bp included the drought and salt-response region, and ABA-response elements might be located in the region between -1 597 and -1 198 bp. Taken together, these results identified some potentially important regulatory element in the TaRBCS11 promoter, and confirmed that the cis-regulatory region that responsible for repression by drought, salt, ABA and light. The results provide important information for further study on the mechanism of PhANGs expression regulation.

Abscisic acid (ABA) can promote the strawberry (Fragaria × ananassa) fruit development and ripening, and inactive ABA glucose ester (ABA-GE) can be converted into free ABA by β-glucosidases (BG), therefore, BG is involved in the fruit development process. But there is no genetic evidence to reveal the mechanism of BG involved in fruit development. To understand the function of BG in the strawberry developmental stages, in this study, Sweet Charlie strawberry was used as test materials, and three genes encoding BG that were FaBG1, FaBG2 and FaBG3 were isolated through molecular cloning of PCR. Firstly, bioinformatics and temporal expression analysis of FaBGs were performed during strawberry fruit development stages, and the ABA contents were also measured. Secondly, molecular regulation of FaBG3 gene expression levels affected fruit endogenous BG activity, and fruit phenotypic changes, physiological indexes such as ABA content, pigments content, soluble solids content, and the fruit hardness were analyzed. In addition, ABA signal pathway, the pigment metabolism and cell wall metabolism related gene expression levels were analyzed. The results showed that BG protein in strawberry contained a region of BglB region, which was a specific region could hydrolyze other complexes. Three genes encoding the BG protein in strawberry were found, and had different expression levels. During the ripening of strawberry fruits, ABA content increased gradually, the expression level of FaBG1 gene was very low, in general the expression amount of FaBG2 gene was decreased, and only FaBG3 gene expression level was consistent with the ABA content and fruit development. Therefore, FaBG3 played a more important role in the regulation of ABA content. Using a technique of RNA interference to reduce the gene expression level of FaBG3 in strawberry fruit also downregulated FaBG1 and FaBG2 genes expression level, suggesting that ABA content was downregulated by FaBG3-interfere, and FaBG1 and FaBG2 expression were also influenced. FaBG3 gene expression levels down-regulation led to the reduction of fruit endogenous beta-glucosidases activity, which influenced the ABA accumulation. Meantime, ABA content changes also affected the physiological changes in strawberry fruit, such as pigment and soluble solid content were reduced, and the fruit hardness were up-regulated, which finally influenced the fruit ripening process. On the contrary, FaBG3 overexpression promoted the strawberry fruit ripening process. In FaBG3-RNAi fruit, the genes expression levels that related to pigment metabolism, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxy-lase (F3H), UDP Glc-flavonoid 3-O-glucosyl transferase (UFGT) and dihydroflavo-nol-4-reductase (DFR) genes, fruit softening, such as pectin methylesterase (PE), expansin (EXP), polygalacturonase (PG) and pectate lyases (PL) genes, fruit aroma metabolism, such as quinone oxidoreductase (QR) and alcohol acyltransferase (AT), and ABA signal pathway, such as pyrabatin resistance (PYR), ABA insensitive 1 (ABI1), ABI3, ABI4 and ABI5 genes were also down-regulated, however, a negative factor of ABI1 which involved in ABA signaling pathway was up-regulated. Due to the expression levels changes of these key genes related to fruit development and ripening, at last, the fruit development process were delayed. The fruit physiological and molecular index changes could reveal the mechanism of BG in regulation of strawberry fruit development.

