The solute carrier family 25 (SLC25) members participate in regulation of anthocyanin biosynthesis. The SLC25 family member SLC25A4 is adenine nucleotide translocase 1 (ANT1), which plays important roles in growth and development, and response to stresses of plant. To enrich the research of SLC25A4 in wheat (Triticum aestivum) and explore biological functions of SLC25A4 in the regulation of anthocyanin biosynthesis, the expression of SLC25A4 was analyzed based on transcriptome sequence results at different developmental stages of 'Guizi 1' wheat grains and was further verified by qPCR. The results demonstrated that the expression of TaSLC25A4-7A, TaSLC25A4-7B and TaSLC25A4-7D1 genes was significantly higher at 25 days post anthesis (DPA) and 35 DPA than that at 10 DPA (P<0.05), particularly qPCR analysis showed that the dynamic change of TaSLC25A4-7A gene expression was consistent with that of anthocyanin accumulation. Furthermore, TaSLC25A4-7A gene overexpression transgenic tobacco (Nicotiana tabacum) lines were obtained, qPCR results showed that the expression level of anthocyanin synthesis related genes (NtDFR, Nt4CL, NtCHI and NtF3H) was significantly higher in transgenic lines than that in wild type (P<0.05), which demonstrated that TaSLC25A4-7A involved in the biological process of regulating anthocyanin synthesis. The results of carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment to transgenic lines and wild type demonstrated that the TaSLC25A4-7A overexpression transgenic lines were more sensitive to CCCP. The above results demonstrated that TaSLC25A4 gene (especially TaSLC25A4-7A) is involved in anthocyanin synthesis, which provides reference for further exploring the biological function of the TaSLC25A4 gene in anthocyanin accumulation and stresses response of wheat.

Ostrinia furnacalis, Mythimna separata and Helicoverpa armigera are the main lepidopteran pests to corn. In order to assess the resistance of transgenic cry1A. 301 maize to these insect pests, two events of CM8201 and CM8204 were evaluated through in vitro tissues laboratory bioassay and artificial infestation in field trials. The results of laboratory bioassay showed that the larval mortality rate of O. furnacalis, M. separate and H. armigera was above 95% when the larvae fed on the heart leaves, silks and grains of CM8201 and CM8204 after 5 d. Hence, the heart leaves, silks and grains of CM8201 and CM8204 showed high resistance to these pests. The results of insect identification on field showed that at whorl stage, as far as the resistance to O. furnacalis be concerned, the mean values of feeding grade of CM8201 and CM8204 were 1.17 and 1.18, respectively. The pest grade was 1. The resistance level was high. As far as the resistance to M. separate be concerned, the mean values of feeding grade of CM8201 and CM8204 were 1.38 and 1.35, respectively, and the resistance type was high. At silking stage, as far as the resistance to O. furnacalis be concerned, the average damage level of female ear of CM8201 and CM8204 was 1.27 and 1.30, respectively. As far as the resistance to H. armigera be concerned, the average damage level of female ear was 0.15 and 0.20, respectively. In conclusion, the transgenic insect-resistant maize CM8201 and CM8204 showed good insecticidal effects against 3 major maize lepidopteran pests at all stages of maize growth. This study provides material basis for the breeding of new insect-resistant transgenic maize varieties.

Dehydration responsive element binding protein (DREB) transcription factors play an important role in plant development and response to various stresses. The study on its DREB transcription factors of Brachypodium distachyon would promote the elucidation of molecular mechanism of the stress resistance of DREB transcription factors in gramineous crops. The early research work on the transcription profiles from the microarray study of DREB transcription factors showed that BdDREB-30 (GenBank No. XM_003561067.4) responds to various stresses. In order to further explore its function, the expression patterns of BdDREB-30 gene in different tissues and under 7 abiotic stresses were studied by qPCR, and the function of its promoter were studied by GUS staining. The results showed that the expression of BdDREB-30 was the highest in stems and its expression under drought and H2O2 stresses was significantly higher than that of the control. The results of multi-sequence alignment with other proteins showed that BdDREB-30 protein contained the typical AP2/ERF domain of DREB/CBF subfamily and the characteristic sequence of A-1 subclass. Evolutionary analysis showed that BdDREB-30 was relatively close to the homologous of wheat crops such as barley (Hordeum vulgare) and rye (Secale cereale). The analysis results of cis-acting elements of promoter showed that the promoter of BdDREB-30 gene contained many light response elements, plant hormone response elements and stress response elements. In order to further study the expression characteristics of the promoter, it was constructed into plant expression vector and transferred into tobacco (Nicotiana tabacum). The plant leaves of transgenic tobacco under low temperature, drought and salt stresses were stained with GUS. GUS staining showed that the promoter of BdDREB-30 gene was strongly induced under drought stress, indicated that the promoter of BdDREB-30 gene was a drought inducible promoter. This study provides a theoretical basis for the study of the molecular mechanism of BdDREB-30 gene in response to stresses.

