Utilization of heterosis is an important breeding method, which can greatly improve the yield, disease resistance and stress resistance of tomato (Solanum lycopersicum). The application of male sterile line can reduce the workload of artificial emasculation and it is increasingly adopted by seed companies. However, breeding of male sterile lines is usually time-consuming, which limits the application of male sterile lines. The application of modern gene editing technique in important genes related to stamen development would help better understand the underlying molecular mechanism and should shorten the breeding period of tomato GMS (genetic male sterility) lines. Tomato SlAP3 (Solanum lycopersicum APETALA3) is a homologous gene of AP3 in Arabidopsis thaliana regulating floral organ development. Mutation of this gene causes abnormal stamen development in tomato, resulting in male sterile phenotype. This study used CRISPR/Cas9 genome editing system to create mutation on SlAP3. Two target sites with PAM (protospacer adjacent motifs) sequence were selected from the exon of SlAP3, and CRISPR/Cas9 expression vectors were constructed. Transgenic tomato plants were generated using Agrobacterium tumefaciens. The DNA sequences around the 2 target sites were amplified and sequenced. The flower organ phenotype in gene edited plants was observed. Twenty and eighteen transgenic tomato plants were obtained by transforming two vectors respectively. The DNA sequence around the target sites was amplified, and sequence alignment showed that different deletions of 1~9 nucleotides occurred upstream of PAM, resulting in amino acid deletion and early termination of the SlAP3 protein. The observation revealed that plants carrying homogeneous mutations have homeotic phenotypes and reduced organ number in petals and stamens. These results indicated that the vectors constructed with CRISPR/Cas9 genome editing system could specifically target the tomato SlAP3 gene, resulting in mutation of the gene and the formation of a stamen homeotic mutant phenotype, which provide theory guide and technical support for using CRISPR/Cas9 system to develop tomato male sterile line.

Heavy panicle type is one of the main objectives of super rice breeding, and has the optimal combination for different yield factors per each plant, and is the basis for the high yield of unit area. Heavy panicle variant, Javanica 22 (Oryza sativa ssp. javanica), is a natural variation derived from Xiangdali (Oryza sativa ssp. javanica). Investigation in Javanica 22 physiological traits and genetic characteristics contributes to clarify its utilization value. In this study, the main physiological characters, the appearance quality, eating and cooking qualities (ECQs) of Javanica 22 and its wild type Xiangdali were compared. Indica or Japonica characteristic of Javanica 22 was identified by reported 20 pairs of typical SSR markers. According to the above result of identification, cluster analysis was carried out and SSR fingerprints of 4 varieties/lines were constructed. Glutinous property was experimented by molecular marker Wx M1. The results indicated that the weight of single panicle of Javanica 22 was 7.70 g, belonging to a variant of heavy panicle type. Compared with Xiangdali, the physiological traits of Javanica 22, especially the filled grain number, seed setting rate and single panicle weight were significantly improved (P<0.01), but 1000-grain weight was significantly decreased (P<0.01). The appearance quality and ECQs were also improved. Genetic analysis of Indica or Japonica characteristic showed that Javanica 22 had 8 pairs of markers with Japonica characteristics and 12 pairs of markers with Indica characteristics, belonged to mixed characteristics of Indica and Japonica but partial to Indica, yet its wild type Xiangdali belonging to mixed characteristics of Indica and Japonica but partial to Japonica. Cluster analysis also showed that Javanica 22 belonged to the same group with Jingnuo 6 (Oryza sativa ssp. indica) and its genetic similarity coefficient was 0.63; While its wild type belonged to the same group with Jingnuo 8 (Oryza sativa ssp. japonica), and its' genetic similarity coefficient was 0.81. Glutinous identification showed that the second exon of its waxy gene had nucleotides insertion of 23 bp, which was recessive wxwx genotype. Therefore, Javanica 22 was a typical glutinous rice with lower apparent amylose content (1.42%) than its wild-type, Xiangdali (3.80%). Multiple loci variation in Javanica 22 were found by comparing the DNA fingerprints of variant and wild type in this study. The heavy panicle variant Javanica 22 had more excellent physiological traits than its wild type and its genetic background had the characteristics of Indica and Japonica but partial to Indica, which could be used as an excellent germplasm in super rice breeding and as a bridge parent in hybridization of Indica and Japonica. The conclusion has definite theoretical significance in super rice breeding.

