Wild rice has nearly 1 times more alleles than cultivated rice (Oryza sativa), so it is of great significance to improve rice yield and quality by using wild rice excellent genes. In this study, a set of rice chromosome fragment replacement lines were constructed using annual wild rice (Oryza nivara) as donor parent and indica hybrid rice 'Fuhui 676' ('FH676') as recipient parent. After one hybridization, three backcrosses, and multiple generations of self-cross, 502 BC₃F₇ individuals were selected for whole genome detection using rice high density whole genome SNP chip. Finally, 474 lines containing the genome fragments of the donor parents were used as the establishment population. The results showed that the homozygosity of the whole line was high, and the number of SNP markers in the genetic map was up to 1 429, and the average number of SNP markers in each chromosome was 119. Seventy-two imported lines containing ≤4 fragments were selected as the coverage map of the whole genome, and the imported fragments basically covered the whole genome. The total length of target segment substitution in overlay map substitution line population was 581.84 Mb. The total length of the substitution fragment in the whole genome was 306.56 Mb, covering 82.75% of the genome. Plant height and effective tiller spike of the whole line were investigated in Sanya, Hainan province and Youxi, Fujian province. Combined with genotype, QTL analysis of plant height and effective tiller spike was performed by using complete interval mapping. A total of 9 QTLs affecting plant height were detected in the 2 environments, distributed on chromosomes 2, 3, 7, 8 and 11. Only qPH-3 on chromosome 3 was detected in both environments; A QTL qPH-8-1 significantly correlated with plant height was identified, with LOD value of 16.79 and phenotypic contribution rate of 9.16%. Fourteen effective tiller QTLs were detected, distributed on chromosomes 2, 3, 4, 5, 6 and 7, and qETN-7-3, qETN-7-4 and qETN-7-8 were detected in both environments. A QTL, qETN-7-6, with a LOD value of 58.28 and a phenotypic contribution rate of 32.86%, significantly correlated with the number of effective tillers was identified. This study provides the theoretical basis for fine QTL localization of plant height and tiller.

The SR (serine/arginine-rich) protein family is a class of key regulators in plants, which is involved in plant growth and development, and responsive to abiotic stresses. In this study, GmRSZ21 (GenBank No. XP_014634806.1), the subfamily of RSZ (Zn-knuckles-type arginine/serine-rich protein), was cloned from soybean (Glycine max) and bioinformatics and expression analysis were carried out. The results showed that GmRSZ21 gene contained 4 exons and 3 introns, and the total length of the coding region was 561 bp. GmRSZ21 encoded 186 amino acids, among which arginine accounted for the highest proportion (19.4%). The relative molecular weight of GmRSZ21 protein was 21.36 kD, and its secondary structure was mainly random coil (57.53%), without transmembrane domains or signal peptides. The GmRSZ21 protein was predicted to be an unstable hydrophilic protein and located in the nucleus. Amino acid sequence comparison showed that the GmRSZ21 protein was highly conserved among different species. Phylogenetic analysis showed that the GmRSZ21 protein had the closest evolutionary relationship with its homologues in wild soybean (G. soja). The GmRSZ21 gene promoter contained 20 cis-acting elements, among which, the light response elements were most abundant. Expression analysis showed that GmRSZ21 was expressed in all tissues, with the highest expression in stems and the lowest expression in pods at the onset of bulging; the GmRSZ21 expression could be induced by drought and salt stress, and it was hypothesised that this gene might be participated in abiotic stresses in the soybean. This study provides a reference for the functional analysis of the SR protein family and its application in soybean molecular breeding.

Cauliflower (Brassica oleracea var. botrytis), originating from Europe, has a relatively narrow genetic diversity in China's germplasm resources, urgently requiring studies on germplasm innovation and evaluation techniques. The creation of double haploids (DH) using microspore culture not only shortens the breeding cycle but is also a crucial means for germplasm innovation. Understanding and evaluating the genetic diversity of cauliflower microspore progeny is a key issue to be resolved for the application of DH technology in germplasm innovation and breeding. In this study, 29 pairs of SSR primers with clear and stable band patterns and good polymorphism were selected, with PIC range of 0.29 to 0.57 and average value of 0.42. Using these 29 primers, the marker genotyping study was conducted on a cauliflower breeding germplasm population consisting of 66 DH lines and 18 advanced inbred lines, totaling 84 materials. The results showed that the genetic distances of the materials mainly ranged from 0.3 to 0.6, with an average of 0.56; the genetic similarity coefficients ranged from 0.50 to 0.97, with an average of 0.57. At a genetic similarity coefficient of 0.55, the materials were divided into 4 groups. Through the analysis of the relationships of DH materials, it was found that high genetic diversity could be obtained in the DH progenies of F₁ hybrid, confirming that DH breeding could serve as an important tool for germplasm innovation in cauliflower. This study provides theoretical and technical support for the evaluation and utilization of genetic diversity in cauliflower.

