Brucellosis is one of the most serious infectious diseases in sheep (Ovis aries) and goats (Capra hircus) husbandry. In goats, mutations located at upstream regulatory region of interferon regulatory factor 3 (IRF3) gene have benefit for their resistance to brucellosis infection. IRF3 gene is highly homologous between sheep and goats, and same mutations of IRF3 can be obtained by gene editing in sheep. Aiming to obtain IRF3 edited cell colonies and supply donor cells to generate cloned Brucella resistant sheep by nuclear tranfer. The upstream open reading frame (uORF) region of sheep IRF3 gene was modified by gene editing mediated by CRISPR/Cas9 and prime editing (PE) technology. According to the reference sequence of sheep IRF3 in NCBI database, primers were designed, and the DNA sequence of IRF3 gene in Hu sheep was cloned; Small guide RNA (sgRNA) targeting IRF3-uORF and primer editing sgRNA (pegRNA) sequence were designed based on cloned sequences. All sgRNAs were constructed into the expression vector. Fetal fibroblast cell lines derived from Hu sheep were transfected by CRISPR/Cas9 or PE expression vectors. After monoclonal cell screening, two positive monoclonal cell lines with 10 bp deletion in IRF3-uORF region were successfully obtained by CRISPR/Cas9 but no positive colonies were obtained by PE. This study provide new ideas for combining somatic cloning to obtain new Brucella-resistant sheep germplasm at animal level.

Lipoxygenase (LOX) is an important oxidoreductase in plants, which is widely involved in plant growth, development and response to biological and abiotic stresses. At present, the members of maize (Zea mays) LOX family and their roles in jasmonic acid (JA) signaling pathway are still not clear. In order to explore the function of LOX family genes in maize, bioinformatics methods were used to identify maize LOX family genes, and analyzed their phylogenetic evolution, conserved motifs, tissue expression specificity and expression patterns under biological and abiotic stresses. The expression of maize LOX family genes inoculated with Fusarium graminearum and induced by methyl jasmonate (MeJA) were detected by qRT-PCR. The results showed that maize genome contained 13 LOX family genes, which could be divided into 9-LOX, 13-LOX Type Ⅰ and 13-LOX Type Ⅱ, and all of them contained conservative lipoxygenase motif. The expression levels of ZmLOXs showed significant differences in different tissues or under different biological and abiotic stresses (P<0.05). After F. graminearum inoculation and MeJA treatment, the expression levels of 13 LOX family genes in maize were significantly up-regulated (P<0.05). These results suggested that maize LOX family genes might have important functions in maize growth and development and response to different biological and abiotic stresses. This study provides a reference for further understanding the function and mechanism of LOX family genes in maize.

Sugarcane (Saccharum officinarum) is an important sugar and biofuel crop, and its yield is affected by various biotic and abiotic stresses. S. spontaneum is one of the important parents in sugarcane cultivation and breeding. GRAS (GAI-RGA-SCR) transcription factors play important roles in the regulation of plant photomorphogenesis, seed germination, root growth and defense responses. In this study, 173 members of the GRAS gene family were identified from the genome of Saccharum spontaneum using the Hidden Markov Model (HMM) configuration file of the GRAS domain, which were distributed on 30 chromosomes of S. spontaneum. Phylogenetic analysis by maximum likelihood method showed that 173 GRAS genes of S. spontaneum could be further divided into 8 subfamilies. Motif and gene structure analysis showed that 173 GRAS genes contained at least one conserved GRAS domain at the C-terminal, and 61.2% of GRAS genes contained at least one intron. The analysis of cis-acting elements in the 2 000 bp sequence promoter upstream of 173 GRAS genes found that all GRAS promoter region had cis-acting elements in response to biotic and abiotic stresses, indicating that they might be involved in the regulation process of a variety of stress. The spatiotemporal transcriptional expression analysis found that the members of the GRAS family from S. spontaneum significantly expressed at different stages of stem and leaf development. qRT-PCR verified the expression of 9 genes in mature stem and leaf tissues, and the results were consistent with transcriptome data, suggesting that their family members played different roles in the growth and development regulation of S. spontaneum. The expression of SsPAT1.8-1 and SsDELLA6-2 were induced by the pathogen of sugarcane pokkah boeng disease; SsPAT1.8-2, SsPAT1.9, SsPAT1.10, SsDELLA2, SsSCR2, SsDELLA1-2, SsSCL3.1-2 and SsHAM9 were induced by Sugarcane mosaic virus (ScMV); SsSCL3.2-1 was induced by drought stress. qRT-PCR results showed that after ScMV infection, SsSCR2 was up-regulated in leaf +1 of resistant variety 'B48', SsHAM9 was up-regulated in leaf +1 and leaf -3, and SsLISCL11-1 was down-regulated in leaf -3. It is inferred that these GRAS family genes might play an important regulatory role in sugarcane response to different stresses. These results lay a foundation for further analyzing the function and regulation mechanism of GRAS family genes in sugarcane, and provide valuable gene resources for stress resistance breeding of sugarcane.