microRNA171b (miR171b) is a member of miR171 family and plays an important role in plant growth and development, response to abiotic and biological stresses. To reveal the function of miR171b in eggplant (Solanum melongena) response to Verticillium dahliae, using eggplant cultivar 'Suqi No. 1' as the test material in this study, the pri-miR171b gene was cloned and its expression vector was constructed. The miR171b transgenic eggplant plants were regenerated through Agrobacterium tumefaciens-mediated genetic transformation and analyzed their resistance to Verticillium wilt. The disease resistance analysis showed that the miR171b overexpression line was more resistant to the infection of V. dahliae with disease index of 18.5, which was about 1/3 to 1/2 of the control, while miR171b antisense inhibition lines were more sensible with disease index of 68.4 and 72.6, which were 1.5~1.6 times of the control. The quantitative analysis of pathogen internal transcribed spacer (ITS）showed that the content of V. dahliae in the vascular tissues of miR171b overexpression line was significantly lower than that in the control (P<0.05), and its content in miR171b antisense lines was significantly increased (P<0.05). Antioxidant enzyme activity analysis showed that the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzyme in miR171b overexpression line were higher than those in the control, while their activities were lower in the antisense lines than in the control. The above results showed that miR171b was involved in the defense process of eggplant against Verticillium wilt, and played a positive regulatory role. This study provides a theoretical basis for the breeding of eggplant cultivars with the Verticillium wilt resistance.

Salt stress is one of the main abiotic stresses that restrict plant growth and development. In this study, the halophyte Sesuvium portulacastrum seedlings were treated with 400 and 800 mmol/L NaCl for 0, 6,12, 24, 48 and 72 h, and the changes of physiological indexes in roots and leaves and the expression level of salt-related genes were analyzed. The results showed that Sesuvium portulacastrum seedlings withered at the beginning of stress, while the growth gradually stabilized with the extension of stress time when treated with 400 mmol/L NaCl. While the seedlings wilted and died gradually with the extension of stress time under 800 mmol/L NaCl. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) in leaves and roots all increased significantly under 12 h treatment. The accumulation of soluble sugar and proline increased obviously, and then the enzyme activity and osmotic regulation substances contents decreased. The relative conductivity increased first and then decreased. Four salt-tolerant genes were selected for real-time fluorescence quantitative verification. The expression levels of salt-related genes, plasma membrane Na+/H+ antiporter gene salt overly sensitive 1 (SOS1), protein kinase gene CBL-interaccting protein kinase 8 (CIPK8), Calcineurin B-like 10 (CBL10) and plasma membrane H+-ATPase gene (AHA1), were up- regulated in the roots and leaves under salt stress. The expression levels of SpSOS1, SpCIPK8, SpCBL10 and SpAHA1 genes under 800 mmol/L NaCl reached the highest at 12 h after treatment, and the expressions were 6.80, 124.32, 25.93 and 9.52 times of those under 400 mmol/L NaCl at the same time. This study provides a theoretical basis for the study of physiological indexes and related gene expression regulation of plants under salt stress.

