The concept of 'optimum plant types' in the Green Revolution provides a new idea for the design and breeding of maize (Zea mays) plant type. Over-long and over-wide leaves will lead to decreased yield due to draping, while broad leaves can keep the leaves upright without draping, increase the light receiving area of the population, and effectively improve the photosynthetic efficiency. The classic maize (Zea mays) mutant Crinkly Leaves 1 (cr1) showed crinkled leaves and dwarf plants. The following results were obtained after cytological analysis, mapping cloning, gene editing and expression analysis of the mutant. The pavement cells of the mutant's leaves were deformed and irregularly arranged, and the mapping cloning results showed that the target gene ZmCYP90D1 was located within 0.9 Mb at the end of the short arm in chromosome 3. After functional analysis and exon sequencing of 29 coding genes in the region, it was found that the third exon of ZmCYP90D1 was missing in cr1 mutant. Further genome sequencing revealed that a 1.2 kb transposon was inserted at the splicing site at the end of the third exon of the cr1, resulting in the missplicing of the mRNA of ZmCYP90D1 and the loss of the third exon. The ZmCYP90D1 (Zm00001d039453) gene encoded cytochrome monooxase, which was involved in the biosynthesis of brassinolactone and located in the endoplasmic reticulum. Gene editing produced 5 homozygous knockout lines, all of which showed crinkly leaf and short plant phenotype. Two of the knockout lines, ko1 and ko2, were analyzed, and ZmCYP90D1 was confirmed as the target gene of cr1. RNA-seq showed differentially expressed genes were mainly concentrated in biological processes such as cell division and differentiation, cell wall formation and modification, hormone regulation response, fatty acid anabolism and so on. The identification of cr1 laid a foundation for further analysis of the molecular mechanism of brassinolide synthesis pathway regulating maize leaf development.

The primary objectives of high-yield breeding for foxtail millet (Setaria italica) encompass the optimization of plant height, enhancement of panicle weight, and augmentation of panicle grain weight. In order to investigate the genetic variation of plant height, panicle weight and panicle grain weight of foxtail millet, this study utilized 'Aininghuang' as the maternal parent and 'Jingu 21' as the paternal parent to established a recombinant inbred line (RIL) population, and the RIL population was cultivated at 7 locations over a span of 3 years (2019~2021) in Shanxi province, R-based software package SEA v2.0 was employed to analyze the main gene + multi-gene hybrid genetic model for RIL population traits such as plant height, panicle weight and grain weight. The results demonstrated significant correlations among plant height, panicle weight and grain weight, and the correlation coefficient between panicle weight and grain weight was 0.98 (P≤0.001). The optimal model for plant height was determined to be PG-AI with the polygenic heritability of 97.56%; Similarly, the optimal model for panicle weight was identified as PG-A with the polygenic heritability of 84.04%, primarily influenced by additive effects resulting in an additive effect value of -2.95 indicating negative genetic impact; The most suitable model for grain weight was MX2-ED-A, which was controlled by 2 dominant epistatic major genes along with additive polygenes contributing to mixed inheritance pattern, with major gene heritability accounted for 62.53% and polygene contributed approximately 50.52%, the additive effect of the second major gene was dominant, and the additive epistatic interaction effect value was -11.50, showed a negative genetic effect. The optimal genetic models of plant height and panicle weight in foxtail millet were similar, exhibited a polygenic nature with substantial heritability and little environmental influence; In contrast, the inheritance of grain weight was primarily controlled by a combination of major gene effects and polygenic factors. This study offers valuable insights for the genetic enhancement and precise mapping of genes associated with plant height, panicle weight, and grain weight.