Histones are essential components of chromatin and play an important role in developmental regulation, and resistance to stress. In order to explore the characteristics under cold stress in eggplant (Solanum melongena), in this study, the genome-wide identification of Histone genes and bioinformatics analysis were carried out in eggplant, the transcriptome data were mined to analyze the expression pattern of SmHistones in 2 different resistant eggplant (epl008 and eplck) leaves under low temperature stress. The results showed that a total of 40 SmHistones genes were identified in eggplant, which were unevenly distributed on 9 chromosomes. The members of SmHistone gene family were classified into 5 subgroups (H1, H2A, H2B, H3 and H4). Members of the same groups had similar physical and chemical properties, gene/protein structure, and conserved motifs. Intraspecific collinearity analysis showed that there was a collinear relationship among 6 pairs of SmHistones. The results of promoter elements analysis indicated that a large number of abiotic stress response cis-acting elements, such as low temperature responsive elements and drought responsive elements existed in promoter sequences of Smhistones. Transcriptome and qRT-PCR analysis revealed that 36 Smhistones were differentially expressed under low temperature stress and were significantly upregulated in eggplant epl008. The induced expression of SmHistone3 under low temperature stress was significant, revealing that it may be closely related to the cold metabolic response. This study provides a theoretical basis in breeding of eggplant with high tolerance to low temperature stress.

High temperature stress is one of the primary abiotic stresses that restrict fruit tree production. Grape (Vitis vinifera) is a cultivated fruit crop with high economic value worldwide, and its growth and development are often affected by high temperature stress. Studying the response mechanism of grapes to high temperature stress is of great significance for understanding the domestication of grapes to high temperature stress. In this study, 'Xinyu' grape leaves were collected for high-throughput sequencing and small RNA (sRNA) libraries were constructed for the control group and the high-temperature stress treatment group, respectively. A series of miRNAs related to high temperature stress response were identified, and their functions in high temperature tolerance response were analyzed. A total of 171 known miRNAs and 251 new miRNAs were identified, among which 6 known miRNAs and 21 new identified miRNAs were differentially expressed under high temperature stress. A total of 297 target genes were predicted for these differentially expressed miRNAs. The expression of 10 selected miRNAs and their target genes was validated through qRT-PCR, indicating that these target genes had a significant response to high temperature stress. Gene function and pathway analysis indicated that these genes might play an important role in high temperature stress tolerance. The results of this study provide a theoretical basis for further understanding the high-temperature stress response during fruit tree domestication and the breeding of grape varieties resistant to high-temperature stress.

Trihelix are transcription factors that can specifically bind to the light-responsive element GT element, and are widely involved in plant growth and development and response to adversity stress. In this study, the Trihelix transcription factor family members were identified in the strawberry (Fragaria vesca) genome, and further conducted bioinformatics and expression analysis under stress. The results showed that the strawberry Trihelix transcription factor family consisted of 26 members, distributed on chr01~chr07, among them, the largest number of members were distributed on chr06, with 7 Trihelix transcription factor family members. The results of the subcellular localization prediction showed that the FvTrihelix transcription factor family members were mainly localized in the nucleus and cytoplasm. Phylogenetic analysis showed that FvTrihelix proteins were divided into 5 subfamilies, of which Group2, Group3, Group4 and Group5, contained 1, 3, 11 and 11 FvTrihelix members, respectively. Cis-acting element analysis showed that the members of this family were mainly related to exogenous hormones, drought and low temperature response elements. qRT-PCR results showed that the relative expression of FvTrihelix14 were significantly higher than the control under 4 ℃ condition (P<0.05), being 8 times that of the control (CK). The expression level of FvTrihelix15 under 10% PEG and 4 ℃ treatments showed significant differences compared to the CK (P<0.05), being 12 and 14 times that of the control, respectively. In summary, the members of the strawberry Trihelix transcription factor family are able to respond to various stress conditions. This study provides a theoretical basis for the screening and application of stress-resistant genes in strawberries.