Litsea cubeba is one of the important woody spice species in southern China and is extremely susceptible to biotic and abiotic stresses during growth process. WRKY transcription factors play important roles in plant growth and development, stress response and other physiological processes. In this study, 41 LcWRKY genes were identified based on bioinformatics analysis, sorted into lines and explored for their expression specificity in different tissues. Phylogenetic development revealed that members of the LcWRKY family were divided into 2 taxa, with the 2nd taxon containing 5 subclasses (Ⅱ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d, and Ⅱ-e). The results of conserved motif and structural domain analysis of the LcWRKY genes confirmed the accuracy of the phylogenetic clustering analysis. Protein interaction network co-expression analysis showed that LcWRKY36, LcWRKY38, and LcWRKY39 were at the centre of the common thread, presumably playing key roles in the regulation of Litsea cubeba in response to disease and low phosphorus stress. Analysis of transcriptome data from different tissues revealed that 41 LcWRKY members differentially expressed in different tissues, with 10 genes, including LcWRKY1, LcWRKY3 and LcWRKY8, showing high expression in roots, with LcWRKY8 being 25-fold more expressed in roots than that in leaves, presumably playing an important role in root development and stress. In addition, the relative expression of LcWRKY29, LcWRKY10, LcWRKY15, LcWRKY22, and LcWRKY1 were higher in fruits, which were presumed to be involved in the biosynthesis of secondary metabolites such as citral in bilberry fruits. The present study provides a basis for further analysis of the functions of WRKY transcription factors in L. cubeba.

Aquaporins (AQPs) are highly conserved membrane proteins that facilitate the passive movement of water and other small hydrophilic molecules across biological membranes, playing essential roles in the physiological processes of seed germination, flowering, and fruit development, etc. In this study, 28 APQ gene family members (PpAQP1~PpAQP28) were identified from the peach (Prunus persica) genome, which could be categorized into 5 subfamilies, including plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and uncategorized X intrinsic proteins (XIPs) based on their phylogenetic relationships. Analyses of the chromosomal assignment, gene structures and conserved motifs of these PpAQP members were further performed. Combining with the transcriptome and qPCR detection, tissue-specific expression profiles of these PpAQP genes and their expression patterns at various fruit development stages were analyzed. These results demonstrate the comprehensive expression differentiations of PpAQPs among and within the sub-families, 8 PpAQPs were highly expressed in fruits with their expression high peaks in the early stage of fruit development, suggested their potential roles in the first rapid growth period of fruit development. This research offers a reference for subsequent functional studies of AQPs in fruit development.

Stearyl-CoA desaturase (SCD) is the key enzyme of monounsaturated fatty acid biosynthesis, which plays an important role in the lipid synthesis and oxidation dynamic balance. To explore the qinghai plateau yak SCD gene polymorphism and its correlation with growth traits, DNA sequencing method was used for gene polymorphism analysis,linkage disequilibrium analysis and the method of general linear model were used for correlation analysis. The results showed that there were 2 SNPs in SCD gene (g. 6614C>A and g.6660C>T), there were 2 genotypes in g.6614C>A and 3 genotypes in g.6660C>T. χ2 test showed that the g.6614C>A balanced in the Hardy Weinberg lattice state, g.6660C>T was in a state of imbalance. g.6614C> A was low polymorphic (PIC<0.25), and g.6660C>T was moderately polymorphic (0.50>PIC>0.25). And growth character correlation analysis found that the body weight, body height, body oblique length and tube circumference of CA genotype at g.6614C>A locus were significantly higher than those of CC genotype (P<0.05), but there was no significant difference in chest circumference between CA genotype and CC genotype. g. 6660C>T site of TT genotype individuals had no significant difference with CT and CC genotype, individuals weight, body height and chest circumference of TT genotype were significantly higher than CC and CT genotype (P<0.05), while TT genotype tube circumference was extremely significantly higher than CC genotype (P<0.01). Haplotype and linkage disequilibrium analysis showed that there were 4 haplotypes in the 2 loci, and there was no strong linkage between the 2 loci (r2=0.027). The combined haplotype analysis showed that H1H3 haploid type was the optimal combination. In conclusion, SCD gene polymorphism was related to some growth traits of Qinghai plateau yak, which can be used as candidate genes for marker-assisted selection in yaks. This study provides reference for improving yak breeding.