In tobacco production, the nicotine content of tobacco leaves in China is high to varying degrees, resulting in inconsistent chemical composition of tobacco leaves, which affects the industrial availability of tobacco leaves and causes a large backlog of tobacco leaves in stock. Therefore, effective reduction of nicotine content in flue-cured tobacco leaves has become an important issue in tobacco production. Based on the fact that mechanical injury such as topping can stimulate nicotine synthesis in tobacco plants by activating the jasmonic acid (JA) signal, this study explored the possible pathway of salicylic acid (SA) antagonizing JA signal to regulate nicotine synthesis in tobacco plants by using the antagonistic effect of SA and JA in signal transduction. Using Nicotiana tabacum cv. 'Yunyan 87' as the experimental material, the tobacco plants were subjected to mechanical damage treatments, such as topping and leaf picking, when they were growing to full flowering stage. The effects of SA on the expression of nicotine biosynthesis-related genes and the content of total nitrogen and nicotine in the tobacco plants were analyzed, and the transcriptomic analysis of the tobacco roots treated with JA, SA and JA+SA was performed. The results showed that multiple mechanical injuries further induced the expression of nicotine biosynthesis genes and the conversion of nitrogen to nicotine biosynthesis pathway. In contrast, SA could antagonize JA signaling, inhibit the expression of nicotine biosynthesis genes, hinder the conversion of nitrogen to nicotine biosynthesis pathway, and reduce the nicotine content in tobacco plants. The transcriptomics-based co-expression network analysis showed that 14 transcription factors belonging to the bHLH, AP2-EREBP and WRKY families, respectively, were not only significantly induced by SA, but also their expression patterns were negatively correlated with nicotine biosynthesis genes. In addition, promoter cis-element analysis revealed that the promoters of nicotine biosynthesis-related genes were widely distributed with cis-acting elements that could be recognized by bHLH, AP2/ERF and WRKY, which provided the possibility that bHLH, AP2-EREBP and WRKY transcription factors could repress the transcription of target genes by binding to the promoters. This study provides a reference for effective regulation of nicotine synthesis and improvement of tobacco leaf quality.

The OVATE family protein (OFP) is a plant-specific regulatory protein that plays a significant role in regulating plant morphogenesis and secondary cell wall formation. However, little information about the OFP gene family in Chinese fir (Cunninghamia lanceolata) is available. In this study, ClOFP genes were identi-fied based on the full-length transcriptome. Bioinformatics characteristics and evolutionary relationships were analyzed using online service. Besides, relative expression patterns of all members in different tissues and or-gans were determined by real time fluorescence quantitative PCR (qRT-PCR). Furthermore, the subcellular lo-calizations of all proteins were determined by transient transfection assays. The results showed that 6 ClOFP genes were identified from RNA-seq data, with a typical OVATE domain and contained no introns. The phylo-genetic analysis revealed that ClOFP were distributed in 5 subgroups. In addition, relative expression patterns of different tissues and organs varied differently. ClOFP1 was strongly expressed in young leaves and the rela-tive expressions in stems were approximately decreased with an increase of lignification. ClOFP2 dominantly expressed in female cone. ClOFP3 strongly expressed in root. The relative expression of ClOFP2/3 in low lig-nified stem (S1) was higher than that in others. ClOFP4 dominantly expressed in female cone, male cone and leaves. ClOFP5 was strongly expressed in male cone and the relative expressions of ClOFP5 in stems were ap-proximately increased with an increase of lignification. ClOFP6 was strongly expressed in stems and female cone. Moreover, the relative expressions of ClOFPs in leaves were decreased with an increase in maturity. The results of subcellular localization showed the localization of ClOFP1 in the cytoplasm, ClOFP2/3 in the plas-ma membrane and nuclear localization of ClOFP4/5/6. This suggested that ClOFPs might involve in different regulatory pathways in Chinese fir. Hence, ClOFPs might involve in regulating the development of cone, leaf, stem and other organs, as well as lignin synthesis. The research results provides a crucial theoretical basis for further research on organogenesis and wood formation of Chinese fir, At the same time, it also provides poten-tial genetic resources for molecular breeding of C. lanceolata.