The late up-regulated in response to Hyaloperonospora parasitica gene 1(LURP1) plays a key role in the response against pathogenic oomycete H. parasitica and resistance to stress in plants. In order to elucidate the biological function of the potato (Solanum tuberosum) LURP1 gene and to clarify its position in the protein interaction network, the bait vector pGBKT7-StLURP1 was constructed by homologous recombination and used to screen the potato cDNA library via the yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) system in this research. The result showed that the positive rate of Y2H library screening was about 70%, and finally 88 positive clones were yielded, which was corresponded to 12 StLURP1 interacting proteins with complete ORF. The authenticity of the interactions was verified by retest. Blast alignment and Gene ontology (GO) annotation analysis demonstrated that StLURP1 could interact with the proteins such as DnaJ family protein, oxidoreductase/transcriptional repressor, glycerophosphodiester phosphodiesterase (GPGD), polyphenoloxidase, proline-rich protein, heavy metal transport/detoxification domain-containing protein, actin depolymerizing factor (ADF), nonspecific serine/threonine or tyrosine protein kinase, et al. The result indicated that StLURP1 may be involved in multiple biological processes, including lipid metabolism, biotic and abiotic stress responses, actin depolymerizing, heavy metal ions transport, protein folding and other biological processes. The present study would provide a theoretical foundation for further studying of biological function and role of StLURP1 gene.

Bacterial soft rot disease causes severe damage to Chinese cabbage (Brassica rapa ssp. pekinensis), and Pectobacterium carotovorum (Pc) is a major pathogen. Based on defense-related genes in Chinese cabbage to soft rot disease, molecular markers were developed in this study, which would be very important for molecular marker-assisted breeding and studying defense mechanism of Chinese cabbage to soft rot disease. Firstly, the ESTs (expressed sequence tags) database related to soft rot resistance of Chinese cabbage with independent property rights were analyzed by bioinformatics method, and 56 pairs of specific PCR primers were designed and used to test the polymorphism between resistant line (A19-2) and sensitive line (A32-2) of Chinese cabbage to soft rot disease. The genotypes of the F₂ population (containing 142 Chinese cabbage individuals) generated by F₁ self-breeding were recorded using the markers and the segregation analysis was conducted. All the 56 pairs of specific PCR primers obtained successful PCR-amplification. There were 30 markers which had polymorphism in the resistant and sensitive parents, including 11 EST-PCR markers and 19 EST-CAPS (cleaved amplified polymorphic sequence) markers, and 9 pairs could match with more than one restriction enzymes. There were 14 dominant markers and 16 co-dominant markers. In addition, the genotypes of 142 individuals of the F₂ population were statistically analyzed using the above 30 marker loci. The result showed that 24 loci (80%) had the segregation ratio of 3∶1 or 1∶2∶1, and 6 loci (20%) had the genetic distortion (P<0.05) which was inconsistent with Mendelian ratio. This research developed defense-related EST-PCR and EST-CAPS molecular markers of Chinese cabbage to soft rot disease based on the ESTs resources of Chinese cabbage, which have certain significance in the study on utilization of ESTs resources, localization of defense-related genes and molecular marker-assisted breeding.

The chalcone synthase gene (CHS) were the first committed enzyme gene of flavonoid biosynthesis, CHS condensed 3 molecules of malonyl-CoA and 1 molecule of p-coumaroyl-CoA to form naringenin chalcone. The study was to investigate the relationship between GhCHS1 gene and the formation of fiber color through virus induced gene silencing (VIGS) in colored cotton ZongXu1 (Gossypium hirsutum). The expression of GhCHS1 gene in interference lines (GhCHS1i) was measured by qRT-PCR, and the anthocyanins contents in the fibers and seedcoat, leaves and cotton kernels were determined. Bioinformatics analysis indicated that GhCHS1 proteins were mainly distributed in vacuoles, and the secondary structure of GhCHS1 proteins was mainly composed of random coil and alpha helix. 24 CHS members were screened in the known genomes of Gossypium, belonging to 7 types according to homology, and 6 types of tertiary spatial structures. GhCHS1 (GenBank No. LOC107897841) was predominantly expressed in the developing fibers and leaves of Zongxu1. The expression of GhCHS1 gene was increased in the early developing fibers (0~9 DPA, day post anthesis), reached the peak at 9 DPA, and then decreased from 12 DPA to 20 DPA. The GhCHS1 gene was interference by VIGS technology, and a large number of transgenic interference lines were obtained. Phytoene desaturase (GhPDS) was silenced by VIGS as the positive control, vector-empty CK was used as the negative control, and Zongxu 1 WT was used as the wild type control. The GhCHS1i lines had slightly curly leaves the same as the phytoene desaturase gene interference (GhPDSi) and vector-empty plants compared with the WT plants. The GhCHS1 expression level was significantly decreased in the GhCHS1i plants, the fiber color of GhCHS1i plants was significantly lighter compared with the brown fiber of WT Zongxu1, and different from the white fiber of upland cotton C312. The expression level of GhCHS1 gene in the developing fibers of GhCHS1i lines was significantly lower than that in the WT, the fiber color of the GhCHS1i lines was lighter brown. The expression level of GhCHS1 gene was indicated the positive correlation with the fiber color. Compared with the WT, the GhCHS1i lines had the lower the expression level of GhCHS1 gene, the fiber color appeared significantly lighter (P<0.05). The analysis of anthocyanin content showed that the less the anthocyanin content of fiber and seed coat, cotton kernel, the fiber color became significantly lighter. The results indicated that GhCHS1 gene was involved in the biosynthesis and accumulation of anthocyanins in colored cotton leaves, fiber and seed coat, cotton kernel, and also played an important role in the fiber pigmentation. This study would have important reference value for the formation mechanism of fiber color formation in colored cotton.