The quality and yield of cashmere determine the economic value of cashmere goats (Capra hircus), and hair follicle is the direct tissue to regulate the growth and properties of cashmere. The properties of cashmere can be improved by regulating the development of hair follicle. Wingless-type mouse mammary tumor virus integration site family, member 5A (Wnt5A) gene is an key member in the signaling pathway relates with hair follicle development and play a key role in hair follicle development, but its research in goat is still very limited. In order to well know the molecular genetic characteristics of goat Wnt5A, the variation of this gene was detected by combining PCR-single strand conformation polymorphism (PCR-SSCP) and sequencing in Zhongwei goats, Chaidamu Cashmere goats and Longdong Cashmere goats. The tissue expression characteristics of Wnt5a in Liaoning Cashmere goats and Longdong Cashmere goats were analyzed by qPCR. The results showed that, in the 3 populations, there were 2 mutation sites in the Wnt5A exon 4 amplification region, which were c. 334c>A and c. 213a>G, respectively. Three alleles (A, B and C) were formed and showed five genotypes (AA, AB, AC, BB and BC). In Chaidamu cashmere goat and Longdong cashmere goat, A was the dominant allele, AB was the dominant genotype, while B in Zhongwei goat was the dominant allele and BB was the dominant genotype. Two mutation sites c. 685-5 C > T and c. 685-55 G>A were detected in Wnt5A exon 6, forming 2 alleles D and E, forming 3 genotypes DD, DE and EE. E was the dominant allele and DE was the dominant genotype in the 3 populations. Six haplotypes of H1~H6 were constituted by 2 amplified regions, and the most common one was H2. Wnt5A gene was expressed in all these 7 tissues of Liaoning Cashmere goat and Longdong Cashmere goat, with differences between breeds. At the same time, the expression difference in skin was significant, which speculated that resulted from the correlation between Wnt5A gene expression and the cashmere traits. By comparing the variation and expression characteristics of Wnt5A gene in different goat breeds, the molecular genetic characteristics of the gene were analyzed, which provided basic data for enriching the content of goat genome and the development and utilization of the gene.

There are many stresses during the rearing stage, but transport stress is unavoidable, such as capture, crowding, fasting are the most common transport stressors. These stressors can lead to reduced production performance of goat (Capra hircus) and even cause disease and huge economic losses. Since intestinal function is closely related to production performance, the changes of tight junction proteins related to intestinal barrier function under transport stress were explored. In order to investigate the effects of transport stress on tight junction proteins Claudin-1, Occludin and zonula occluden-1 (ZO-1) in goat small intestine, 18 goat were selected in this experiment and randomly divided into control group (transport 0 h group), 2 h transport group and 6 h transport group. The changes of Claudin-1, Occludin and ZO-1 genes and their protein expressions in goat small intestine were studied by immunohistochemistry and qPCR. Immunohistochemical results showed that Claudin-1, Occludin and ZO-1 proteins were mainly expressed in the cytoplasm of intestinal villi epithelial cells and intestinal gland epithelial cells of goat duodenum, jejunum and ileum. At the same time, the distribution of Claudin-1, Occludin and ZO-1 proteins changed among different groups in the same intestinal segment. The results of qPCR showed that compared with the control group, the expression levels of Claudin-1, Occludin and ZO-1 in the duodenum transit group for 2 and 6 h were significantly decreased (P<0.0001); The expression levels of Claudin-1, Occludin and ZO-1 in the transport 2 h group and 6 h group of jejunal were significantly increased (P<0.001); The expression levels of Claudin-1, Occludin and ZO-1 in the 6 h ileal transport group were significantly increased (P<0.01). It indicated that transport stress could affect the expression of Claudin-1, Occludin and ZO-1 genes and their proteins in goat small intestine. This study provides basic information to explore the mechanism of transportation stress and develop products related to transportation stress relief.