Pyruvate dehydrogenase kinase 1 (PDK1) could be induced by hypoxia inducible factor 1α (HIF-1α) and participated in the regulation of glycolysis. The expression of vascular endothelial growth factor (VEGF) which is the target gene of HIF-1α up-regulated by PDK1. Furthermore, the PDK1 could promote the remodeling of lung structure. To reveal the expression and distribution characteristics of PDK1, HIF-1α and VEGF in the lungs of yaks (Bos grunniens) at different ages, and to explore their possible regulatory role in the adaptation of yak lungs to the hypoxia environment of the plateau, in this study, the lung tissues of healthy yaks aged newborn, 6-month-old, 3-year-old, and 6-year-old were collected. Hematoxylin-eosin (HE) staining, immunohistochemical staining, optical density analysis, qRT-PCR and Western blot were used to investigate the distribution and expression of PDK1, HIF-1α and VEGF in lung tissues of yaks in different age groups. The results of HE staining showed that the smooth muscle of the terminal bronchiole wall was thickened with age. The immunohistochemical and densitometric analysis results showed that PDK1, HIF-1α and VEGF were mainly distributed in the single-layer ciliated columnar epithelium of terminal bronchioles and their branches, as well as in the associated arteries and alveolar walls of yaks in different ages. Moreover, qRT-PCR results showed that the relative expressions levels of PDK1 and HIF-1α were the highest in the 6-year-old group, followed by the 3-year-old group. And there was no significant difference in the relative expression of VEGF among the 4 age groups. Additionally, the Western blot results showed that the expression of PDK1 protein in the 6-year-old group was significantly lower than that in newborn, 6-month-old and 3-year-old groups. The expression of HIF-1α and VEGF protein in the 6-year-old group was significantly higher than that in the newborn, 6-month-old and 3-year-old groups. Consequently, the protein expression of PDK1, HIF-1α and VEGF were negatively correlated with age. Expression of PDK1 increased and then decreased with age, while HIF-1α and VEGF showed an increasing trend. It was speculated that PDK1, HIF-1α and VEGF proteins could regulate glucose metabolism and vascular remodeling processes in the lung under hypoxia. In conclusion, PDK1, HIF-1α and VEGF might play an important role in the development of yaks lung and participate in the regulation of yaks adaptation to plateau hypoxia environment. This study provides basic data for comparative study of the adaptation mechanism in yaks of different ages to high altitude hypoxia.

Epidermal growth factor (EGF) and its receptor (epithelial growth factor receptor, EGFR) can regulate the growth and survival of endometrial epithelial cells to a certain extent, hypoxia-inducible facto-2α (HIF-2α) is one of the main factors regulating the internal environment of the uterus and the adaptation of animals to the hypoxic environment, all of which can affect the development and normal function of the uterus. In this study, the expression of EGF, EGFR, and HIF-2α in the yak (Bos grunniens) uterus was localized and detected by immunohistochemistry (IHC), qPCR and Western blot. The expression of EGF, EGFR, and HIF-2α in the yak uterus at different stages of life was analyzed by qPCR and Western blot to investigate the molecular mechanisms involved in the reproductive development of yaks. IHC results showed that EGF, EGFR, and HIF-2α were mainly distributed in the cytoplasm, uterine gland, vascular endothelial cells, and lymphocytes of the yak endometrial epithelium in 3 periods of the uterus. The qPCR results showed that the relative expression of EGF in the luteal phase was significantly higher than that in the follicular phase and gestational stage (P<0.05), and the relative expression of EGFR and HIF-2α in the follicular phase was significantly higher than that in the luteal phase and pregnancy (P<0.05). The results of Western blot showed that the relative expression of EGF, EGFR, and HIF-2α was significantly higher than that in the follicular stage and luteal phase during pregnancy (P<0.05). In conclusion, EGF, EGFR, and HIF-2α were widely distributed in uterus, and the expression of the 3 were different at different times, suggested that they might play an important role in yak reproduction. This study further elucidates the changes of expression of EGF, EGFR, and HIF-2α in yak uterus from estrus to pregnancy, and provides basic information to study the role of the three factors in the development mechanism of endometrial of Yak at different stages of reproduction cycle.