Salvia miltiorrhiza is a traditional Chinese herbal medicine, and its bioactive ingredients mainly include tanshinones and salvianolic acids. In order to explore the effects of multi-gene co-overexpression on the growth and accumulation of active substances in S. miltiorrhiza, in this study, the tissue expression and protein subcellular localization of cobacyl pyrophosphate synthetase gene 1 (SmCPS1) and cytochrome P450 monooxygenase gene (SmCYP76AH1) were analyzed, and pYLTAC380H-SmCPS1-SmCYP76AH1, an co-overexpression vector of SmCPS1 and SmCYP76AH1, was constructed using a multi-gene stacking expression system, and then the overexpression vector was transformed to S. miltiorrhiza clean seedlings by Agrobacterium tumefaciens-mediated method. Through callus induction culture, resistance and green fluorescence signal screening, genome integration identification and gene transcription level detection after transformation, double-gene co-overexpressed S. miltiorrhiza lines were obtained, and the content of tanshinone, photosynthetic and fluorescence characteristics of the double-gene co-overexpression lines were further determined. qRT-PCR showed that SmCPS1 and SmCYP76AH1 were expressed in the roots, stems and leaves of S. miltiorrhiza, both of which were highly expressed in the roots. Subcellular localization analysis showed that SmCPS1 was localized in chloroplasts and cytoplasm, and SmCYP76AH1 was localized in endoplasmic reticulum. The contents of tanshinone Ⅰ, tanshinone ⅡA and cryptotanshinone in roots of SmCPS1 and SmCYP76AH1 double-gene co-overexpression lines were extremely significantly increased (P≤0.01). Photosynthetic and fluorescence characteristics of leaves showed that the transpiration rate and stomatal conductance decreased significantly in SmCPS1 and SmCYP76AH1 co-overexpression lines, and the intercellular CO₂ concentration (Ci) and net photosynthetic rate (Pn) varied with the expression level of SmCPS1 and SmCYP76AH1. The initial fluorescence (F₀), maximum fluorescence (F_m) and non-photochemical quenching coefficient (NPQ) increased significantly in co-overexpressed lines, but the maximum photochemical efficiency (F_v/F_m) of photosystem Ⅱ (PSⅡ) remained unchanged. The multigene transformation method was established in this study and the double-gene co-overexpression lines were obtained which provides experimental materials and methods for further research on the regulation of tanshinone, and provide reference for exploring the function of of SmCPS1 and SmCYP76AH1 in the above ground part of S. miltiorrhiza.

Pectin, a crucial constituent of plant cell walls, assumes a significant role in both plant development and cell wall fortification. The enzymatic catalyst responsible for pectin polymerization is glucuronate isomerase, also known as UDP-D-glucuronate-4-epimerase (GAE). This enzymatic process involves the utilization of UDP-α-D-galacturonic acid (UDP-GalA) as the monosaccharide donor to form the pectin skeleton. To investigate the cell wall resistance mediated by GAE of Antirrhinum majus, 8 AmGAEs genes were identified at the whole genome level. Bioinformatics analysis of their physical and chemical properties, subcellular localization, phylogeny, conserved motifs, gene structure, chromosome localization and cis-acting elements showed that all the members of the AmGAE family were alkaline hydrophilic proteins located in Golgi apparatus. The genes had resistance-related response elements such as defense and stress, methyljasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) in the promoter region. Furthermore, RNA-seq and qRT-PCR were used to analyse and reverify the resistant and susceptible materials to S. sclerotiorum in snapdragon. The results showed that the expression of AmGAE1a, AmGAE3, AmGAE5 and AmGAE2 genes were significantly induced in the resistant material Am6 (R), while the expression of AmGAE7 and AmGAE4 genes was significantly induced in the susceptible material Am1 (S). Among them, the expression patterns of AmGAE1a, AmGAE3, AmGAE7 and AmGAE4 genes in qRT-PCR were consistent with differentially expressed genes (DEGs) by RNA-seq, and thus finally identified as candidate genes. The results provide a theoretical basis and new genetic resources of AmGAE family genes involved in the resistance to S. sclerotiorum for further study.