The misuse of antibiotics seriously imperils the development of duck husbandry and even endangers human health. Therefore, it is urgent to find reliable alternatives to antibiotics. Immunopotentiator can effectively increase the ability of specific immune responses to enhance the defense against microorganisms or pathogens. The aim of this study was to investigate the effects of 5 different immunopotentiators on the thymus apoptosis and immune-related gene expression in Shaoxing ducklings (Anas platyrhynchos). The 150 1-day-old Shaoxing ducklings were randomly divided into 6 groups, and saline, chlorogenic acid, β -D-glucan, astragalus flavone, nonmethylated CpG DNA and chicken immunoglobulin G (IgG), were injected subcutaneously once daily for 3 d, respectively. At the age of 18 d, the mRNA and protein expression of apoptosis and immune-related genes were detected by qPCR and Western blot. The results showed that compared with control group, the mRNA and protein levels of thymus B-cell lymphoma 2 (Bcl2) were significantly decreased during injecting chlorogenic acid, β-D-glucan, astragalus flavone, nonmethylated CpG DNA and chicken IgG, respectively (P＜0.05). And the expression levels of thymus Caspase 3 showed significantly up-regulated in some extent (P＜0.05). In addition, the expression level of thymus inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were increased in some extent during injecting 5 different immunopotentiators, respectively. Meanwhile, compared with control group, the levels of interferon-α (IFN- α), interferon-β (IFN-β), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), retinoic acid-inducible gene-I (RIG-I), toll-like receptor 3 (TLR3) and toll-like receptor 7 (TLR7) mRNA expression were up-regulated in some extent during injecting 5 different immunopotentiators, respectively. Taken together, chlorogenic acid, β -D-Glucan, astragalus flavone, nonmethylated CpG DNA and chicken IgG could be used as immunopotentiators to regulate duck innate immune. This study provides new insight to prevent the duck disease from pathogen infections, and provides reference for reliable alternatives to antibiotics in animal production.

In recent years, grass carp hemorrhagic disease caused by Grass carp reovirus (GCRV) has caused serious losses to the grass carp industry. In order to explore the immune protective effects of recombinant interferon on grass carp and its mechanism, the prokaryotic expression vector PET-30a-rCiIFNγ of Ctenopharyngodon idella interferon γ (IFNγ) was constructed and transferred into Transetta (DE3). The recombinant protein rCiIFNγ inducted by IPTG and purified by nickel column chromatography. The antiviral activity of rCiIFNγ in vitro was detected by cytopathic effect (CPE) observation. The protective efficacy of rCiIFNγ in vivo was evaluated by challenge experiment. The results showed that the molecular weight of rCiIFNγ is about 23kD, and the antiviral titer of purified rCiIFNγ was 2.8×103 U/μg. rCiIFNγ could not only inhibit cytopathic effect induced by GCRV in vitro, but also induced the expression of IFN-stimulated genes (IFN, IRF1, IRF7, Mx, PKR, STAT3 and IFNr) in liver, spleen, kidney and intestine, and significantly reduced the mortality of C. idellus challenged with GCRV. These results indicated that the recombinant interferon IFNγ fusion protein rCiIFNγ was effective in protecting grass carp against GCRV infection. This study provides a theoretical basis for the antiviral application of grass carp interferon γ.

Helicoverpa armigera is an omnivorous pest that causes severe yield loss of many crops. The current planting of Bt crops and the extensive use of chemical pesticides have enabled the cotton bollworm to evolve a defense mechanism that protects itself against pesticides and plants secondary substances. In order to develop an environment friendly insecticide for harmless control of H. armigera, the core sequence HE1, which can respond to 2-tridecanone (2-TD), of the cytochrome P450 CYP6B6 promoter was used as a probe to screen out a positive regulatory domain zinc finger protein 10 like (PRDM10l) gene by using DNA pull down technology. The sequence of HaPRDM10l were cloned from 6th instar larvae of H. armigera, analysed by bioinformatics and its fusion protein expressed by using a prokaryotic expression system. qPCR was used to detect the spatial-temporal expression profile of HaPRDM10l in H. armigera, and its expression pattern in the midgut of the 6th instar larvae of H. armigera was detected after 2-TD treatment. The results showed that the ORF of HaPRDM10l (GenBank No XM_021334552.1) was 2 067 bp, encoded 688 amino acids. The molecular weight and theoretical isoelectric point of the protein were 77.96 kD and 8.84, respectively. HaPRDM10l was expressed at all developmental stages of H. armigera, with the highest expression in 5th instar larvae, and among different tissues the highest expression in the midgut of 6th instar larvae. After the cotton bollworm was treated with different concentrations of 2-TD, both HaPRDM10l and CYP6B6 could respond to the induction of 2-TD. The expression of HaPRDM10l reached the maximum when 10 mg/g of 2-TD treatment for 12 h, which was 7.6 folds of that in the control group, and the expression level of CYP6B6 reached the maximum at 6 h after 5 mg/g 2-TD treatment, which was 4.6 folds that of the control group. Further analysis revealed that there was a positive correlation between the expression of HaPRDM10l and CYP6B6 in the 6 h treatment group (r=0.749, P<0.01). The above results suggested that HaPRDM10l may regulate the expression of CYP6B6 in response to the stress of 2-TD. This study provides a reference for clarifying the mechanism of the H. armigera in response to the stress of plant secondary substances.