The Tibetan antelope (Pantholops hodgsoni) is endemic to the Tibetan Plateau and becomes endang ered species, which is national class Ⅰprotected animals in China. Inter-species somatic cell nuclear transfer (iSCNT) would be a possible rescue strategy for these species. iSCNT has been regarded as a potential alternative for rescuing highly endangered species and can be used as a model for studying nuclear-cytoplasmic interactions. However, iSCNT embryos often fail to produce viable offspring. In this study, the Tibetan antelope fibroblasts were treated by the 4 classic mouse (Mus musculus) transcription factors of octamer-binding transcription factor 4 (OCT-4), sex determining region Y -Box 2 (SOX2), Kruppel-like factor 4(KLF4) and (avian myelocytomatosis viral oncogene homolog (C-MYC) to induce the cell reprogramming. The pluripotent gene expression, cell growth curve and cell cycle of the induced cells were detected. The induced cells (iPS-ZLY) were then used as donors to transfer to the enucleated bovine (Bos taurus) oocytes. The results showed that the induced Tibetan antelope fibroblast cells (iPS-ZLY) had numerically higher cell mass in the G2-M stage than the Tibetan antelope fibroblasts (ZLY)(21.5% vs 16.7%). Cell growth curve indicated that iPS-ZLY cells proliferated faster than the ZLY cells, iPS - ZLY cells gone into the growth of the plateau in 4 d, while the ZLY cells gone into a plateau in 6 d. The karyotypes of both iPS-ZLY and ZLY cells were similar (68.3%vs 69.6%). The pluripotent gene OCT-4 expressed only in the iPS-ZLY. When transfered of the iPS-ZLY and ZLY cells, irrespectively to enucleated bovine oocytes, the iPS-ZLY cells resulted in 2.4% cloned blastocyst development, while the ZLY cells yielded 0.95% cloned blasatocysts. These results suggested that treatment of Tibetan antelope fibroblast cells with transcriptional factors enhanced cell proliferation, induced pluripotent gene expression and improved inter-species cloned embryo development. This was the first report using the 4 mouse transcription factors to induce the Tibetan antelope somatic cells, and then to evaluate the potential development of the iSCNT reconstructed embryos, resulted from the induced cells. This research provides an alternative method to increase the reprogramming of endangered animal cells and probably is beneficial for endangered animal protection.

Premature bolting will cause serious losses to the production of Chinese cabbage (Brassica rapa ssp. pekinensis). The molecular mechanism involved in bolting and flowering is important for the improvement of late bolting Chinese cabbage varieties. The late and early Chinese cabbage-cabbage translocation lines of AT4 series added No. 4 chromosome fragments from cabbage (Brassica oleracea var. capitata) under Chinese cabbage background were used as materials in this experiment, in which a total of 8 differentially expressed transcript-derived fragments (TDF) between late and early bolting translocation lines were screened by cDNA-amplified fragment length polymorphism (cDNA-AFLP). After being sequenced and BLAST, homologous alignment and function analysis indicated that 8 TDFs included 3 types: transcription translation, signal transduction and membrane transport category. In function prediction of TDFs, cellular signal transduction and material transport, material and energy metabolism, stress response, gene regulatory and expression and cell cycle were involved. In order to confirm whether and/or how genes expression changed and to evaluate the relationship between time of bolting and TDFs expression level, the 8 TDFs were further analyzed by qRT-PCR for 3 extra-late bolting Chinese cabbage-cabbage translocation lines, 3 extra-early bolting Chinese cabbage-cabbage translocation lines, one extra- late bolting inbred lines, one extra- early bolting inbred lines, one bolting resistance Chinese cabbage variety and one extra-early bolting Chinese cabbage variety. These materials by vernalization treatment at 0, 7, 14 and 21 d. The results showed that the expression peaks of extra-late bolting lines were significantly higher than that of the extra-early bolting lines in fragments of P65M58-11, P65M58-12 and P75M47-9, respectively. The expression level of P65M58-11 and P65M58-12 were the highest after vernalization treatment for 14 d. In extra-late bolting lines, the expression level of P75M47-9 were the highest in the 7 d of vernalization treatment. But the expression in extra-early bolting lines presented down-regulated trend from vernalization treatment initiation. The expression peaks of 4 TDFs, such as P77M58-9, P77M58-12, P63M49-8-1 and P63M61-4-2, were significantly lower in the extra-late bolting lines than that in extra-early bolting lines. The expression peak of P63M49-8-1 was at 7 d of vernalization treatments in the extra-early bolting lines. However, the expression of extra-late bolting lines showed gradually increased of 7~21 d vernalization treatments. The expression peaks of P77M58-9, P77M58-12 and P63M61-4-2 were at 14th d of vernalization treatments. The expression of P63M49-1-2 had no significant differences between the extra-late and extra-early bolting lines. In the extra-early bolting lines, the expression peak of P63M49-1-2 appeared at 7th d of vernalization treatments. However, in extra-late bolting lines the expression peak appeared at 21th d of vernalization treatments. In this research, we identified 8 genes with different expression level and expression patterns between extra-late bolting lines and extra-early bolting lines for 6 Chinese cabbage-cabbage translocation lines, 2 inbred lines and 2 Chinese cabbage varieties. These results will lay the foundation to further discover genes related to bolting and flowering, validate genes function, and obtain late bolting materials in Chinese cabbage.