As pigs (Sus scrofa) are important livestock animals, the study of porcine pluripotent induced stem cells can contribute to the development of the stockbreeding, T-box transcription factor 3 (TBX3) is important for promoting and maintaining stemness in porcine pluripotent stem cells. To investigate the function and regulatory mechanism of porcine TBX3 promoter, the conservativeness of the promoter region was assessed by sequence comparison and evolutionary tree construction, the potential transcription factor binding sites in the promoter region was also predicted and then the GO and KEGG enrichment analyses of the predicted transcription factors was performed to explain their involvement in the biological processes. Finally, the activity of promoter was verified by dual-luciferase reporter system. The results indicated that the predicted region of the porcine TBX3 promoter exhibited a low degree of commonality. The common region was found to be involved in cell proliferation, differentiation, apoptosis, and other processes. The predicted region was mined for a variety of potential transcription factors. The predicted region was confirmed to have binding sites and to participate in the signal pathway, including stemness maintenance and thyroid hormone signal. The predicted region was also confirmed to have transcriptional activity, with its core transcriptional promoter located within the region of -246/+64 bp. This study provides a reference for understanding the transcription regulatory mechanisms of TBX3 transcription factors in porcine cells.

Suppressor of cytokine signaling 3 (SOCS3) is a protein that responds to signal transduction and regulates milk protein synthesis. αS1-casein is one of the most important allergens in ruminant milk. However, the effect of SOCS3 on αS1-casein synthesis in goats (Capra hircus) is still unclear. To elucidate the gene sequence, structure and expression of SOCS3, and the effect of SOCS3 on αS1-casein expression in goat mammary gland, in this study, Anhui white goat mammary tissue was used as experimental material, SOCS3 gene coding region sequence was cloned by PCR. Bioinformatics analysis was conducted by online software, the expression of SOCS3 in different lactation periods was detected, interference and overexpression of SOCS3 were performed for functional analysis. The results showed that the complete coding region of SOCS3 gene in goats was 690 bp, encoding 229 amino acids. The molecular weight of SOCS3 protein was 25.09 kD, the theoretical isoelectric point was 8.97. There were 34 phosphorylation sites, no signal peptide and transmembrane helix structure, and SOCS3 was an unstable protein carrying positive charge. SOCS3 protein interacted with SOCS2, Janus kinase (JAK), tyrosine kinase 2 (TYK2), insulin receptor substrate 2 (IRS2), leptin receptor (LEPR) and interleukin 6 (IL6) and other proteins. SOCS3 gene of goat was most closely related to sheep, followed by cattle (Bos taurus). The expression of SOCS3 gene was the highest in the early and peak lactation periods. In goat mammary epithelial cells, interference with SOCS3 gene significantly up-regulated αS1-casein expression, and overexpression of SOCS3 gene significantly down-regulated αS1-casein expression. These results indicated that SOCS3 had a negative regulatory effect on αS1-casein synthesis in goats. This study provides a basis for studying the regulatory mechanism of SOCS3 gene in goat αS1-casein synthesis.

Horn is an important trait in goats (Capra hircus), and the potassium inwardly rectifying channel subfamily J member 15 (KCNJ15) gene is an important gene, which affects the growth of horn. The present study selected Chengde polled goat as the test animal and extracted genomic DNA. Then the 2 353 bp sequence (2064 bp upstream~289 bp downstream of the start codon) of KCNJ15 gene of Chengde polled goat was amplified by PCR. The sequence was analyzed by online software, the plasmids with different deletion fragments were constructed and transiently transfected into 293T cells. In order to determine the core promoter region of the KCNJ15 gene, dual luciferase detection kit was used to measure the activity. The sequence of Chengde polled goat (2353 bp) was successfully obtained, and software prediction showed that -423~+289 bp might be the active region in the promoter. At the same time, several transcription factor binding sites and CAAT and TATA regulatory elements were simultaneously predicted. The dual luciferase activity assay showed that -491~+ 289 bp had the highest relative activity value among the 4 fragments, suggesting that this region might be the core promoter region, which was consistent with the results predicted by software. This study provides basic data for in-depth exploration of the molecular regulation mechanism of KCNJ15 gene in goat horn traits.