The expression of Gretchen Hagen 3 (GH3) genes plays an important role in plant growth and development and response to biotic stresses. In this study, the potato (Solanum tuberosum) cultivar 'Qingshu 9' as the materical for identification and bioinformatics analysis of GH3 family members, and their expression patterns were analysed in potato different tissues and in response to salicylic acid (SA), Methyl jasmonate (MeJA) and gibberellin A3 (GA3) and Phytophthora infestans stresses. Promoter cis-acting element analyses showed that StGH3 family members contained a large number of hormone-related acting elements, speculated that it had potential function in response to biotic and abiotic stresses. Tissue specific analysis showed that StGH3.4 and StGH3.5 genes were highly expressed in roots, StGH3.8 was highly expressed in stems, and StGH3.10 and StGH3.16 were highly expressed in leaves. StGH3 family members were regulated under SA, MeJA, GA3 and P. infestans stress treatments and showed different expression patterns. The StGH3.1, StGH3.5, and StGH3.8 genes responded more strongly to the P. infestans treatment.This study provides a scientific basis for subsequent studies of StGH3 genes function.

Fruit and vegetables are highly susceptible to mechanical wounding during harvest and postharvest handling, and the wounds induce the production of several phytohormones at the wounds, but involvement of jasmonic acid (JA) in potato (Solanum tuberosum) wound stress has not been reported. In this study, based on transcriptome analysis of pre-wounded potato tubers, the genes related to JA synthesis and signaling were screened, the expression of these genes and related enzyme activities were analyzed,the JA content at the wounds was determined. The results showed that the wound significantly upregulated the expression levels of genes related to JA synthesis at the tuber wound, including lipoxygenase (LOX) family members StLOX1.5, StLOX2, StLOX3.1 and StLOX6, propadiene oxidative synthase (AOS) family members StAOS1, StAOS2 and StAOS3, and propadiene cyclohexylase (AOC) family members StAOC and 12-oxo-dienoic acid reductase (OPR) family members StOPR1, StOPR2 and StOPR3. The wound also significantly increased LOX and AOS activities and significantly increased JA levels at the tuber wounds. In addition, the wound significantly upregulated the expression levels of key JA signaling genes, the jasmonate ZIM domain (JAZ) family members StJAZ1, StJAZ2, StJAZ3, StJAZ4 and StJAZ7. In conclusion, the wound activates JA synthesis and signaling in potato tuber wounds. This study provides a theoretical basis for the study of the response mechanism of potato wound stress.

Laccase (LAC) is a glycoprotein oxidase that influences lignin synthesis, regulates the levels of phenolic compounds in plants, and participates in plant defense mechanisms against pests and diseases. In order to obtain a new tomato (Solanum lycopersicum) germplasm with high resistance to Botrytis cinerea and in compliance with transgenic safety, in this study, based on the pGM626-Act1 vector constructed in the laboratory in the previous stage, a gene-deleter system with the fruit-specific E8 promoter driving the FLP gene and the Act1 promoter driving the Eucommia ulmoides laccase 1 (EuLAC1) gene in a plant expression vector was designed and constructed. Agrobacterium-mediated transformation technology was used to genetically transform the tomato variety 'Micro-Tom' and 5 positive tomato transgenic plants overexpressing EuLAC1 were successfully obtained. The results of resistance analysis showed that the onset time of EuLAC1 overexpressing tomato plants was significantly delayed after inoculation with B. cinerea, which was in sharp contrast with wild type and empty vector control plants. Additionally, the lesion diameter in tomato plants overexpressing the EuLAC1 gene was significantly smaller than that in the control group (P<0.05), which comprised the wild type and empty vector tomato plants. The result showed that overexpressing the EuLAC1 gene in tomato plants markedly improved their resistance to B. cinerea. Overexpression of the EuLAC1 gene also increased the activity of protective enzymes and the expression level of disease process-related protein (PR) genes in tomato plants. Deletion analysis of exogenous genes in transgenic tomato fruits revealed that no exogenous genes were detected in 22 out of 40 tomato fruits, with an exogenous gene deletion efficiency of 55%. This study provides a new technical method for the development of new disease-resistant tomato germplasm that meets transgenic safety standards.