FK506-binding proteins (FKBPs) are known as the receptor for the immunosuppressant drug FK506. FKBPs are ubiquitously distributed in all living organisms and highly conserved during evolution. FKBPs have peptidyl-proly cis-trans isomerase (PPIase) activity and can be used as molecular chaperone to have a function in protein folding. Plant FKBPs take part in diversity of cellular processes including plant growth and abiotic stress responses. In this study, TaFKBP62c-2B was isolated from wheat (Triticum aestivum) by RT-PCR and the expression pattern of this gene was analyzed through qRT-PCR. The subcellular localization of TaFKBP62c-2B was also investigated by using the green fluorescent protein (GFP) method. The sequencing analysis showed that TaFKBP62c-2B gene consisted of 1 926 bp CDS, 37 bp 5' UTR and 176 bp 3' UTR, and encoded 642 amino acids (GenBank accession No: KU350629). The amino acid sequence analysis demonstrated that TaFKBP62c-2B had 3 conserved FK506 binding domains (FKBd), which were respectively named 62c-2BⅠ, 62c-2BⅡand 62c-2Ⅲ, and 3 tetratricopeptide repeats (TPR), so TaFKBP62c-2B belonged to multiple-domain FKBPs. The amino acid alignment analysis showed that 62c-2BⅠ was the most similar conserved domain with HsFKBP12a and should be the key domain for the PPIase function of TaFKBP62c. The 9 homologus genes from 9 different species of Aegilops tauschii, Triticum urartu, millet (Setaria italic), barley (Hordeum vulgare), Brachypodium distachyon, sorghum (Sorghum bicolor), maize (Zea mays), soybean (Glycine max), rice (Oryza sativa indica Group) of TaFKBP62c-2B were searched and found in NCBI database and the identity of amino acid between 9 homologous genes and TaFKBP62c-2B ranged of 86%~96%. The highest identity of amino acid was between EMS49100.1(T. urartu) and TaFKBP62c-2B, while the lowest was between soybean (XP_014634970.1) and TaFKBP62c-2B. Then phylogenetic tree of FKBP62c was constructed using these 9 genes and TaFKBP62c-2B. The expression pattern disclosed the expression level of TaFKBP62c-2B was increased immediately under heat stress, it came to the peaks at 2 h and droped gradully after that. TaFKBP62c-2B was responded to drought stress, the expression came to the peaks at 1 h and droped, it came to another peak at 8 h. Tissue expression pattern analysis showed that TaFKBP62c-2B expressed in multiple tissues. Stem accumulated higher TaFKBP62c-2B transcription compared with the spike, spikelet, lemma and palea, while root and leaf had the least TaFKBP62c-2B transcription among all the tissues. According to the prediction database, TaFKBP62c-2B was localized to endoplasmic reticulum (ER). The subcellular location were validated by means of fusing TaFKBP62c-2B with N-terminal GFP and then expressed in wheat protoplats using PEG transfection method. The green fluorescence were mainly observed in cytoplasm, and were not seen in chloroplasts. TaFKBP62c-2B should be localized to ER. In this study, the TaFKBP62c-2B gene was isolated, which the expression pattern and subcellular localization were detected. All above results pave the way to further function analysis of TaFKBP62c-2B.