In the preparation of animal vaccines such as avian influenza using the canine (Canis lupus familiaris) kidney cell line Madin-Darby canine kidney (MDCK), it was found that the MDCK cell line has tumorigenicity. In this study, highly tumorigenic and non-tumorigenic MDCK cells were selected for RNA-seq high-throughput sequencing. The differentially expressed circular RNA (circRNA) were obtained, and their source genes were analyzed by GO and KEGG. The StarBase and mirTarBase databases were used to predict target microRNA (miRNA) and mRNA of tumorigenic circRNA. Total RNA of tumorigenic and non-tumorigenic MDCK cells were extracted using TRIzol method, and the expression levels of 6 differential circRNAs were verified by qPCR. The results showed that 3 491 circRNAs were identified with the length of 200~800 nt. Among the screened out 27 differentially expressed circRNAs, 18 were up-regulated and 9 were down-regulated. GO, KEGG and Reactome enrichment analysis revealed that differentially expressed circRNAs were associated with ATP activity and amino acid metabolism-related enzymes, linked to adhesion, migration, and various metabolic pathways. The network construction of competing endogenous RNA (ceRNA) illustrated 4 key circRNA-miRNA-mRNA regulatory network related to MDCK cell tumorigenicity, novel_circ_002222/novel-m0145-3p/dynactin 1 (Dctn1), novel_circ_002222/miR-197-x1/Dctn1, novel_circ_001205/novel-m0580-5p/annexin A11 (ANXA11), and novel_circ_003257/novel-m0675-3p/RNA-binding protein 48 (RBM48). The above results indicate that differentially expressed circRNAs related to tumorigenicity in MDCK cells are mainly enriched in transmembrane material transport and energy metabolism-related pathways, which might play a role in energy uptake and metabolism in MDCK cells. This study provides a research foundation and technical support for revealing the mechanism of circRNA involvement in MDCK cell tumorigenesis and establishing genetically engineered non-tumorigenic MDCK cell lines.

As functional marine oligosaccharides, algae oligosaccharides (AOS) and red algae oligosaccharides (ROS) had a variety of biological activities, they gradually showed excellent characteristics and potential. In this study, to explore the role of marine functional oligosaccharides in aquaculture, the growth performance, muscle nutrient composition and serum immunity function of Oncorhynchus mykiss were investigated by adding AOS or ROS in diets. The experiment was divided into 5 groups: The control group was fed a basic diet, while the experimental groups were supplemented with either 0.25% or 0.5% AOS or ROS in the basic feed, respectively. The experimental period was 37 d. The results showed that 0.25% of AOS had significant effects on the weight gain rate (WGR) and specific growth rate of O. mykiss (P<0.05). In terms of nutrition, both oligosaccharides could significantly increase the content of crude lipid, essential amino acids and flavor amino acids in back muscle (P<0.05), and the high-dose group was superior to the low-dose group. At the same time, the content of unsaturated fat acid was increased, and the content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were significantly increased when the addition amount was 0.5% (P<0.01). In terms of immune function, both could improve the activities of lysozyme, acid phosphatase, alkaline phosphatase, superoxide dismutase, catalase, glutathione peroxidase and total antioxidant capacity in the serum of O. mykiss, the high-dose group was generally outperforming the low-dose group. This study indicated that feeding AOS or ROS to O. mykiss had a promoting effect on growth, which is helpful in improving the nutritional and flavor quality of fish meat. It also improved the immunity and disease resistance of O. mykiss, and has potential applications value in the addition of aquatic feed.