Soft rot disease which is caused by Erwinia beijingensis is the major disease in Pleurotus eryngii. Until now, the pathogenic mechanism of E. beijingensis is unknown. In the present study, the whole genome information of E. beijingensis was obtained by Nanopore and Illumina platform, the gene function annotation and secretion system composition were analyzed. The protein interactions of secretion system were predicted by string website (https://www. string-db. org/), and the effector proteins were predicted by SecRet6 and effectvet3 software. The genome size of E. beijingensis was 4.11 Mb, with GC% of 50.17%, encoding 3 815 genes. E. beijingensis displayed type Ⅰ, type Ⅲ, type Ⅳ and type Ⅵ secretion system (T1SS, T3SS, T4SS and T6SS). T1SS was composed of 3 proteins, including inter membrane (IM) component, membrane fusion protein (MFP) and outer membrane (OM) protein channel, among which 2 pairs of proteins have interactions. It is speculated that 36 proteins were secreted through T1SS. T3SS consisted of 9 Hrc proteins and 11 Hrp proteins, with 88 pairs of protein interactions, and 154 effector proteins were predicted to be secreted by the T3SS. T4SS consisted of 9 VirB proteins with 21 pairs of protein interactions and 11 effector proteins. E.beijingensis had 2 T6SS, T6SS-1 was composed of 13 T6SS proteins and 11 hypothetical proteins, 104 protein interactions were formed between T6SS proteins. T6SS-2 included 11 T6SS proteins and 8 hypothetical proteins, 55 pairs of protein interactions were found in T6SS-2. It was predicted that 6 effector proteins secreted into host cell by T6SS. 58 secretion system genes could be expressed during infection, and 34 genes showed significant different expression. This study analyzed the composition, protein interactions and effector proteins of secretion systems in E. beijingensis from the genomic level, which provided basic data for the analysis of pathogenic genes and theoretical basis for further analysis of pathogenic mechanism.

Plant chitinase and β -1, 3-glucanase, two important defensive factors in the plant defense system, can hydrolyze the main components of pathogenic cell wall chitin, β -1, 3-glucan and peptidoglycan, thus effectively inhibit the growth of pathogens. In addition, they also have synergistic antimicrobial effects and are widely used in plant disease resistance genetic engineering. In this paper, the research progress on the structure, classification, biological characteristics, expression regulation mechanism and the synergistic disease resistance of plant chitinase and β-1,3-glucanase were reviewed. This study will provide references for systematically understand the action mode of plant chitinase and β -1, 3-glucanase, and for the genetic improvement of plant disease resistance.

The stigma exsertion is an important trait for the positional male-sterile types of tomato (Solanum lycopersicum), with a longer style than the stamens and it was also used in hybrid seed production without artificial emasculation with high seed purity, low seed production cost and easy sterility maintenance, which has been a hot spot in the use of tomato male sterility in recent years. The research progress of QTL or genes related to stigma exsertion in tomatoes was reviewed in this paper, and the application status of stigma exsertion in tomatoes was clarified, as well as the direction and countermeasures of future research and application, aiming to provide references and insights for accelerating breeding of male sterility traits in tomato.

Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin angiotensinogen system (RAS) and a functional receptor of coronavirus such as SARS-CoV-2. To enrich the research content of ACE2 in ducks (Anas platyrhynchos) and explore the relationship between the ACE2 and avian coronavirus, the full-length coding sequence of the duck ACE2 gene was amplified by PCR, and the eukaryotic expression system was constructed by connecting homologous recombination with pcDNA3.1(+ ) vector. The optimal screening concentration of geneticin (G418) was determined for Chinese hamster ovary (CHO) cells, after transfecting of cells, selected monoclonal cell lines for G418. Western blot and immunofluorescence were used to identify the obtained cell lines. The cell line for expressing duck ACE2 protein was established, determined the enzyme activity of ACE2 recombinant protein. The results showed that the duck ACE2 gene was successfully cloned, and its coding region was 2 435 bp; the pcDNA3.1(+ ) -ACE2 plasmid was successfully constructed, and single enzyme digestion verified that a single band appeared at about 7 863 bp. The recombinant plasmid was successfully transfected into CHO cells by Lipofectamine, and exogenous ACE2 recombinant protein was expressed. Three monoclonal cell lines named pcDNA3.1(+ ) -ACE2-1, pcDNA3.1(+ ) -ACE2-2, pcDNA3.1(+ ) -ACE2-3 were successfully obtained, respectively. The recombinant ACE2 protein extracted from the monoclonal cell line showed high enzymatic activity. This study successfully constructed a duck ACE2 eukaryotic expression system and obtained 3 cell lines that can stably express ACE2 recombinant protein. This study provides a basis for the study of duck ACE2 protein and coronavirus and other biological functions and enriches the related theories of the protein.

Bacillus subtilis is a bacterial species wildly used in food industry, feed fermentation and bioengineering. This is owing to its safety and excellent ability of heterologous protein expression. In particular, the expansion of studies proved the use of B. subtilis 168 as the host strain for genetic engineering. However, the transformation efficiency of B. subtilis from the natural environment was extremely low, which greatly limited the application and modification of genetic engineering. In this study, wild-type B. subtilis C6 (BS-C6) was selected as the original bacteria for genetic engineering, which was isolated from the intestinal tract of Reticulitermes labralis and stored in our laboratory. The engineering bacteria B. subtilis C6-comk (C6- comk) and B. subtilis C6-comks (C6-comks) were constructed via double-crossover homologous recombination. Competence transcription factor (comK) is a key regulatory protein that affects genetic competence and DNA uptake in B. subtilis. The C6-comk strain was obtained by replacing the original promoter of the comK gene with xylose-inducible promoter (PxylA). On the other hand, the comK gene was amplified from the genome of B. subtilis 168, and then fused with the xylose-inducible promoter (PxylA). The overlap product was inserted into the extracellular serine protease (epr) site of wild-type B. subtilis C6 by using the homologous recombination approach. The super-competent C6-comks strain was successfully obtained. The results showed that the C6-comk strain transformed with plasmids and PCR products could not obtain positive clones, indicated that the method needs further improvement. Fortunately, the desirable phenotypes of C6-comks strain were observed. The results showed that the plasmid transformation efficiency of C6-comks strain was (4117±363) CFU/µg, the efficiency was improved by about 8 folds (P<0.01) compared to BS-C6 (wild type). Notably, the transformation efficiency of PCR products was (442±52) CFU/µg, transcending BS- C6 by about 73.7 folds (P<0.01). Furthermore, the qRT-PCR results showed that the expression of key genes for competence formation were significantly increased compared to BS-C6. The comK, comGB, comGF, comFA and comFC was improved 77, 1 654, 1 180, 885 and 108 folds (P<0.01), respectively. In contrast, the gene expression levels of flagellar basal-body rod protein (flgB), xylose isomerase (xylA) and xylulokinase (xylB) were only 64% (P<0.05), 12% (P<0.01) and 11% (P<0.01) compared to BS-C6. The gene expression levels of C6-comks strain and C6-comk strain were also compared. The results showed that the gene expression levels of comK, comGB, comGF, comFA and comFC were significantly increased by 1.5, 451, 403, 797 and 100 folds (P<0.01) in C6-comks, respectively; while xylA and xylB were significantly reduced by 5.2 and 6.6 folds (P<0.01). There was no treatment effect on flgB gene expression between C6-comks and C6- comk strains (P>0.05). Taken together, this study successfully generated super-competence B. subtilis from the natural environment and analyzed the reasons for the difference of transformation efficiency, which provided a valuable reference for genetic engineering application of wild-type B. subtilis as a host cell.