Blue eggshell is an important economic trait in the poultry, as it presents the thicker eggshell and plays more important role in filtering solar radiation and damage resistance than the brown eggshell and the white eggshell. Now the genetic basis underlying blue eggshell in the chicken (Gus gallus) has been uncovered. However, its genetic mechanism remains unknown in ducks (Anas platyrhynchos domesticus). In this study, to comprehensively analyze the transcriptome basis of the blue eggshell in ducks, homozygous blue-shelled and homozygous white-shelled Shaoxing ducks were bred by using the test cross, and transcriptome sequencing of uterus from the 2 genotypes was performed using the Illumina HiSeq 2500 platform with 3 biological replicates per genotypes. After removing sequencing adaptors and the low-quality reads, we totally obtained 288 008 582 clean reads, and 71.62% of them were successfully mapped to the duck genome. 93.04% of the mapped reads were uniquely aligned to the duck genome. These mapped reads were assembled into 64 587 transcripts, including 19 589 novel ones. Based on these RNA-seq data 5 known alternative splicing event, including Skipped exons (SE), Retained intron, Alternative 5' splicing site (A5SS), Alternative 3' splicing site (A3SS) and Mutually exclusive exon (MEX) were also analyzed, and 20 302 alternative splicing events were identified. Among these alternative splicing event, SE was found to be the major type, with the times of 8388, followed by 4891 A5SS and 3649 A3SS, and MEX was the rarest events, happening 603 times. In this study, reads per kilobase per million of mapped reads (RPKM) was calculated for gene expression. Based on the RPKM value, 21 829 genes were expressed (RPKM≥1), and most of these genes were expressed between 1 to 100 RPKM. Further differential expression analysis between the 2 genotypes showed that 1 768 genes exhibited differential expression (P＜0.05,｜fold change｜≥1.5), including 700 up-regulated and 1 068 down-regulated in homozygous blue-shelled ducks when compared with the white-shelled individuals. Among these differentially expressed genes we found that several genes were related with the main pigments (Protoporphyrin-IX, biliverdin, and biliverdin zinc chelate) of the eggshell, such as biliverdin reductase A (BLVRA), uroporphyrinogen Ⅲ synthase (UROS), hemoglobin subunit alpha-1 (HBA1), heme binding protein 1 (HEBP1), and so on. To verify the gene expression data from the RNA-seq, Real-time PCR was carried out for 4 randomly selected genes, including ferrochelatase (FECH), heme oxygenase 1 (HMOX1)、RasGEF domain family member 1B (RASGEF1B) and polycystic kidney disease 2 (PKD2). Results showed that all of the 4 genes exhibited consistent expression pattern in Real-time PCR with that of RNA-seq. Further Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these differentially expression genes took part in numerous functions and pathways, including functions associated with heme and bile metabolism, such as heme binding, tetrapyrrole binding, primary bile acid biosynthesis and bile secretion. This study provides the first transcriptome basis of eggshell color in ducks, and the differentially expressed genes identified in this study will provide valuable resources for the further genetic analysis of the eggshell color in ducks.

Mitogen-activated protein kinase (MAPK) cascades are important pathways of signal transduction to response the changes from external environment in plants. In this study, SlMPK3 gene, cloned from Solanum pimpinellifolium, was transgenetically overexpressed in cultivated tomato (S. lycopersicum) M82 using the agrobacteria-mediated method. T1 generation was used to investigate the role of Solanum lycopersicum mitogen-activated protein kinases 3 (SlMPK3) under chilling stress. The SlMPK3 transcript was up-regulated rapidly under low temperature stress in leaves. Moreover, the relative expression fold was higher in transgenic plants as compared to the control plants. Cold tolerance index was significantly higher than control (0.8 and 0.37, respectively) after identification of cold resistance in seedling. After low temperature treatment, leaves from the control plants appeared a