The worldwide cultivated freshwater economic crayfish Cherax quadricarinatus is also extensively farmed in various regions of our country. To investigate the genetic diversity and structure of different regions and artificially bred populations of C. quadricarinatus in China, This study conducted analysis on 10 regional aquaculture populations and 3 generations of selectively bred populations using a panel of 15 and 20 microsatellite markers. The results revealed that among the 10 aquaculture populations, the average number of alleles (Na) ranged from 5.867 to 6.867, while the average number of effective alleles (Ne) ranged from 3.090 to 3.857. Additionally, all populations exhibited a Shannon index (H') greater than 1, with both mean observed heterozygosity (Ho) and mean expected heterozygosity (He) exceeding 0.5. The average polymorphism information content (PIC) ranged from 0.531 to 0.595. In addition, the number of migrants per generation (Nm) in 10 different regions ranged from 11.343 to 32.526. The genetic differentiation index (Fst) ranged from 0.008 to 0.027, the genetic distance (D) ranged from 0.024 to 0.090, and the genetic similarity coefficient (I) ranged from 0.914 to 0.977. The cluster map constructed based on D showed that Chaoshan and Nantong, Zhuhai, Taiwan and Pinghu groups cluster into a branch, Wuxi and Huaian, Hainan and Zhuji groups cluster into a branch, and Shanwei groups cluster into a branch alone. Analysis of the selectively bred populations over 3 generations revealed a significant decrease in genetic diversity parameters. In which, Ho decreased from 0.663 to 0.638, H' decreased from 1.449 to 1.372, Na decreased from 3.509 to 3.303, and PIC decreased from 0.640 to 0.635, indicating a decrease in these parameters due to artificial selection. Furthermore, with increasing generations, the parameter I gradually declined from 0.961 to 0.931 while D steadily increased from 0.044 to 0.07 between consecutive generations. However, the I between adjacent generations increased from 0.957 to 0.960, and Nm increased from 26.262 to 29.895. Fst decreased gradually, with a value of 0.009 between the first and second generations and a value of 0.008 between the second and third generations. These results further indicated that consecutive artificial selection led to a decrease in genetic similarity and an increase in genetic distance between generations, also resulting in an increasing level of genetic differentiation. All the above research results indicated that there was no significant genetic differentiation among different regional populations of C. quadricarinatus in China, and the genetic diversity levels were high in all regions, making them suitable as foundation populations for breeding. After 3 generations of selective breeding, the genetic diversity level of the selectively bred populations decreased slightly but remained at a relatively high level. The genetic variation and genetic differentiation levels were relatively low, indicating that they still possess strong breeding potential. These findings provide important theoretical support for the selective breeding of C. quadricarinatus.

Excessive application of chemical fertilizers and pesticides and soil continuous cropping have seriously restricted the production of pepper (Capsicum annuum), so it is very important to improve the soil environment so as to increase the yield of pepper. In order to screen rhizosphere growth-promoting bacteria of pepper and study their promoting effect, the rhizosphere soil from pepper in Qiqihar city was used as experimental materials. The growth-promoting bacteria in the rhizosphere of pepper was screened out using functional media, and identified by 16S rDNA sequence. The seed germination and pot experiments were conducted to verify the growth-promoting effect of the strains with strong growth-promoting ability. A total of 51 strains of pepper rhizosphere growth-promoting bacteria were isolated, which belonged to 12 genera. The results showed that among the 51 growth-promoting bacteria, 8 strains had the ability of phosphorus resolving, 35 strains could produce gibberellin, 33 strains could secrete auxin (IAA), and all 51 strains had the ability of phosphorus-solubilizing, potassium releasing and nitrogen fixation. Bacillus tequilensis K10 could significantly promote pepper seed germination and increased the number of root hairs of pepper young roots. B. siamensis G19-1 and Acinetobacter johnsonii N9-2 significantly increased above ground fresh weight, root fresh weight, aboveground dry weight, root dry weight and plant height (P<0.05), and the ability of bacteria to solubilize phosphorus, release potassium, fix nitrogen, produce gibberellins and IAA was significantly positively correlated with aboveground fresh weight, aboveground dry weight, and root dry weight of pepper plants (P<0.05). In conclusion, this experiment screened 3 growth-promoting bacteria K10, G19-1 and N9-2 as candidate strains for pepper microbial fertilizer, and will provide strain resources and technical support for the development of pepper microbial fertilizer.