The number of scapes is an important index to measure the quality and price of narcissus (Narcissus tazetta) bulbs. Because it is hard to strict control ethylene concentration, the traditional method for narcissus bulbs fumigation may cause bad effect on flower bud induction. In this study, transcriptome with deep sequencing was used on the ethylene (ETH)/1-methylcyclopropene (1-MCP) treated narcissus to reveal the molecular mechanism of the floral initiation of narcissus. A scientific method of narcissus bulbs treatment have been obtained in our study. Narcissus bulbs were incubated with 200 μL/L of ethylene for 5 h in a sealed container and these bulbs were treated again two days later. Ethylene treatment promotes the floral bud differentiation of narcissus and doubles the number of scapes and flowers. The results of physiological and biochemical indexes showed that exogenous ethylene application promoted DNA replication, gene expression, protein synthesis, sugar metabolism, plant hormone signal transduction, increased the level of protein, peroxidase (POD) activity, indole-3-acetic acid (IAA), and zeatin (ZA) level. De novo transcriptome and RNA-Seq analysis of three treatment groups showed that 65 898 unigenes were obtained in all samples, including 55 793(control group), 57 321(1-MCP), and 64 350(ETH), with average length is 692 bp. Gene Ontology (GO) analysis showed that exogenous ethylene promotes the expression of most genes in all terms. Through differential expression analysis, 62 differentially expressed genes (DEGs) related to flowering were screened out: four genes in starch and sucrose metabolism, nine genes in polyamine synthesis and transport, eleven genes in lignin synthesis and transport, thirty-one flowering-related genes, and seven other regulation genes. Key genes of COL14, FRL3, VRN1, FPA in the photoperiod pathway, vernalization pathway, autonomous pathway had significant difference in expression compared to the control group. Twelve genes correlated with flowering were selected and confirmed by qRT-PCR analysis and the expression profiles were consistent with the RNA-Seq results. These DEGs might have vital function on the floral bud differentiation of narcissus. In this study, a scientific technique of narcissus bulbs treated with ETH has been conducted, and the physiological, biochemical and molecular mechanism of ethylene had been discussed. The study will be helpful for better understanding the floral bud differentiation of narcissus, as well as guides the narcissus production.