symptom of leaf wilting and necrosis, leaf chlorosis and leaf yellowing. However, the transgenic plants were still keep normal symptom without obvious yellowing. After 120 h treatment at 4 ℃ low temperature, the relative electrolyte leakage was 74.66% in wild type plants, that of wild type plants was 74.66%. The malondialdehyde (MDA) content was 6.76 μmol/g FW on average in the leaves of transgenic plants, which was 30.81% lower than control at 120 h low temperature treatment. Hydrogen peroxide (H2O2) content was significantly higher in wild type plants leaves than that of the transgenic plants (P＜0.05)( 2.02 and 1.57, respectively) at 120 h. At the same time, the antioxidant enzymes activity, soluble sugar and soluble protein content increased significantly (P＜0.05) in the transgenic plants and wild type plants. At 120 h after low temperature treatment, the activities of super-oxide dismutase (SOD), peroxidase (POD) and peroxidase (CAT) in the leaves of transgenic plants were 29.40%, 24.24% and 29.40%, respectively higher than control. The content of soluble protein and soluble sugar gradually increased with the increasing of the treatment time in the transgenic plants and wild type plants after low temperature. After 120 h treatment at 4 ℃, the soluble protein content in the transgenic plants was 40.02 mg/g FW (OE-4), 42.21 mg/g FW (OE-6) ，40.33 mg/g FW (OE-7), respectively, which were significantly higher than that of control (36.86 mg/g FW)(P＜0.05). The soluble sugar content was 51.87 mmol/g FW average in transgenic plants, which was 21.82% higher than that in control at same treatment-point. This study showed that overexpression of SlMPK3 gene could enhanced the low temperature tolerance in transgenic tomato seedlings to some extent. The present study lays a solid foundation on further investigations about the functions of tomato SlMPK3 gene. At the same time, this study supplies a new germplasm for the cultivation of new low temperature tolerance transgenic tomatoes.

H3N8 influenza viruses have a broad host range, which are commonly found in wild birds and domestic poultry, and have been isolated from some mammals. Moreover, H3N8 influenza viruses have the ability to occasionally infect and transmit among other species. In recent years, avian influenza surveillance data indicates that the number of isolated H3N8 subtype Avian influenza virus (AIV) has a tendency to increase year by year. To understand the biological characteristics of the H3N8 AIV, two H3N8 viruses (A/duck/Guizhou/S1092/2013(H3N8)(DK/GZ/S1092/2013) and A/duck/Guizhou/S1145/2013(H3N8)(DK/GZ/S1145/2013)) were isolated from Guizhou province at 2013 for phylogenetic analysis, infectious experiment in mice (Mus musculus) and receptor-binding specificity analysis. The phylogenetic analysis results indicated that the 2 viruses displayed obviously genetic diversity, and all genome of the viruses had different origins except for hemagglutinin (HA) gene. The mice study suggested that the 2 viruses could effectively replicate in the lung and nasal turbinate of mice without pre-adaptation. The virus titers of DK/GZ/S1092/2013 in lung and nasal turbinate were 4.00 and 5.50 log10EID50/mL (EID50: Fifty percent embryo infection doses), respectively, and the virus titers of DK/GZ/S1145/2013 in lung and nasal turbinate were 4.25 and 3.58 log10EID50/mL, respectively. In addition, the 2 H3N8 influenza viruses displayed low virulence to mice, and mice did not display obvious clinical symptoms after infection with the viruses. In the observation period, DK/GZ/S1092/2013 only caused 1.8% bodyweight loss and DK/GZ/S1145/2013 only caused 0.8% bodyweight loss. The receptor-binding specificity analysis demonstrated that the 2 viruses had the ability to bind both sialic acid (SA) α2,3-Gal receptor from poultry and SA α2,6-Gal receptor from human, which indicated that the 2 H3N8 subtype AIV posed potential threat to infect human. In conclusion, this study systematically researched the biological characteristic of the 2 H3N8 subtype AIV, and the results indicated that the H3N8 influenza viruses posed potential risk to infect mammals. This study plays an important role in the prevention and control of H3N8 subtype AIV.