MYB transcription factors play a crucial role in the accumulation of anthocyanins in peach (Prunus persica) fruit. A deeper understanding of MYB transcription factors is beneficial for elucidating the molecular regulatory mechanism of secondary metabolic networks in peach, providing a theoretical basis for genetic improvement, and developing new varieties rich in anthocyanins. In this study, in order to investigate the role of PpMYB113 in the accumulation of anthocyanins in peach flowers and fruits, the PpMYB113 gene (GenBank No. XM_007216468.2) was cloned using red flesh peach as the experimental material. The bioinformatics and expression pattern analysis was performed, and the function of PpMYB113 gene was studied through transient transformation in tobacco (Nicotiana benthamiana) and peach flesh. The results showed that the open reading frame of PpMYB113 gene was 720 bp, encoding 239 amino acids. The phylogenetic tree analysis indicated that the PpMYB113 protein sequence had the closest phylogenetic relationship with the MYB transcription factors of the S6 subfamily in Arabidopsis thaliana. The expression of PpMYB113 gene was analyzed in petals, branches, leaves, bark, fruit skin, and fruit flesh of peach by using semi-quantitative RT-PCR and RT-qPCR. The results indicated that PpMYB113 gene was highly expressed in petals, fruit skin, and fruit flesh, while it was not expressed in branches, leaves, and bark. At the same time, PpMYB113 was overexpressed in tobacco leaves and white fruit flesh using the transient transformation system. The overexpressed tobacco leaves and white fruit flesh showed red color phenotype and the anthocyanin contants were significant increase. In the overexpressed tobacco leaves and white fruit flesh areas, the expression of PpMYB113 was significantly higher than that of the control, and the expression of ANS (anthocyanidin synthase) and UFGT (UDP glucose: flavonoid-3-O-glucosyltransferase) genes were significantly up-regulated. Based on these findings, it was concluded that PpMYB113 plays a positive promoting role in the accumulation of anthocyanins in peach. These results provide a foundation for further investigation into the molecular regulatory mechanism of MYB transcription factors in the accumulation of anthocyanin in red flesh peach.

Zinc finger-homeodomain (ZF-HD) proteins are plant-specific transcription factors composed of a zinc finger (ZF) domain and a homeodomain (HD). The ZF-HD family plays a crucial role in plant growth and development by regulating the expression of target genes through binding to their promoters. In this study, the VvZF-HD11 gene was isolated from the leaves of the grape (Vitis vinifera) variety 'Zuoyouhong' and assigned the GenBank accession number PP735488. The full length of the coding region of this gene was 969 bp, encoding 322 amino acids. The conserved domains of the VvZF-HD11 protein were the zinc finger domain ZF at the N-terminus (72~127 aa) and the homeodomain domain HD at the C-terminus (202~258 aa). Comparison of the amino acid sequence and phylogenetic analysis indicated that VvZF-HD11 shares the highest homology with wild tobacco (Nicotiana tabacum) NaZF-HD9. Further analysis through qPCR showed that the expression of the VvZF-HD11 gene was induced by various abiotic stress factors and signaling molecules. Specifically, VvZF-HD11 expression peaked under specific conditions, such as low temperature and salicylic acid (SA) induction at 3 h, heat stress and H₂O₂ treatment at 6 h, salt stress, H₂S and abscisic acid (ABA) treatment at 12 h, and drought stress at 9 h. Moreover, transient transformation of VvZF-HD11 into grape leaves followed by heat stress at 45 ℃ for 4 h demonstrated that overexpression of VvZF-HD11 enhanced the leaves' ability to resist heat stress. By detecting physiological indicators and the expression levels of genes related to heat stress, it was found that VvZF-HD11 could enhance the antioxidant enzyme activity of grape leaves, reduce membrane oxidative damage, and promote the expression of genes related to heat stress, thereby improving the plant's ability to resist heat stress. Based on the above results, it was speculated that VvZF-HD11 might be involved in regulating the stress response of grape. This study establishes a foundational theoretical framework for the genetic enhancement of grape varieties exhibiting tolerance to heat stress.