WRKY transcription factors play important roles in various stress response. However, the phosphorus (P) deficiency in the tropical and subtropical forest soils has severely posed challenges for the productivity. Therefore, screening the related low-P stress genes, as well as to reveal the molecular regulation mechanisms of low phosphorus tolerance were necessary, which will provide a theoretical basis for the molecular breeding and create novel germplasms with high quality and stress resistance. WRKY transcription factors (TFs), a plant-specific TF family, play important and unique roles in biological regulations involving in stress defenses, development, and metabolite synthesis, however, little has been known about their roles in response to phosphorus starvation in masson pine (Pinus massoniana). To further understand their roles in low phosphorus (P) stress, currently, the full-length sequence of PmWRKY164 was cloned by rapid amplification of cDNA ends (RACE) methodology. Homologous analysis, multiple alignments, as well as related bioinformatics analysis were performed. Quantitative real-time PCR (qRT-PCR) was used to detect the temporal and spatial expression patterns of PmWRKY164. Finally, the pBWA(V)HS-PmWRKY164 expression vector was introduced into tobacco through Agrobacterium-mediated procedure. The result showed that PmWRKY164 (GenBank No. MH579749) was obtained, whose full-length cDNA was 1 977 bp and the corresponding lengths of open reading frames (ORF) was 1 164 bp, which encoded 387 amino acids, including a single WRKYGQK conserved domain and C₂H₂ (C-X_4-5-C-X₂₃-HXH) zinc-finger motif and belonged to GroupⅡb of WRKY family. Homology analysis showed that PmWRKY164 had higher similarity with other plants in the conservative region of the WRKY family. Phylogenetic analysis revealed that PmWRKY164 was mostly close to the WRKY transcription factor of Taxus wallichiana var. chinensis and Picea abies. Spatiotemporal expression analysis showed that PmWRKY164 constitutively expressed in root, stem and leaf, with the highest expression in leaf, followed by root and lowest in stem. In root and stem, PmWRKY164 showed the lowest and highest expression at the 36 d and 60 d after treatment, respectively; in leaf, the expression level was the highest at 60 d and the lowest at 12 d after the stress. Except that the expression level in leaves showed an upward trend, the expression levels of roots and stems showed a rising-decreasing-increasing trend. Further, twenty transgenic tobacco plants were obtained using the A. tumefaciens mediated. 3 different transgenic lines, i.e. Line-2, Line-3 and Line-8 as well as the wild-type lines, were subjected to different concentrations of low-P stresses. Except that the content of malondialdehyde (MDA), the content of phosphorus, Apase activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity in transgenic tobacco were significantly higher in comparison with those of the wild type. This study provided new information and ideas for further exploring the function of PmWRKY164 and would provide a theoretical basis for the molecular breeding in prolonging of phosphorus stress of P. massoniana.

The heart fatty acid-binding protein (FABP3), which involved in the regulation of fatty acid absorb and intracellular transport, associated with tenderness and juiciness via effecting on intramuscular fat (IMF) content in muscle. In this study, the expressions of FABP3 gene were detected using qRT-PCR in tissues of Gannan yak (Bos grunniens). The variations of FABP3 gene were screened by single strand conformation polymorphism (SSCP) so as to evaluate their effects on carcass and meat quality traits of yak. The results showed that the FABP3 gene expressed in all of 16 tissues investigated and their relative expression level with the order of heart>longissimus dorsi>rectum>ileum>pancreas>jejunum>others tissues in Gannan yak, and that significantly higher expression was detected in heart than other tissues (P<0.05). Seven mutations, including one mutation in 5'-UTR and intron 1 and 5 in intron 2 of FABP3 gene were identified in 4 yak populations. Variations in 5'-UTR-intron 1 had effect on carcass weight, tenderness and cooking meat percentage of Gannan yak (P<0.05). Allele B₁ was associated with an extremely significant increase in carcass weight and a decrease in Warner-Bratzler shear force (P<0.01), and allele C₁ was associated with a extremely significant decrease in carcass weight and cooking meat percentage (P<0.01). These results enrich the molecular genetic data of meat quality traits of yak.

β-defensins are the endogenous antibacterial peptide of the organism. Saccharomyces cerevisiae mannan can induce ovine Ruminal epithelial cells (RECs) β-defensin-1 (SBD-1) expression. However, its mechanism of inducing SBD-1 expression is unclear, which limits the development and utilization of mannan preparations. In order to explore whether mitogen-activated protein kinase (MAPK) (p38, ERK1/2, JNK) and Nuclear factor κB (NF-κB) are involved in the expression of SBD-1 in ovine RECs induced by mannan. Firstly, the effects of mannan on the expression of p38, ERK1/2, JNK and NF-κB were examined by qPCR and Western blot. Then the phosphorylation levels of p38, ERK1/2, JNK, IκB and p65 were detected by Western blot after stimulation of RECs with mannan for 5, 15, 30, 45 and 60 min, respectively. Finally, qPCR and ELISA were used to detect the effects of specific inhibitors of p38, ERK1/2, JNK and NF-κB on mannan-induced SBD-1 expression to determine the involvement of p38, ERK1/2, JNK and NF-κB pathways in the expression of SBD-1 induced by mannan. The results showed that mannan stimulation significantly increased the mRNA levels of p38, ERK1/2, JNK and NF-κB in RECs and the phosphorylation levels of p38, JNK, ERK1/2, IκB and p65 (P<0.01). And mannan stimulated RECs different times and activated phosphorylation of p38, ERK1/2, JNK, IκB and p65. Moreover, treatment with p38, ERK1/2, JNK and NF-κB inhibitors significantly decreased the expression of SBD-1 induced by mannan (P<0.01), and the inhibitory effect of p38 inhibitor on SBD-1 was the most obvious. These results indicate that the S. cerevisiae mannan-induced SBD-1 expression in RECs is mediated by MAPK and NF-κB signaling pathways, and the p38 signaling pathway may play a major role. This study explains the function of mannan to promote immunity from the mechanism of mannan-induced defensin expression, and provides a theoretical basis for better development and utilization of mannan preparations.