Cluster of differentiation 4 (CD4) is a co-receptor and signal transduction molecule which is mainly expressed on the CD4+ cell membrane. CD4 molecule participates in the recognition of major histocompatibility complex (MHC) class Ⅱ molecules, and plays an important role in the development and differentiation of T lymphocyte subsets, and in the process during CD4+ cell antigen recognition. This study was to clone the coding sequence (CDS) of the CD4 gene and its spliceosome in pigs (Sus scrofa), and reveal the expression feature of CD4 mRNA in porcine tissues. Based on the mRNA sequence of the porcine CD4 gene (GenBank accession number: AY515292), the coding sequence of the CD4 gene was amplified in Large White pigs using reverse transcription-PCR (RT-PCR), and the expression profiling of CD4 alternative splicing was analyzed in different tissues. Meantime, the expression level of CD4 mRNA was analyzed in different tissues of Large White pigs, as well as in three immune tissues of Large White, Landrace and Songliao Black pigs by quantitative Real-time PCR (qRT-PCR). The results showed that two different CD4 coding sequences were identified (GenBank accession number: KC333253 and KC333254) by RT-PCR amplification, cloning and sequencing, and the complete type (1 375 bp) and splicing type (1 252 bp) CD4 encoding 457 and 397 amino acids, respectively; both the two CD4 coding sequences were expressed in porcine peripheral blood, thymus, lymph node and spleen, and the expression level of complete type CD4 was higher than splicing type. The qRT-PCR results showed that the expression level of the porcine CD4 gene in spleen was significantly higher than in liver, stomach, kidney, heart and muscle (P＜0.05); there was no significant difference in the CD4 mRNA level in the same immune tissue of Large White, Landrace and Songliao Black pigs (P＞0.05), and the expression level of the CD4 gene in thymus was significantly higher than that in spleen and lymph node among the three pig breeds (P＜0.05). This study provides a basis data for further investigating the structure and function of the porcine CD4 gene.

Fatty acyl amino acids, an endogenous compounds from the dehydration synthesis of fatty acids and amino acids, plays an important role of anti-inflammatory, cell proliferation and cell apoptosis. Skeletal muscle protein deposition is promoted by extracellular signal-regulated kinase (ERK) in mitogen-activated protein kinase (MAPK) signaling pathway and ribosomal protein (rpS6) in mammalian target of rapamycin (mTOR) signaling pathway, while muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFBx) in forkheadbox O1 (FoxO1) signaling pathway increase protein degradation. In addition, fatty acyl amino acids could be detected in skeletal muscle, which also express their receptors. To identify the effects of N-Oleoyl glycine (OLGly) on the proliferation, differentiation and protein deposition of mouse (Mus musculus) myoblast cell line (C2C12), cell counting kit-8 (CCK-8) was used to calculate cell numbers and detected creatine kinase activity to identify the differentiation level of C2C12. Moreover, qRT-PCR was adopted to test the mRNA expression level of proliferating cell nuclear antigen (PCNA), cell cycle protein dependent kinase inhibiting factors (p27 and p21), myogenic detemination gene 5 (Myf-5) and myogenic regulatory factor (MyOG), which were the marker genes of proliferation and differentiation of C2C12 cells. In this study, total protein content and the protein synthesis level (puromycin incorporation) were tested. Western blot was used to detect the expression level of mTOR, ERK, S6, FoxO1, MAFBx and MuRF1. The results showed that 0.2, 2 or 20 μmol/L of OLGly had no significant effect on the cell numbers and the activity of creatine kinase, as well as the marker genes expression of proliferation and differentiation of C2C12 cells. Interestingly, OLGly could significantly enhance the protein contents of C2C12 myotubes in dose and time-dependent manners. 20 μmol/L of OLGly significantly enhanced the total contents of protein and the incorporation contents of puromycin (P＜0.05). However, the same dose of the mixture of oleic acid and glycine had no significant effect on protein synthesis. Together, these results indicated that the effect of OLGly on protein synthesis in C2C12 myotubes was not mediated by its metabolites. Furthermore, the protein expression of the p-ERK and p-S6 were significantly elevated in response to 20 μmol/L OLGly treatment for 30 and 60 min (P＜0.05). However, both OLGly and the mixture of its motabolites, oleic acid and glycine, unable to alter the protein expression of p-mTOR. For the protein degradation, OLGly had no effects on the protein expression of p-FoxO1, MAFBx and MuRF1 and the mRNA expression level of MAFBx and MuRF1. In summary, the present evidences confirmed that OLGly promoted protein synthesis, while had no effect on the proliferation and differentiation of C2C12 cells. The upregulation of ERK and S6 might be involved in the process for OLGly induced protein synthesis. The present results will provide the experimental basis for the development of novel drugs to promote skeletal muscle growth and protein deposition.