MYB transcription factor is one of the gene families with the largest number of members in plants, among which R2R3MYB plays an important role in anthocyanin synthesis. The study aimed to identify R2R3MYB gene family members at genomic level and analyze their functions in anthocyanin synthesis in Ginkgo biloba. The sequence information was obtained by homology alignment, and conserved domain analysis, evolutionary tree construction and conserved motif analysis were carried out by bioinformatics analysis software. The gene expression level was detected by qPCR. The gene function was studied through tobacco (Nicotiana benthamiana) leaf transformation. 78 R2R3MYB family members were identified in G. biloba genome, which were divided into 7 branches in the phylogenetic tree. One gene, Gb_34086, homologous to AtMYB75 of Arabidopsis thaliana, was selected and its expression was up-regulated by light induction. Gb_34086 protein was located in the nucleus and had transcriptional activation activity. The overexpression of Gb_34086 gene in tobacco leaves could enhance anthocyanin accumulation under light. This study preliminarily proved that Gb_34086 played a positive regulatory role in anthocyanin synthesis, which provides a basis for further study of the molecular mechanism of Gb_34086 and improving the the anthocyanin production of Ginkgo biloba.

Lavender (Lavandula angustifolia) is an important spice cash crop in the world. The essential oil extracted from the inflorescence is rich in aromatic compounds. These compounds mainly rely on the methylerythritol-4-phosphate (MEP) pathway in plant plastids for metabolic synthesis. In the MEP pathway, 1-deoxy-D-xylose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), as the first 2 key rate-limiting enzymes, play a decisive role in the smooth progress of the whole metabolic process. The LiMYC1 (myelocytomatosis 1) gene cloned from Lavandula × intermedia was targeted, and its function in the synthesis of volatile terpenoids was verified. In this study, The pCAMBIA3301 and pTRV2 plant expression vectors were constructed, and overexpression and gene-silencing plants were obtained using Agrobacterium-mediated genetic transformation methods. The expression of LiMYC1, LiDXS and LiDXR genes in transgenic plants were detected, and volatile terpenoids in leaves were qualitatively and quantitatively analyzed. The results showed that in the LiMYC1 gene overexpression line, the expression of LiDXS and LiDXR increased significantly, and the contents of linalool and caryophyllene also increased to varying degrees. On the contrary, in the LiMYC1 gene silenced strain, the expression of LiDXS and LiDXR were significantly reduced, and the content of caryophyllene was reduced, but the content of linalool was abnormally increased. The above results indicated that the LiMYC1 gene positively regulated the MEP pathway of terpenoid synthesis, affected the expression of key enzymes LiDXS and LiDXR, and had a certain regulatory effect on the biosynthesis of caryophyllene and linalool. This study has important guiding significance for analyzing the molecular mechanism of lavender aroma formation and optimizing the economic value of lavender.

Trehalose-6-phosphate synthase (TPS) is the crucial rate-limiting enzyme for the trehalose biosynthesis, and plays a significant role in the stress responsiveness. To investigate the function of TPS regarding growth and development, stress response in Casuarina equisetifolia, physicochemical features, chromosomal localization, phylogenetic tree, conserved motifs, gene structure, cis-regulatory elements and collinearity were analyzed in this study. Their expression patterns in different tissues, and exposed to drought, salt stress and abscisic acid (ABA) treatment were determined. Results indicated that 7 CeTPS genes were randomly distributed in 4 chromosomes, encoding 826~998 amino acids that were hydrophilic. The CeTPS proteins were divided into 2 subfamilies through phylogenetic analysis, showing relatively close to dicotyledonous plants, and harbored gene duplications. The promoter regions of CeTPS genes were rich in cis-acting elements involved in hormonal responsiveness, growth and development, and stress responsiveness. CeTPS genes showed tissue-specific expression, and were differentially expressed under drought, salt stress, and ABA treatment. Especially, CeTPS4 might play an essential role in stress responsiveness. The results lay a foundation for in-depth study of the CeTPS genes associated with abiotic stress responsiveness.