Mammals coat color molecular mechanism has always been hotspot in animal genetics and breeding, variety of coat colors in nature arouse the interest of many researchers; horses (Equus caballus) with different complex colors, which is indispensable to the horse registration in breeding, and the molecular mechanisms of horse coat color is very important. Recent years, most researches are focused on breeds, few researches are from the perspective of the individuals and hair follicle pigment synthesis. Since Tobiano Mongolian Horse has unique coat color, the formation mechanism must be more complex. Pigment synthesis involves a large number of genes and complex regulatory networks. Tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) which is now called dopachrome tautomerase (DCT) are 3 major genes at downstream of melanogenesis pathway. Paraffin-embedded tissue sections were stained with hematoxylin-eosin (HE) to analyze hair follicle structure and pigment distribution. qRT-PCR was used to detect the mRNA expression levels of TYR, TYRP1 and DCT genes in different color skins. The results showed Western blot (WB) and immunofluorescence (IF) were used to quantitatively locate their corresponding proteins. The pigment distribution in different color (black and white) skin tissues and the length of hair follicle dermal papillae was significantly different, meanwhile, the thickness of dermis was different, and the diameter of hair follicle hairball was slightly different. The mRNA and protein expressions of 3 genes were significantly different in different color skin tissues, and the protein expression was basically located in the hair matrix of hair follicle dermal papillae. The results showed TYR, TYRP1 and DCT, downstream genes of melanogenesis, might affect the synthesis of pigment in the different color skin tissues of Tobiano Mongolian horses, and can be the candidate regulatory factors in the process of hair pigment deposition. It will present information for the in-depth study of the molecular mechanism of Tobiano Mongolian Horse coat color and provide reference for other mammals.

Myostatin (MSTN), also known as GDF-8 (growth-differentiation factor 8), is a member of the transforming growth factor-beta (TGF-β) super-family, which participates in various process of body growth regulation. As a negative regulatory of muscle growth, its mutation will lead to the excessive proliferation of muscle cells and muscle hypertrophy. To study how MSTN mutation affect its protein structure and biology functions, the multi-level structure and function changes of MSTN were predicted and analyzed by bioinformatics methods, based on our MSTN gene knock-out mice (Mus musculus). The results showed that the nt 175~180 sequence in the third exon of the MSTN gene was missing, the expression level of the MSTN decreased which led to deprived the MSTN protein in muscle, the expression level of myogenin determination gene (Myod), myogenic factor 5 gene (Myf5) and myogenin gene (Myog) increased significantly, activin typeⅡreceptor b3 gene (ActR2b3), the receptor protein of the MSTN decreased and the muscle hypertrophy. Analysis of the deletion mutation MSTN protein structure, it lost the Y (309) and C(310) amino acids, which elongated the beta pleated sheet and changed the protein structure. The functional prediction result showed that, the mutant MTSN protein of these mice lost a protein bonding site, which reduced its bonding capability to the receptor and impaired its biological functions. This study showed that the third exon nt 175~180 of MSTN in mice was the core sequence that affected the function of MSTN protein, and resulted in the muscle hypertrophy phenotype, which would provide a good mouse model for studying the function of MSTN gene.