The development of fish cell line provides an important tool to study virology, immunology, genetics, oncology, developmental biology, toxicology, clinical medicine and biotechnology in aquaculture. Many fish cell lines have been established from freshwater and anadromous species, but few in marine fishes. As a widely cultivated marine fish species with high economic value in Europe and China, the number of cell lines was inadequacies in the research of turbot (Scophthalmus maximus). To expand quantity of marine fish cell lines, also provide alternative tool for gene function research or the isolation of the virus, a new cell line, Scophthalmus maximus heart cell line (SMH), was established from turbot heart in the research. Tissue block method was used and the heart tissues were collected aseptically, and washed 3 times with phosphate-buffered saline (PBS) and minced into small pieces (1 mm3) by surgical scissors in DMEM-F12 medium (pH 7.2). Then tissue pieces were washed again with PBS until it was neat without blood. It had been cultured in 25 cm2 cell-culture flask which should be inverted at 24 ℃, then returned the flask 4~5 h later. After completion of the above steps, 3 mL DMEM-F12 complete medium was added into the flask, which was supplemented with 20% FBS, 50 mmol/L 2- mercaptoethanol (2-Me), 10 ng/mL basic fibroblast growth factor (bFGF), 100 IU/mL penicillin and 100 μg/mL streptomycin, 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes). When cells grew into a conflent monolayer, they were subcultured by trypsin solution (0.25% trypsin and 0.03% EDTA in PBS) at a split ratio of 1∶2. The cultured SMH cells, fibroblastic in morphology, had been subcultured to passage 58 in 230 d in a good proliferating state. The SMH cells at passage 23 were cryopreserved in liquid nitrogen. After 3 months, the cells could undergo cryopreservation with proliferated to 80% confluency in 5~6 d. The morphology and proliferation ability of SMH cells were the same before and after cryopreservation after thawing. The growth curve of SMH cells at passage 30 suggested that the SMH cells were at latent stage on the beginning of 2 d and went into logarithmic stage of 3~4 d. And their doubling time was calculated to be 41.28 h at passage 30. Transfection experiment demonstrated that SMH cells transfected with pEGFP-N3 plasmid could express green florescence protein (GFP) with higher transfection efficiency. The green florescence signals were observed every 12 h under a Nikon Eclipse T2000-U florescence microscope. GFP expressed successfully in cells and achieved maximum fluorescence value at 96 h. Using geneticin (G418) to screen SMH cells which transfected by recombinant plasmid, showed a bunch of cells with higher efficiency of fluorescence. Chromosome analysis revealed that the SMH cell line had a normal diploid karyotype with 2n=44, which occupied 64% in the 100 metaphase cells counted at passage 42. The SMH cells sequence of cytochrome oxidase subunitⅠ(COⅠ) gene showed a 99% consistency with turbot (GenBank Accession No. KJ205427). Above all, the species of SMH could be identified from turbot by mitochondrial DNA COⅠ gene. The establishment of the SMH can provide experimental material for genetic research capabilities or more, and importantly, could progress the study of this cell line in virus isolation and cell-pathogen interaction study as more fish virus susceptibility to SMH cell line needing to be detected.

Starch is one kind of abundant biomass resources which is widely used in lots of industries such as food, brewing and pharmaceutical industries. Pullulanase (pulA, EC 3.2.1.41) is one of the starch-debranching enzymes which catalyzes the hydrolysis of α-1,6 glycosidic bonds in starch, and improves the utilization of starch. Hence, it has a more profound reason to develop pullulanase in our own way. In the previous study, a pullulanase high-producing strain, Klebiella variicola strain 7, was obtained from the soil near a starch factory. The gene encoding pullulanase (GenBank No. KJ146839) from Klebiella variicola strain 7 was expression in Escherich coli BL21(DE3). In order to improve expression quantity, secretion efficiency and properties of pullulanase, Mutants of M1 and M2 were built using the method of genetic engineering of removing N- terminal amino acids of pullulanase to improve expression quantity, secretion efficiency and properties of pullulanase. The results showed that, the optimum temperature of both M1 and M2 were 45 ℃, and the optimum pH of M1 was 6.0, while the optimum pH of M2 was 5.6. The half-life and specific activity of M2 were 37 minutes and 582.204 U/mg, which were 6.17- and 1.6- fold that of M1. With pullulan as substrate, Kinetic studies showed that the Vmax, Kcat, Km and Kcat /Km of M2 was 0.001 3 μmoL/(mL·s)-1, 191.80 s-1, 0.30 mg/mL, 693.30, respectively. Compared with M1, the substance affinity increased, and the catalytic efficiency was 2- fold that of M1. This study provides a new way and thinking for improving the specific activity and half-life period of enzymes by N-terminal amino acids truncation.