Huperzia serrata, a member of the Lycopodiaceae family, contains huperzine A (Hup A), a highly selective acetylcholinesterase inhibitor used to treat and alleviate symptoms in Alzheimer's disease (AD). The DOF family, a transcription factor family unique to plants, plays a significant role in plant growth, development, and responses to abiotic stress. Based on the full-length transcriptome data of H. serrata, this study identified the DOF gene family (HsFOF), analyzed its protein physicochemical properties, conserved domains, subcellular localization, secondary and tertiary structures, and regulatory networks. By utilizing RNA-seq data, the expression patterns of HsDOF were analyzed across various tissues and under conditions of high temperature and drought stress. The results showed that a total of 19 HsDOF genes were identified; The number of amino acids encoded the protein ranges from 213 to 574, with a molecular weight of approximately 23.89 to 60.50 kD and an isoelectric point of 5.15 to 9.93; The subcellular localization prediction results showed that all HsDOFs were located to nucleus. The secondary structure was mainly displayed as α-helix and irregular curl, with 7 types of tertiary structure models used in the 19 HsDOF proteins. Phylogenetic analysis showed that the DOF protein of Arabidopsis thaliana and H. serrata could be divided into 9 subgroups, with HsDOF distributed in 2 of them. Expression pattern analysis showed that the HsDOF genes showed differential expression in different tissues. Under high temperature stress, 6 HsDOF genes were upregulated or downregulated by more than 2 times at 1 or more time points. Under drought stress, 6 HsDOF genes were upregulated or downregulated by more than 2 times at 1 or more time points, of which 3 were upregulated; 2 downregulated. The research results provide a theoretical basis for further exploring the biological functions and molecular breeding of the HsDOF gene family.

Transmembrane protein 18 (TMEM18) is an important candidate gene for animal growth and development, encoding a transmembrane protein with important biological functions. In order to explore the expression pattern of TMEM18 gene in sheep (Ovis aries) tissues, the correlation between it's polymorphism and feed conversion ratio (FCR), and molecular genetic markers for breeding were searched. Based on a population of 845 Hu sheep with clear pedigrees and complete phenotypic records, this research utilized qRT-PCR technology to detect the expression levels of the TMEM18 gene in 10 tissues of Hu sheep and employed Sanger sequencing technology to scan for SNPs in this gene, which were then analyzed for association with feed efficiency traits. The results showed that the relative expression of the TMEM18 gene was significantly higher in the heart than in other tissues (P<0.05), followed by high relative expression in the duodenum, rumen, lungs, and liver. A synonymous mutation polymorphism site, g.517832 T>C, was detected in the third intron of the sheep TMEM18 gene. The results of multiple comparisons showed that the mutation locus was significantly associated with FCR100~120, FCR140~160, FCR80~120, FCR140~180, FCR100~160, FCR80~160, FCR100~180 and FCR80~180 (P<0.05), among them, FCR140~160, FCR100~160, FCR80~160, FCR100~180 and FCR80~180 with CC genotype were significantly higher than those of TT genotype and TC genotype (P<0.05). The results of this study suggested that TMEM18 g.517832 T>C site could be used as a candidate molecular marker to improve feed efficiency in sheep. This study provides reliable genetic materials for the breeding of grain-saving varieties.

Madin-Darby canine (Canis lupus familiaris) kidney (Madin-Darby canine kidney, MDCK) cells have the advantages of standardization and high yield as stromal cells for influenza vaccine production. However, its adherent growth has hindered the scale-up of production and its tumorigenicity has prevented the dissemination of MDCK-based vaccines. This study successfully obtained a non tumorigenic suspension cell line XF06 of MDCK through monoclonal screening and suspension domestication. A nude mouse (Mus musculous) tumor experiment was conducted using XF06 and tumorigenic suspension cell line XF04. 1×10⁷ cells were subcutaneously injected into the back of thymus free nude mice, with 10 mice in each group; Using human (Homo sapiens) cervical cancer cell lines (Hela)(with tumorigenicity) as positive controls and human embryonic lung fibroblasts (MRC5)(without tumorigenicity) as negative controls. The results showed that the screened cell line XF06 did not form tumors in nude mice. The protein expression difference of XF04 and XF06 was compared by data-independent acquisition (DIA) proteomic technology, and the tumorigenic genes were screened, and the expression levels of genes and proteins were verified. The results shows that the gene and protein expression levels of 4 proteins (baculoviral IAP repeat containing 5 (BIRC5), fascin actin-bundling protein 1 (FSCN1), insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1), TNF alpha induced protein 2 (TNFAIP2)) were significantly reduced in non-tumorigenic XF06 cells (P<0.05), which might be involved in the regulation of tumorigenic MDCK cells. This study provides reference for the screening of target genes using gene editing technology to inhibit tumorigenesis.