Many studies have shown that homeobox C8 (Hoxc8) gene plays an important regulatory role in the crested traits of chickens (Gallus gallus), and the crested traits have a significant impact on the early growth performance of the chickens. Therefore, in order to study the correlation between the expression of Hoxc8 gene and the crested traits and growth performance of Wumen crested chickens, this study had taken the Wumeng crested chickens as research objects, measuring the early growth performance and the expression of Hoxc8 gene in different tissues. The results showed that Hoxc8 gene was expressed in all tissues of adult crested Wumeng crested chickens. It had the highest expression in crested bone; higher expression in testis; moderate expression in kidney, chest muscle, leg muscle, tibia and large intestine; and lower expression in heart, liver, ovary, hypothalamus spleen (P<0.01); Hoxc8 gene was differentially expressed in the scalp and back shin tissues of Wumeng crested chickens with 24 weeks of age, with the slight expression in crested chickens and higher expression in non-crested chickens (P<0.01). Hoxc8 gene was differentially expressed in the tissues of female and male Wumeng crested chickens: The expression of scalp and back shin tissue in cocks of 24 weeks old Wumeng crested chickens were significantly higher than that of hens (P<0.01). In adult crested Wumeng crested chickens, the expression of tibia and leg muscle tissue of cocks was higher than that of hens, and the expression of heart, liver, hypothalamus and pituitary tissue of hens were higher than that of cocks; The crested traits had an effect on the early growth performance of Wumeng crested chickens. With the increase of the age of the week, the growth trend of the body weight, body length, chest depth, chest width and tibia length of crested Wumeng crested chickens were better than that of the non-crested Wumeng crested chickens; The early growth performance of cocks with crested and non-crested Wumeng crested chickens were better than that of hens; the early weight gain of Wumeng crested chickens with better shelling weight was better. The above results showed that the crested traits of Wumeng crested chickens were related to the specific expression of Hoxc8 gene in their scalp and back skin tissue. there was a good growth performance of crested Wumeng crested chickens. The results of this study could provide certain guiding significant for the conservation and breeding of Wumeng crested chickens.

Pyruvate kinase (PK) is a key rate-limiting enzyme that catalyzes the last step of glycolysis and plays an important role in animal glucose metabolism. Studies have shown that there were 2 SNPs (T+1817C and G+1818T) in the 3' non-coding region of Ma-type pyruvate kinase (PKMa) gene of Ctenopharyngodon idella. The completely linked SNPs were combined into 3 genotypes, TG/TG, CT/TG and CT/CT (abbreviated as AA, AB and BB), respectively. In order to investigate the PKMa gene polymorphism effect on glucose tolerance, the growth performance of the 3 genotypes were tested and compared in the low-glucose (LD) and high-glucose (HD) diet environments. After 8 weeks feeding trial, the results showed that the growth rate of grass carp in LD group was faster than that in HD group. In the LD population, the body weight of AA haplotype was 4.9% and 6.1% faster than AB haplotype and BB haplotype, respectively (P>0.05). In the HD population, the body weight of the AA haplotype was 36.8% and 30.4% faster than the AB haplotype and the BB haplotype, respectively (P<0.05) and increased by 6.7% compared with LD population AA haplotype. For the specific expression of PKMa gene in brain tissue, the concentration of PK enzyme in brain tissue was detected. It was shown that the PK enzyme concentration in LD population was lower than that in HD population. The AA genotype individuals was significantly higher than that of the AB and BB haplotypes individuals in the LD and HD population, respectively. The results showed that the PMKa gene mutation was associated with the body's tolerance to glucose of C. idella. It is speculated that the expression level of the PKMa enzyme concentration is increased to better utilize the glucose dietary having the AA genotype C. idella. This study provides candidate markers for the breeding of new varieties of glucose tolerant in C. idella.

The wide scale use of zinc oxide nanoparticles (ZnO NPs) makes organisms beings more prone to the exposure to ZnO NPs and its adverse effects.To investigate the toxic effect of ZnO NPs in monocytes/macrophages (MO/MΦ) from red drum (Sciaenops ocellatus), MO/MΦ from red drum were respectively exposed to the different concentrations of ZnO NPs (0, 12.5, 25, 50 μg/mL) for 6, 12, 24 h. Cell activity was determined by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay and lactate dehydrogenase (LDH) release. Cellular uptake of ZnO NPs, intracellular reactive oxygen generation and apoptosis rate were measured by flow cytometry after exposure to ZnO NPs for 6 h. The mRNA expressions of Caspase 9 in MO/MΦ were also examined using qRT-PCR assays.Compared with the control group, the experimental groups showed obvious difference in following aspects: 1) The activity of the cells detected by the MTT assay and the ability of the cells to absorb neutral red all decreased with the increase of ZnO NPs concentration and the extension of exposure time; 2) The release of LDH were increased with the extension of ZnO NPs exposure time and the increase of ZnO NPs concentration; 3) Cellular uptake of ZnO NPs was increased; 4) Intracellular reactive oxygen species (ROS) generation was increased; 5) Cell apoptosis rate showed an increasing trend; 6) mRNA expression level of gene Caspase 9 showed significant up-regulation (P＜0.05). These results demonstrated concentration and time dependent cytotoxicity after exposure to ZnO NPs on MO/MΦ from red drum. Intracellular ROS may be the major factor in cytotoxicity of ZnO NPs. When ZnO NPs entered cells, ZnO NPs increased the level of intracellular ROS which led to apoptosis. The above research provides a theoretical basis to study toxic effect of ZnO NPs to aquatic organisms.