Inhibitor of DNA binding 1 (ID1) is widely present in the body, and affects cell proliferation and apoptosis. To reveal the role of transcription factor ID1 in ovarian follicular granulosa cells of sheep (Ovis aries), the eukaryotic overexpression vector pcDNA3.1-ID1 was constructed and ID1 RNA interference fragment (siID1) was designed and synthesized. The expression level of ID1 gene was detected by qRT-PCR after 24 h of isolated and cultured ovarian follicular granulosa cells treated with follicle-stimulating hormone (FSH). The results showed that the expression of ID1 gene was extremely significantly decreased after FSH treatment (P<0.01). CCK-8 assay was used to detect the proliferation of granulosa cells within 5 d after transfection with pcDNA3.1, pcDNA3.1-ID1, siNC and siID1 in granulosa cells. After 48 h of transfection, JC-1 assay was used to detect the apoptosis of granulosa cells and the expression levels of proliferation and apoptosis genes were detected by qRT-PCR. The result showed that the survival rate of ID1 overexpressed granulosa cells was reduced, the green fluorescence of JC-1 was enhanced, the expression levels of proliferation-related genes cyclin dependent kinase 2 (CDK2)、cyclin dependent kinase 4 (CDK4) and cyclin D2 (CCND2) were significantly down-regulated (P<0.05), the expression of apoptosis-related gene B-cell lymphoma-2 (Bcl2) was significantly decreased (P<0.05), Bcl2-associated X protein (Bax) gene expression was significantly increased (P<0.05), and caspase-3 (Casp3) gene expression was extremely significantly increased (P<0.01). The higher survival rate of granulosa cells and the lower green fluorescence of JC-1 were found in cells with inhibited ID1 expression. The expression levels of proliferation-related genes CDK2 and CCND2 were significantly increased (P<0.01), CDK4 gene was significantly increased (P<0.05), the expression of Bcl2 was significantly increased (P<0.01), and the expression of Bax was significantly decreased (P<0.05). In summary, transcription factor ID1 inhibited the proliferation and promoted the apoptosis of sheep granulosa cells. This study provides a basis for exploring the biological functions of ID1 in follicular granulosa cells and revealing its role in ovarian follicle development.

Glycoprotein 2 (GP2) is a kind of glycosylphosphatidylinositol-anchored proteins (GPI-APs) produced by mucous glands and secretory cells, it can ingest pathogenic bacteria in the intestinal cavity and is of great significance to maintain the homeostasis of the body. In order to obtain the antibody against Bactrian camel (Camelus bactrianus) GP2, the bactrian camel GP2 gene was cloned into pET-28a, constructing the recombinant plasmid pET-28a-GP2, the recombinant plasmid pET-28a-GP2 was then transferred into Escherichia coli BL21 for induction and expression. After the optimization of induction conditions, the expression of GP2 recombinant protein was detected by SDS-PAGE and Western blot. To prepare rabbit polyclonal antibodies against GP2 with target protein, The titer and specificity of the antibody was detected by ELISA and Western blot, immunohistochemical staining was used to understand its expression in ileum. The results showed that the size of GP2 protein was about 65 kD, the optimal induction conditions were 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) 6 h, mainly expressed in the form of inclusion bodies. The polyclonal antibody was used at a titer of 1∶2.56×105 to specifically recognize recombinant protein GP2 as determined by ELISA and Western blot. Immunohistochemical staining results showed that GP2 is mainly expressed in follicular associated epithelium of ileum, and expressed on the cell membrane of microfold cell (M cell). This study provides a reference for the follow-up study of GP2, enriches the relevant theories of mucosal immunity, solves the problem of no commercially available antibodies on the market at present, and provides antibody support for the development of new drugs.