Autophagy plays an important role in plant resistance to pathogen infection. The autophagy-related protein 12 (ATG12), is mainly involved in the formation and extension of the double-layer membrane of autophagy. However, its role in plant pathogens, especially Oomycetes, has been rarely reported. In order to clarify the role of Peronophythora litchii PlATG12 (GeneBank No. OR909654) in the growth and development, and pathogenesis, the PlATG12 knockout mutants were obtained by CRISPR/Cas9 gene editing and PEG mediated protoplast transformation technology, and the in situ complementation was performed. Phenotypic assay results showed that compared with the wild-type strain SHS3 (WT), the average mycelial growth rate of the ΔPlatg12-77 and ΔPlatg12-315 mutants was decreased by 6.50% and 7.49%, respectively, and the average sporangium number was reduced by 33.86% and 34.02%, respectively. Meanwhile, the zoospore release rate of the mutants was significantly lower than those of WT after incubating at 12 ℃ for 0.5 and 2 h. In addition, the oospore production was only 20.25% and 22.00% of the WT, respectively, and the oospore of type Ⅲ and type Ⅳ increased significantly. The pathogenicity of PlATG12 knockout mutants on litchi (Litchi chinensis) leaves was lower than that of WT. However, the phenotypic defects of PlATG12 knockout mutants were rescued after in situ complementation of PlATG12. These results indicated that autophagy-related gene PlATG12 plays a key role in the growth and development, and pathogenesis of P. litchii. This study provides theoretical basis for the functions of autophagy pathway in the pathogenic process of oomycetes.

Bovine viral diarrhea/mucosal disease (BVD/MD) is a contact infectious disease caused by Bovine viral diarrhea virus (BVDV), which will lead to persistent infection, and infected calves (Bos taurus) will continuously expel the virus to the outside. Our research group used RNA- protein interaction detection (RaPID) technology to screen tumor necrosis factor receptor associated factor 2, (TRAF2) and Sushi, Nidogen and EGF like domains 1 (SNED1) genes. In order to determine the effects of TRAF2 and SNED1 genes on BVDV replication, in this study, the small interfering RNA (siRNA) technique was used to knock down the TRAF2 and SNED1 genes in madin-darby bovine kidney cells (MDBK), and the knock-down efficiency of TRAF2 and SNED1 genes and the changes of BVDV 5'UTR RNA level of BVDV infected cells were detected by qPCR. The accumulation of double stranded RNA (dsRNA), an intermediate product of BVDV replication, was detected by immunofluorescence staining. The cytopathic effect (CPE) was observed by fluorescence inverted microscope, and the virus titer of offspring was calculated by Karber method. The results showed that the mRNA levels of TRAF2 and SNED1 were significantly reduced (P<0.05), which indicated that TRAF2 KD and SNED1 KD cells were successfully constructed. In BVDV infected control group (NC), CPE phenomena such as plaque, shedding and death began to appear at 36 h, and CPE phenomena of TRAF2 KD cells and SNED1 KD cells weakened. Compared with NC, after BVDV infected TRAF2 KD and SNED1 KD cells, the level of BVDV 5'UTR RNA decreased significantly at 24, 36 and 48 h (P<0.05). The formation and accumulation of dsRNA, an intermediate product of BVDV replication, were detected by immunofluorescence staining. The green fluorescence intensity in TRAF2 KD and SNED1 KD cells decreased significantly at 36 and 48 h. 48 h after BVDV infected TRAF2 KD and SNED1 KD cells, the virus titer of the offspring decreased extremely significantly (P<0.01). The above results showed that knocking down TRAF2 and SNED1 would inhibit BVDV replication, and this study provides an important basis for revealing the pathogenesis of BVDV.

At present, soil heavy metal pollution has become a global major environmental issue. Copper (Cu) is an essential trace element for plants, but high concentrations of copper have toxic effects on plants. In order to adapt to copper stress environments, plants have developed various copper-tolerant molecular and microbiological mechanisms. This article introduces the role of hyperaccumulating plants in copper-contaminated soil, systematically outlining the morphological, physiological, molecular, and rhizospheric microbiological mechanisms of plant responses to copper stress, including the following: The physiological defense and absorption transport mechanisms of plants against copper; Plant growth promoting rhizobacteria (PGPR) enhancing plant tolerance to copper stress by promoting plant nutrient absorption and secreting growth-regulating substances; PGPR reducing copper damage to plants by inducing systemic resistance and adsorbing accumulated copper ions; The mechanisms of plant responses to copper stress mediated by rhizospheric microbial community structure and function; And the changes in rhizospheric microbial community structure in response to different copper stresses. This article provides scientific support for the future breeding of copper-tolerant plant germplasm and the management and restoration of copper-contaminated soil.