As a main component of the anti-oxidant defense system of pathogenic fungi, peroxidase can remove reactive oxygen species (ROS) derived from plant organisms, and promote the successful infection of host plant cells by pathogens. Related studies on peroxidase (POX) of Fusarium graminearum have not been reported. In this study, 31 Fusarium graminearum peroxidase genes were obtained by searching the database of fungal peroxidase genes using bioinformatics methods, and were divided into 18 subfamilies by phylogenetic analysis and conserved domain analysis. Analysis of the expression pattern of the peroxidase family gene revealed that the FgPOX (Fusarium graminearum peroxidase)-13, FgPOX-17, FgPOX-16, FgPOX-19 and other genes had high expression levels in both mycelium and spore. and the FgPOX-24 and FgPOX-29 had higher expression levels in mycelium, the FgPOX-11, FgPOX-10, FgPOX-4 and FgPOX-23 had higher expression levels in spore. The expression pattern of peroxidase family gene in the process of infection was analyzed. It was found that the expression level of FgPOX-13 was significantly increased and maintained at a high level during the infection of the pathogen. In addition, genes such as FgPOX-17, FgPOX-28 and FgPOX-30 were highly expressed in the early stage of pathogen infection, and genes such as FgPOX-12, FgPOX-10 and FgPOX-23 were highly expressed in the late stage of pathogen infection. Furthermore, qRT-PCR was used to analyze the expression of Fusarium graminearum peroxidase family genes under H₂O₂ stress, the expression levels of FgPOX-1, FgPOX-4, FgPOX-6, FgPOX-9, FgPOX-10, FgPOX-22 and FgPOX-30 were found to be 20 times higher than that under normal conditions, and FgPOX-9, FgPOX-13, etc. with the prolongation of H₂O₂ stress time, the expression level was significantly enhanced, while FgPOX-1, FgPOX-21, etc. with the prolongation of H₂O₂ stress time, the expression level was reduced. This study clarified the number of genes, phylogenetic relationships, conserved domains of Fusarium graminearum peroxidase family, and the expression patterns of different tissues, infection processes and H₂O₂ stress in pathogens, which could provide basic datas for clarifying the function of Fusarium graminearum peroxidase family genes.

Infectious bursal disease virus (IBDV) infection cause IBD in chicken (Gallus gallus), which is an acute, highly contagious and immunosuppressive poultry disease. In order to explore the transcriptome changes in host cells infected by IBDV infection, cellular RNAs extracted from IBDV or mock infected DF-1 cells were used to build a library by using TruSeq^® RNA LT Sample Prep Kit v2, and then performed high-throughput sequencing on the Illumina HiSeq 3000 instrument. The transcriptome changes of DF-1 cells were analyzed at 12 h after IBDV infecting, and significantly altered genes were screened. The results showed that there were a total of 3 417 differentially expressed genes with 1 887 upregulated and 1 530 downregulated ((Fold Change, FC)>2 and P≤0.05). Systematic bioinformatics analysis with Gene Ontology (GO) and KEGG, revealed that IBDV infection could cause significant changes in more than 20 signaling pathways, such as immunity, apoptosis and metabolism in DF-1 cells. Among them, cellular immunity and apoptosis pathway might play important roles in IBDV infection and immune regulation mechanisms. Nine differentially expressed genes were randomly selected for qRT-PCR verification, and the changing trend was consistent with that of transcriptome data. This study provides reference information for revealing the molecular mechanism of IBDV pathogenesis and immunosuppression.