Tobacco brown spot is the main fungal disease that endangers the tobacco industry. Biological control is an important means to control plant diseases. Synthetic community has been a research hotspot in the field of biological control because of its good growth-promoting and stress-resistant functions in agricultural production.The rhizosphere soil of healthy tobacco (Nicotiana tabacum) plants in the tobacco field with severe brown spot disease in southern Shaanxi was used as the material, and the strains with significant antagonistic effect on Alternaria alternate were isolated and screened by dilution separation, plate confrontation method and mycelial growth rate method. The growth-promoting strains were screened by measuring the ability of bacteria to produce indole acetic acid (IAA) and growth-promoting pot experiments. The antibacterial strains were screened by measuring the activities of phenylalaninammo-nialyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) defense enzymes in tobacco leaves, and then the selected strains were identified by morphological, physiological and biochemical characteristics and molecular biological methods. Then, the Box-Benhnken response surface method was used to optimize the ratio of bacteria to finally construct a multi-functional biocontrol bacteria group. The screened antagonistic strain Xh628A (GenBank No. PQ269269) and growth-promoting strain X5616A (GenBank No. PQ269268) were identified as Bacillus velezensis, and the induced antibacterial strain Xh649A (GenBank No. PQ269267) was identified as Acinetobacter pittii. The optimal addition ratio of the optimized flora was determined to be Xh628A:Xh649A:X5616A=5.0:1.0:4.5, and the average mycelial inhibition rate of the flora was 77.24 %, compared with the single strain, its growth-promoting and resistance-inducing ability has been improved. This study provides a research basis for the subsequent development of efficient, stable and environmentally friendly multifunctional biological pesticides, and provides a scientific method for the construction and ratio optimization of multifunctional biological flora.

Salmonella anatum is a major pathogen that causes salmonellosis in ducks (Anatidae). rfaH encodes the transcriptional anti-termination factor RfaH, which is essential for bacterial transcription, infection, and other biological functions. This study investigated the effect of the rfaH gene on various biological aspects of S. anatum, including growth, motility, biofilm formation, and cell infection. To deeply understand the biological roles of the rfaH gene in S. anatum, an rfaH gene deletion strain ΔrfaH and the complement strain CΔrfaH derived from the S. anatum isolate strain SA01 were generated using Red homologous recombination technology. The sequencing results showed that the rfaH gene deletion strain ΔrfaH and the complement strain CΔrfaH of SA01 were successfully constructed. After the deletion of the rfbH gene, the growth rate remained unchanged, yet the O-antigen synthesis was impaired, leading to a significant decrease in bacterial motility, an enhancement in biofilm formation, an increased adhesion and invasion capacity against DF-1 cells, and a weakened proliferation ability in RAW264.7 cells. The findings indicated that the rfaH gene was involved in bacterial motility, biofilm formation, and cell infection. This study establishes a solid platform for future research on the biological functions of the rfaH gene and the development of Salmonella-based delivery vectors for vaccine applications.

Cotton (Gossypium spp.) is an important cash crop and the cotton industry plays an important role in rural revitalization, economic development and people's livelihood security in China. Since the founding of the People's Republic of China, the basic research, agricultural production and industrial development of cotton in China have undergone significant changes and progress. Among that, the varieties breeding and utilization of cotton take up an important position in the development of cotton industry strain. Rapid development of modern molecular biology has significantly promoted the innovation of cotton biological breeding technology, and improves the iterative upgrading from traditional breeding to molecular breeding of cotton. At the same time, the innovative study and development of valuable cotton products such as cottonseed and gossypol have effectively promoted the development of innovative value and comprehensive utilization of cotton. On the whole, the basic research and the integrated development of the whole industry chain have made significant progress, which has improved the quality and market competitiveness of China's cotton. Driven by scientific and technological innovation, China's cotton breeding industry would strengthen the structural adjustment and optimization, and industrial upgrading to achieve higher-quality development.