Oxidative stress mediates apoptosis of goose (Anser cygnoides) follicular granulosa cells (GCs), thus affecting laying performance. To investigate the effects of vitamin E (VE) on goose follicular GCs, an oxidative stress model was established by adding H2O2 (100 μmol/L), and the effects of oxidative stress on apoptosis in goose follicular granulosa cells were examined in vitro. Four different concentrations of proanthocyanidins, resveratrol, gallic acid and VE were added to the model, and the best reliever and optimum concentration were screened out. Furthermore, ELISA, qPCR and immunofluorescence detection techniques were used to explore the effect of the best alleviator VE on the apoptosis of goose follicle GCs mediated by oxidative stress. The results showed that 100 μmol/L H2O2 treatment for 12 h extremely significantly increased the level of apoptosis in follicular GCs (P<0.01). Among the 4 different antioxidants, 40 μmol/L VE had the best alleviation effect on the oxidative stress model (P<0.01). The addition of VE significantly inhibited the release of lactate dehydrogenase (LDH) (P<0.05), and reduced the expression of oxidative stress-related genes superoxide dismutase 1 (SOD-1), SOD-2, catalase (CAT), cyclooxygenase 2 (COX-2) and reactive oxygen species (ROS) content (P<0.05). Besides, GCs proliferation viability increased significantly (P<0.05) and there was a phenomenon of dose dependence. The expression levels of anti-apoptotic genes B-cell lymphoma/leukemia-2 (Bcl2) were significantly increased (P<0.05), but pro-apoptotic genes cysteinyl aspartate specific proteinase 3 (Caspase3), Caspase9 and tumor protein p53 (p53) were significantly decreased (P<0.05), as well as the number of apoptotic cells were significantly reduced after VE treatment (P<0.05). In conclusion, the goose follicle GCs had obvious apoptosis under oxidative stress. Adding VE could inhibit oxidative stress and relieve goose follicle GCs apoptosis. The study can provide reference for improving goose laying performance.

As highly competitive endogenous RNA (competing endogenous RNA, ceRNA), circular RNAs (circRNAs) regulate cell differentiation, proliferation, apoptosis and energy metabolism through the sponge effect of miRNA. Lycium ruthenicum is rich in anthocyanin, which has remarkable antioxidant activity and can effectively reduce the hypoxic injury of cardiomyocytes. In this study, RNA-seq technology and bioinformatics methods were used to screen differentially expressed circRNA, miRNA and mRNA, and construct circRNA- miRNA-mRNA regulatory network. qRT-PCR was used to screen ceRNA relationship pairs that might be involved in the regulation of L. ruthenicum anthocyanins in hypoxic-induced H9c2 rat cardiomyocytes. The results showed that the differentially expressed 1 570 mRNAs, 5 miRNAs and 4 circRNAs were hypoxic- related molecules, which might be involved in the regulation of L. ruthenicum anthocyanins in hypoxic- induced H9c2 rat (Rattus norvegicus) cardiomyocytes. Based on pearson correlation analysis and target relationship prediction, the ceRNA network was successfully constructed, and 2 candidate circRNA-miRNA- mRNA relationship pairs were screened by qRT-PCR. This study provides basic data to further explore the mechanism of circRNA and its target genes involved in regulating the protective effect of anthocyanin in L. barbarum on cardiomyocytes.