Infectious bursal disease (IBD) is an acute, highly contagious infectious disease caused by Infectious bursal disease virus (IBDV). The incidence rate of IBD in susceptible young chickens (Gallus gallus)can reach 80%~100%, and the mortality rate is as high as 30%~35%. To test the changes of long noncoding RNA (lncRNA) in host cells infected with IBDV, the expression profile of lncRNA in IBDV-infected and mock-infected chicken fibroblast line DF-1 cells were analyzed by high throughput sequencing. The results showed that there were 958 differentially expressed lncRNA. Among them, 728 were up-regulated and 230 were down-regulated. Target genes of the differentially expressed lncRNA were predicted. Functional annotation or enrichment analysis was performed by Gene Oncology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG). GO analysis suggested that target genes of the differentially expressed lncRNA involved in immune response, cell death, response to cytokine and I-κB kinase/NF-κB signaling. KEGG enrichment analysis suggested that these genes functioned in various cellular signaling pathways, including Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and apoptosis. Differentially expression of 9 randomly selected lncRNAs were verified by real-time quantitative polymerase chain reaction (qRT-PCR). The results were consistent with high throughput sequencing data. This is the first report of the differentially expression of lncRNA in DF-1 cells upon IBDV infection. This study provides a basis for further research on key lncRNA related to the host immunity and IBDV pathogenesis.

Endo-β-N-acetylglucosaminidase (ENGase) can cleave the N-linked oligosaccharides of glycoprotein and is an essential tool enzyme for protein deglycosylation. This study obtained the genome data of Enterococcus faecalis from the NCBI database and amplified the ENGase gene endoE (endoglycosidase E) by PCR. Then the expression vector pET-28a-endoE was constructed by homologous recombination and transformed into Escherichia coli BL21 star (DE3) competent cells. The recombinant protein was efficiently expressed in E. coli, and the deglycosylation properties of EndoE was systematically analyzed. The results showed that recombinant protein was expressed in soluble form in E. coli, and the yield was 45.6 mg/L after purified by Co²⁺ affinity chromatography and anion-exchange chromatography. The deglycosylation activity analysis indicated that the recombinant EndoE could hydrolyze the N-linked glycan of both natural and denatured ribonuclease (RNase B), ovalbumin (Ova) and immune globulin G (IgG). Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) proved that recombinant EndoE could remove the N-linked oligosaccharides in RNase B. In addition, in the temperature range of 20~50 ℃ or pH range of 4.0~6.0, EndoE had ideal hydrolytic activity. It also had tolerance up to 100 mmol/L DL-dithiothreitol (DTT), 1 mol/L NaCl, and 2% Triton X-100. Compared to the other reported ENGase from Enterococcus faecalis, EndoE has a broader range of substrates and is a prospective tool enzyme in protein deglycosylation study.

As a necessary technique in the obtaining of transgenic and gene editing pigs (Sus scrofa), embryo transfer technology has an important influence on the cloning efficiency. In this paper, the key points of pig cloned embryos transfer, such as the selection of recipient sows, the concurrent nature of receptor sow estrus and transplanted embryos, the operation method, the anesthesia method, the time of transfer, the number of transplanted embryos and the post-operative nursing, are reviewed and discussed. This paper provides reference for the operation of pig somatic cell cloning embryo transfer.

Sweet potato leaf curl virus (SPLCV), is one of the most important diseases on sweet potato (Ipomoea batatas), that infection reduced the yields of the most sweet potato growing area. Rapid and accurate detection of SPLCV is essential for diagnosis, prevention and control of the disease. In this study, a set of primers for loop-mediated isothermal amplification (LAMP) detection was designed based on the coat protein gene (cp) selected from the sequencing results of SPLCV,design a set of LAMP specific primers, with SYBR Green Ⅰ for evaluation indicator, optimize the reaction conditions, a rapid and visualized LAMP method for detection of SPLCV was established, this method were completed under isothermal conditions at 65 ℃ for 1 h. Specificity test results showed that SPLCV could be specifically detected by this method, and there was no cross reaction with other DNA pathogens such as other sweet potato DNA virus, Tomato yellow mosaic leaf curl virus (TYLCV) and Tobacco leaf curl virus (TLCV), indicated that the method had good specificity. The sensitivity results showed that the minimum detection limit of this method was 1 pg/μL of the SPLCV genomic DNA, which was 10 times of ordinary PCR. It showed that this method had high sensitivity and good repeatability. The technique of LAMP of SPLCV established in this study had the advantages of rapid amplification, high efficiency, good specificity, simple operation, no need for special instruments, visual observation results with the naked eye, and is suitable for rapid detection of SPLCV samples in the field and grass-roots departments.