Echinacea purpurea is a widely used medicinal plant worldwide with significant economic value,known for its effects in enhancing immunity, antibacterial, and anti-inflammatory properties. The active ingredient chicoric acid is a crucial substance for exerting the medicinal effects of E. purpurea and serves as a key indicator for evaluating the quality of medicinal herbs. The production of active ingredients is not only regulated by its own genetic factors but is also significantly influenced by environmental factors such as light, temperature, and microorganisms in the synthesis and accumulation of active components in E. purpurea. This article mainly elucidated the efficacy of E. purpurea and its active ingredients, further discussed the synthetic pathways of the main active ingredient chicoric acid, summarized the regulatory mechanisms of the genetic characteristics and various environmental factors on the synthesis of chicoric acid, and finally provided an application outlook on the current status of enhance the accumulation of its active ingredients. This review provides new directions for the rational development of E. purpurea resources and is of great importance for improving the quality and clinical efficacy of medicinal plants.

The plantago asiatica mosaic virus (PlAMV) and the Lily mottle virus (LMoV) are currently the two most serious viral pathogens affecting the lily (Lilium spp.) industry in China. The aim of this study is to develop a rapid and efficient detection system for these 2 viruses. Firstly, total RNA was extracted from diseased lily plants, and cDNA was obtained through reverse transcription. Subsequently, a dual-synchronous detection system was optimised using cDNA as a template, and the specificity and sensitivity of the detection system were evaluated. The results demonstrated that the established dual-synchronous detection system amplified specific segments of 910 and 521 bp for PlAMV and LMoV, respectively, with no amplification for other common viruses, indicating strong detection specificity. The lowest detection limits for PlAMV and LMoV were 967.5 fg and 59.0 pg, respectively, which were 50 times and 5 times lower than the lowest detection limits of single-gene RT-PCR detection (PlAMV 19.35 fg, LMoV 11.8 pg). Sequence analysis of the amplified products with sequencing segments from 19 PlAMV isolates and 21 LMoV isolates from different sources revealed similarities of 86.71% to 99.40% for PlAMV and 94.75% to 99.49% for LMoV. The dual-synchronous detection system was employed to examine 19 field samples, and the results indicated that 3 samples were positive for both PlAMV and LMoV, 2 samples were positive for PlAMV, 4 samples were positive for LMoV, and the remaining 10 samples were not detected, which was consistent with the results of gene chip detection. In conclusion, the detection system this study established is capable of detecting both PlAMV and LMoV viruses simultaneously, with reliable results and broad application prospects.

Peroxisome proliferator activated receptor α (PPARα) is an important transcriptional regulator involved in lipid metabolism in animals, such as fatty acid oxidation, lipoprotein assembly and transport, lipid catabolism in the liver. So it is particularly important to study the role of PPARα in the lipid metabolism of chicken (Gallus gallus). In order to obtain its effective antibody, the chicken PPARα recombinant protein expressed in Escherichia coli was used to prepare antiserum in Sprague-Dawley (SD) rats (Rattus norvegicus) immunized with the PPARα protein. The titer of the antibodies was detected by Western blot and ELISA. The results showed that PPARα recombinant protein could be soluble expressed in E. coli induced by 0.06 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) at 15 ℃, and the purity of the recombinant protein obtained by Ni²⁺ affinity chromatography was about 89%. The ELISA results showed that the titer of the prepared PPARα antibodies was approximately 1:512 000. The sensitivity of Western blot detection of recombinant protein by antibodies is about 100~200 pg, and endogenous PPARα protein in chicken liver could also be detected. This study provides a reliable antibody for further analysis of the regulatory function of PPARα on the related genes about lipid metabolism in chickens.