Tripartite motif containing 25 (TRIM25), a member of the TRIM family, plays an important role in the host antiviral immune response. To understand the molecular characteristics of TRIM25 in rainbow trout (Oncorhynchus mikiss) and its expression changes during infection with Infectious hematopoietic necrosis virus (IHNV), in this study, the full-length cDNA sequence of TRIM25 (GenBank No. OL692831) was obtained by rapid amplification of cDNA ends (RACE) technique and bioinformatically analyzed. At the same time, the qPCR technique was used to detect changes in TRIM25 expression in different tissues of healthy rainbow trout and at different time points (0, 6, 12, 24, 48, 72, 96, 120 and 144 h) after infection with IHNV in the intestine, liver, spleen, skin, head kidney and gill. The results showed that the full-length sequence of the rainbow trout TRIM25 cDNA, 4 745 bp, had an ORF of 2 028 bp and encoded 675 amino acids. Bioinformatics analysis showed that the TRIM25 protein had a molecular weight of 76.04 kD, a theoretical pI of 8.79, an instability coefficient of 49.2 and an average hydrophilic coefficient of -0.591, indicating TRIM25 was an unstable hydrophilic protein. Analysis of the structural domains of TRIM25 protein revealed that the protein included RING, B-boxes, B-Box C-terminal domain and PRY-SPRY structural domains. The homology and phylogenetic tree analysis showed that rainbow trout had the highest homology (94.96%) with salmon (Oncorhynchus keta) and was closely related, but had low homology with mammals and amphibians. qPCR showed that TRIM25 was widely expressed in all tissues of healthy rainbow trout, with its highest expression in liver, followed by that in head, kidney and brain, and the lowest expression in skin. After infection with IHNV, the expression of TRIM25 gene in liver, head kidney, spleen, intestine and skin peaked at 48 h, and gill reached the peak at 96 h. Expression of each tissue at the peak was extremely significant compared to the control (P<0.01), with the most significant changes in expression in head and kidney and spleen, which were 5.72 and 4.33 times of the control group, respectively. The above results suggested that TRIM25 played an indispensable role in the immune response of rainbow trout against IHNV infection. This study provides basic data for an in-depth study of the role of TRIM25 in the regulation of antiviral immunity in fish.

The farming of Whitmania pigra was experiencing with the problems of single food type and food shortage supply, which limited the development of farming. Therefore, developing the various types of food for W. pigra was becoming urgent. The aim of the present study was to evaluate the effect of mixed feeding to W. pigra with Chlorella vulgaris and Sinotaia quadrata on growth, digestion, immune and pharmaceutic quality. The healthy W. pigra (initial weight (3.512±0.002) g) was divided into 3 groups randomly, with triplicates. They were feed with C. vulgaris individually (Algae group, CV), C. vulgaris and S. quadrata together (algae and snail mixed group, CV+SQ) as well as S. quadrata individually (snail group, SQ) for 30 d. The results showed that CV+SQ group obtained significantly higher final body weight (FBW), weight gain (WG), feed intake (FI) compared to other groups (P＜0.05), and specific growth rate (SGR) and feed conversion ratio (FCR) were significantly better than CV group (P＜0.05). For body composition, the leucine and lysine contents in CV+SQ group was significantly higher than those in SQ group (P＜0.05). The aspartic acid content was in opposite tendency. In addition, CV+SQ group exhibited significant higher α -amylase and antithrombin activities than other groups (P＜0.05), higher lipase and protease activities than CV group (P＜0.05), as well as higher superoxide dismutase (SOD) and catalase (CAT) activities than SQ group (P＜0.05). SDS-PAGE result indicated that the band of peptide with around 30 kD was in higher density in the CV+SQ group than in other groups. The transcription levels of insulin-like growth factors-1 (IGF-1), inorganic pyrophosphatase (PPase), α-glucosidase (α-GLU), SOD, alkaline phosphatase (AKP), CAT and hirudin (WP) in CV+SQ group were significantly higher than those in SQ group (P＜0.05). In conclusion, mixed feeding to W. pigra with C. vulgaris and S. quadrata promoted the growth, digestive capacity, pharmaceutic quality and immune capacity. This study provides a theoretical basis for optimizing feeding strategies of the farming of W. pigra.