Production of biofuel and mass cultivation of microalgae mainly depends on the nature of microalgae strain. The aim of this study was to screen microalgae strains for swine manure wastewater treatment and lipid production from natural water area. Through multi-step screening, three experimental protocols including streak plate method, single-cell isolated offspring and capillary theory of separation, were used for separating and purifying the algal strains from 51 sites. As a result, 118 microalgaes were obtained, among which 71 facultative heterotrophic microalgae strains were screened including 33 strains which were proved to be tolerant to swine manure wastewater, among which 17 strains were adapted well in swine manure wastewater without further acclimation. Some algae strains were preliminarily identified by morphological observation as Chlorella sp. and Scenedesmus sp.. All candidate strains were grown in swine manure wastewater for 7 days to evaluate their specific growth rate and lipid content. The 13-6, 19-4, 20-6 and 34-2 strains showed faster specific growth rates than other candidate strains during 7 days with the average specific growth rates of 0.147, 0.162, 0.177 and 0.154 d-1, respectively. The total lipid content of the candidate algae which grew in swine manure wastewater ranged from 7.4% to 29.2%. Among them, the 19-4, 24-1 and 34-2 strains showed higher lipid content for 19.7%, 22.9% and 28.8%, respectively, which suggested that these candidates could accumulate higher lipid content than other strains. Two strains (C. sorokinlana 19-4 (GenBank No. KU948990) and Chlorella sp. 34-2 (GenBank No. KU948991)) were chosen for other studies because of their ability to adapt to swine manure wastewater for high growth rates (0.162 and 0.154 d-1) and high lipid content (19.7% and 28.8%). The maximal biomass concentrations of C. sorokinlana 19-4 and Chlorella sp. 34-2 in swine manure wastewater in 30 L photobioreactor reached 0.78 and 1.12 g/L, and algal growth rate reached 0.153 and 0.149 d-1, and lipid content reached 18.73% and 29.27%, respectively. Total nitrogen and total phosphorus were reduced during the cultivation period of the inoculation treatments, whereas no obvious variation was observed in the corresponding negative control. Total nitrogen was reduced 60.24% from 90.81 to 36.11 mg/L for Chlorella sp. 34-2, and 70.56% from 90.81 to 26.73 mg/L for C. sorokinlana 19-4. Total phosphorus was reduced 85.07% from 4.02 to 0.60 mg/L for Chlorella sp. 34-2, 90.98% from 4.02 to 0.36 for C. sorokinlana 19-4 during the 7 days. The production of biodiesel made from microalgae mainly influence by the fatty acid component. The fatty acid compositions of Chlorella sp. 34-2 cultured in swine manure wastewater and BG11 medium were investigated. It was found that C16:0, C18:2n6c and C18:3n3 were the major fatty acids, which accounted for more than 83.97% of the total fatty acids. Their fatty acid composition met the requirements of standards of the raw materials for biodiesel production. This species has great potential for use in the treatment of swine manure wastewater and biodiesel production.

As one of most cutting-edge technologies of life science, synthetic biology has great potential to develop into valuable applications in several research fields such as medical science, pharmaceuticals, chemical industry, agricultural science, biofuels, bio-materials and bioremediation. However, it may also bring up potential risks, which attracts great attentions from not only research community but also the society at large. This paper will first review how the concept of synthetic biology has developed; the potential risks and biosafety concerns raised from its research and applications; and the existing regulatory frameworks from the key players of synthetic biology around the world. Based on this knowledge, we will conduct a comprehensive analysis on the potential implications of synthetic biology on the human and environment.