Heat shock protein (DnaK) is a highly conserved stress protein produced by organisms under the influence of unfavorable factors, it has dual roles of carrier and adjuvant, however, the DnaK protein of Mycoplasma synoviae (MS) is less studied. In this study, in order to prepare a monoclonal antibody (MAb) of MS DnaK protein and preliminarily characterize its antigenic epitope, the recombinant plasmid was constructed and the recombinant protein was expressed. The recombinant protein of Mycoplasma synoviae, DnaK (rMSDnaK protein) was purified. BALB/c mice (Mus musculus) were immunized with rMSDnaK protein. Mice with high antibody levels were selected, their spleens were isolated, and the collected splenocytes were fused with myeloma cells (SP2/0 cells), after which positive hybridoma cells in good condition were screened by limited dilution and indirect ELISA, and ascites were collected and purified after injection into the peritoneal cavity of quinacrine mice. The rMSDnaK was successfully expressed identified by SDS-PAGE. Indirect ELISA was used to screen one hybridoma cell line, named 6G6, which could secrete anti-DnaK antibody and had good stability. The MAb 6G6 subtype was identified as IgG3 subclass. The potency of the purified MAb was determined by indirect ELISA to be 1:102 400. Western blot results showed that MAb 6G6 reacted only with rMSDnaK protein. Immunofluorescence assay (IFA) results showed that MAb 6G6 reacted fluorescently only with MS. Western blot results demonstrated that the prepared truncated MS DnaKΔ3-3 was able to react with rMSDnaK protein, which proved that the antigenic epitope region recognized by the monoclonal antibody was amino acids 506~596 at the C-terminus. In this study, the anti-MS DnaK MAb was successfully prepared and its antigenic epitope was identified, which provides biological materials for the subsequent establishment of MS detection by double-antibody sandwich ELISA for MS detection.

Swine influenza (SI) is an acute infectious disease of pigs (Sus scrofa) caused by Swine influenza virus (SIV). In recent years, 3 SIV subtypes, namely H1N1, H1N2 and H3N2, are most prevalent in China. This study aimed to develop virus-like particles (VLP) of SIV H1 and H3 subtype and determine their morphological structure and biological characteristics. Genes encoding the SIV surface protein—hemagglutinin (HA) of H1 and H3 subtypes were synthesized and ligated unto the expression vector. The recombinant plasmids was obtained and transfected to HEK-293T cells for protein expression. Meanwhile, the gene encoding the 2,4-dihydroxyprotenine synthase (LS) derived from the super pyretic Aquifex aeolicus was synthesized, and both a signaling peptide and immunoglobulin-bin-binding domain sequence in streptococcal protein G (pG) were introduced at the N-terminus of the sequence. The pG-LS recombinant plasmids were constructed and expressed separately through Escherichia coli and HEK-293T cell expression systems. After the conjugation of the 2 proteins, the morphology and the formation of VLP could be observed by transmission electron microscopy. Average particle diameters of the conjugated proteins were measured with dynamic light scattering method while the biological properties of the complex protein were analyzed by SDS-PAGE, Western blot and hemagglutination assay. SDS-PAGE results indicated that both recombinant proteins (H1-Fc and H3-Fc) were correctly expressed; at the same time, Western blot showed that both prokaryotic and eukaryotic systems resulted in successful expression of pG-LS nanoparticles. Together with SDS-PAGE analysis, it was shown that pG-LS expressed in HEK-293T cells had higher purity, and henceforth used for the subsequent construction of pG-LS-HA virus-like particles. Electron microscopy analysis of the conjugated proteins showed that the pG-LS-HA forms spherical virus-like particles, with a diameter around 80 nm. Further immunological analysis and particle diameter measurement both showed the successful construction of VLPs. The results of hemagglutination assays indicated HA-Fc alone could not agglutinate erythrocytes, while the virus-like particles prepared from 2 conjugated proteins showed exponential affinity, which could efficiently agglutinate red blood cells of different species. This study successfully constructed pG-LS nanoparticles and 2 pG-LS-HA VLPs, where HA proteins successfully displayed at the VLP surface. Both developed pG-LS-HA had stable property and biological function, with a potential of large-scale production. In the meantime, this study found that both H1 and H3 HA could be effectively displayed to form VLP, which shed light on the future use of multi-subtype influenza virus HA protein co-presentation strategy to develop novel, multivalent VLP vaccine candidates.