Anguillid herpesvirus (AngHV) is an important viral pathogen of eel (Anguilla), it causes“mucus sloughing and hemorrhagic septicemia disease”on cultured eels, which brought huge economic losses to the farmers. The study of immunogenic protein of AngHV is of great significance for the development of immunological diagnostic technology and subunit vaccine of AngHV. In order to obtain the immunogenic proteins of AngHV, in this study, mass spectrometry analysis on the viron proteins was conducted. The obtained protein ORF36 was further analysed by bioinformatics software. Then the sequence of ORF36 was cloned into expression vector pET-30a, transformed into Escherichia coli BL21, and induced by isopropyl β-D-thiogalactoside (IPTG) for prokaryotic expression. After that, the anti-ORF36 polyclonal antibody was prepared by immunizing rabbits (Oryctolagus cuniculus) using the expressed recombinant protein. The titer of the polyclonal antibody was tested, followed by the specific detection on the prepared antibody by the eel ovary cell line (EO) cells and host tissues infected by AngHV. The sensitivity and neutralization effect of the antibody on AngHV was evaluated, then the virion localization analysis of ORF36 was conducted. According to the results of mass spectrometry, the main immunogenic protein of the virion was identified to be ORF36. Bioinformatics analysis showed that ORF36 protein had no transmembrane domain or signal peptide. Thirteen B cell epitopes were predicted, which indicated that ORF36 had good immunogenicity. ORF36 was cloned into prokaryotic expression vector pET-30a, and induced for protein expression. SDS-PAGE results showed that high level of expressed ORF36 was achieved in E. coli BL21 (DE3), with the size of 40 kD and mainly existed in the form of inclusion body. ELISA results showed that the titer of the anti-ORF36 polyclonal antibody was 1: 8 000. Western blot results showed that the prepared antibody could specifically recognize infected EO cells, as well as the gill, fin and skin mucus tissues of eels infected by AngHV. Sensitivity evaluation results showed that the antibody could detect a minimum level of 1 000 PFU virions. The neutralization effect test results showed that the antibody significantly reduced the viral titer, revealing the neutralizing effect of the antibody on AngHV. Western blot on the envelope, nucleocapsid, and purified virions demonstrated that ORF36 was the structural protein of virion and localized on the nucleocapsid. In conclusion, the main immunogenic protein ORF36 of AngHV virion was identified, and the polyclonal antibody against ORF36 with virus neutralization effect was prepared, and ORF36 was identified as the nucleocapsid structural protein of AngHV. These results would lay a research foundation for elucidating the role of ORF36 in AngHV infection and development of the viral subunit vaccine.

Carotenoids and apocarotenoids play essential roles in fruit coloration and characteristic aroma formation, plant antioxidant and abiotic stress resistance, as well as in improving human immunity and anti- aging. Sweetpotato (Ipomoea batatas) is the fourth largest food crop in China, revealing the metabolic regulation mechanisms of carotenoids and apocarotinoids in sweetpotato would facilitate the development of high-quality sweetpotato varieties with high nutritional quality, good-looking and wide adaptive to harsh environmental conditions. The complexity of self-incompatibility and partial cross-incompatibility in sweetpotato, results in limitations of traditional hybrid breeding methods in sweetpotato variety improvement and breeding. In this paper, the contents and types of carotenoids in tuberous roots of sweetpotato with different flesh colors were described, the research progress in regulation of carotenoid metabolism-related genes in sweetpotato by molecular biology technology were reviewed, and the possible problems and suggestions in current research on carotenoid metabolism of sweetpotato were summarized. This review provides new ideas for the cultivation of sweetpotato with highly carotenoid-enriched and abiotic stress- resistant sweetpotato.

With the advent of CRISPR/Cas system, plant genome editing has entered a new era of precise editing for any genes of interest. The CRISPR/Cas toolkits have been successfully tested on a wide variety of fruit tree species. According to the statistics of breeding research, researchers have edited about 45 fruit tree genes by CRISPR/Cas genome editing technology, which were mainly used to enhance disease resistance, prolong shelf-time, early flowering, tree dwarf and increase shoot branching. However, the fruit tree genome editing still relies mostly on Cas9-based indel mutation and Agrobacterium-mediated stable transformation. Transient transformation for transgene-free genome editing is preferred, but it typically has very low efficiency in fruit trees, substantially limiting its potential utility. In this review, the CRISPR/Cas system and its related technologies were summarized, and then we introduced the delivery methods of the CRISPR /Cas expression vector into plant genome and the current status of fruit tree genome editing practices using the CRISPR/Cas system, and discussed the problems impeding the efficient application of the CRISPR/Cas toolkits for fruit trees genome editing, as well as future prospects. This review provides reference for research on gene function and molecular breeding of fruit trees.

Nitrogen is the most important element affecting the yield and quality of rice (Oryza sativa). In the process of rice N-efficient breeding, molecular marker-assisted selection for target genes is helpful for efficient and convenient screening of N-efficient rice varieties. Transcription factor TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS) is involved in the regulation of various developmental processes. OsTCP19 is a newly reported nitrogen-efficient gene with 29 bp deletion in its promoter region. In this study, Pro TCP19-F/R was designed to develop molecular markers to identify rice materials. By comparing and analyzing the OsTCP19 promoter region of 33 reference genomic materials, it was found that 29 bp deletion of OsTCP19 promoter region was found in 'Tumba', 'CG14', 'IR64', 'N22', 'Shuhui 548', 'Basmati' and 'ZH11'. Using 'Shuhui 548' as positive control, OsTCP19 genotypes of 23 rice breeding parents, F1 generation and its paternity, and 'Xikeshui 646' hybrid lines were identified. The results showed that 'Xikeshui1288', 'Kasalath', 'IRAT109' had a deletion of 29 bp, which was consistent with 'Shuhui548' and identified as ostcp19 mutant genotype. The test-cross F1 generation of 'Xikehui1288' and 'Xikehui646' contained 2 strips, which were identified as heterozygous type. Two genotypes could be identified by single plant identification of 'Xikehui646'. Therefore, the marker identification method could identify OsTCP19 genotype and seed purity efficiently and accurately, and the identification method is simple, fast and low cost, which has potential popularization value in rice breeding.

Jiaobai is an edible fleshy stem formed by the stem of Zizania latifolia infected by Ustilago esculenta. Gray Jiaobai often appears in field production, which seriously affects the yield and quality of Jiaobai. The infection and proliferation of T-type U. esculenta is the main factor causing gray Jiaobai, normal Jiaobai was formed by the infection of MT type strain, and long-term asexual breeding makes T-type strain and MT strain coexist in Z. latifolia seedlings. Therefore, it is very important for the development of Z. latifolia industry to purify the strains in Z. latifolia seedlings and eliminate T-type strain, so as to control the production of gray Jiaobai from the source. A specific detection method for T-type strain was established, and the detection limit was 0.008 8 ng/µL. Then, through the detection of the relative content of T-type strains in seedlings under different seedling raising methods, it is clear that the shell seedling of "with Jiaobai" seedling was the best way of initial purification, and the seedling raising in the upper part of bolt tube was the best way of seedling expansion. Based on the fact that the adaptability of T-type strain to stress was much higher than that of MT-type strain, finally, a small number of purified Z. latifolia seedlings without gray Jiaobai were obtained by 5 years stress treatment of Z. latifolia seedlings and the detection of T-type U. esculenta. The above results have theoretical and practical guiding significance for the breeding of Jiaobai seedlings.

Haemophilus parasuis (HPS) is the pathogen causing Glasser's disease, which is one of the most serious bacterial pathogens that harm the health of pigs clinically. There are many serotypes of HPS, serotype 4 is the most isolated serotype and the more serious serotype. crp gene encodes cAMP receptor protein (crp) and crp is one of the most important systemic regulatory factors and plays a crucial role in adapting to environmental changes during bacterial infection. In order to study the effect of crp gene on biological characteristics of HPS type 4 (HPS4) such as growth, resistance and virulence, the crp gene deletion strain Δ^crp of HPS type 4 clinical isolate HPS4 was constructed by natural transformation method. Morphological characteristics, growth characteristics, biofilm formation, tolerance pressure, serum bactericidal activity, iron utilization and mice (Mus musculus) infection assays of HPS4 and Δ^crp were studied. The results showed that crp deletion strain Δ^crp of clinical isolate of HPS4 was successfully constructed in this study. After crp was deleted in HPS4, the growth was significantly slowed down, the biofilm formation ability was significantly weakened, the tolerance to osmotic pressure, oxidative stress and heat stress was decreased, the serum survival ability was decreased, the iron utilization was decreased and the virulence of mice was decreased. The results indicated that crp gene had significant effects on growth, resistance and virulence of HPS4. This study provides basic data for further exploring the function of crp gene and screening attenuated strains of HPS4.