Organoid is a tissue substitute that is structurally and functionally similar to the origin organ. At present, many organoid culture systems have been established, however testicular organoid culture is still in the exploratory stage. In this study, testicular tissues of Saanen dairy goats (Capra hircus) were taken, and testicular cells were isolated and cultured by mixed enzyme digestion. Testicular cells were cultured in three dimensions using a mixed medium containing low melting point agarose. The formed organoids were evaluated using light microscope and immunofluorescence detection techniques, and the culture effects of different sources of testicular cells, different numbers of testicular cells and different concentrations of low melting point agarose on testicular organoids were compared. Finally, the model function of the testicular organoid was verified by lipopolysaccharide (LPS) infection. The result showed that cells ranging from 2×10⁵ to 3×10⁵, in 1% low-melting-point agarose were more favorable for the formation of goat testicular organs in goats. The organoids formed possess seminiferous tubule-like structures, with spermatogonial stem cells and Sertoli cells evenly distributed within. Testicular organoid infected with LPS were smaller than normal organoids, exhibited cellular debris, and were accompanied by a significant increase in inflammatory factors, indicating that these organoids could respond to external stimuli and simulate the immune response of organs to a certain extent, making them a suitable model for the in vitro study of testicular diseases. This study not only promotes the development of testicular organoid culture technology, but also provides an important scientific basis and experimental platform for a variety of fields, including reproductive biology, disease modeling, drug screening, regenerative medicine, and personalized medicine.

Pseudomonas plecoglossicida is the main pathogen which causes the visceral white spot disease in large yellow croaker (Larimichthys crocea), leading to serious economic loss. Previous studies have shown that immune stimulating complexes (ISCOMs) have good oral immune effects. To evaluate the oral immune effects of P. plecoglossicida immune stimulating complexes (Pp-ISCOMs) on L. crocea, Pp-ISCOMs vaccine was prepared, which presented a cage-like nanoparticle structure. At 21 d post oral vaccination, the expression of immune-related genes in the head-kidney of immunized L. crocea were detected, and the immune serum titer was measured using agglutination tests. The immune protective effects of Pp-ISCOMs were then determined by challenge assay with P. plecoglossicida. The results showed that the expression of immune or anti-inflammatory factors such as myeloid differentiation factor 88 (MyD88)(P<0.05), interleukin 2 (IL-2)(P<0.001) in the head kidney of Pp-ISCOMs-immunized L. crocea were significantly higher than those of the inactived-P. plecoglossicida-immunized group (Pp) or the blank control group (CK); and the agglutination titer of large yellow croaker serum (21 d post immunization) was 2². After a P. plecoglossicida attack with a dose of 5×10² CFU/g by intraperitoneal injection, the relative percent survival (RPS) of L. crocea vaccinated by Pp-ISCOMs and Pp were estimated to be 73.3% and 40% respectively. The results indicate that the Pp-ISCOMs could improve the resistance of L. crocea to P. plecoglossicida, and suggested a promising prospect for application. This study provides technical support for the prevention and control by immune to visceral white spot disease in L. crocea.

Eugenol has been widely used in fishery anesthesia because of its wide source, low cost, good effect and short residual period, but its safety is still controversial over the world, and it is considered that eugenol compounds are carcinogens or potential carcinogens for rodents. In order to provide an efficient and simple method for the on-site rapid detection of eugenol residues in aquatic products, in this study, a time-resolved fluorescence immunochromatographic technique for eugenol and prepared immunochromatographic test strips were established. The fluorescent microspheres and anti-eugenol monoclonal antibody were labeled with Eu³⁺, and parameters such as microsphere activation time, fluorescent labeling buffer pH, amount of labeled antibody, amount of fluorescent microspheres and T- and C-line encapsulation concentration were optimized, and the immunochromatographic test strips were prepared and evaluated under the optimal conditions. The results showed that the test strips made under the optimal conditions had a good linear relationship between 0.05~0.40 μg/mL with the standard curve equation: y=-2.415lnx+2.589 4, R²=0.994, and the instrumental limit of detection (LOD) of 0.1 μg/mL, which indicated that the strips had a good sensitivity. The recoveries of the spiked samples were betweeen 94.16% and 109.27%, and the coefficients of variation (CV) of intra-batch and inter-batch were from 2.00% to 7.33% and from 2.73% to 6.97%, respectively. The results of the eugenol specimen detection by the newly developed test strips were consistent with those of the traditional high-performance liquid chromatography (HPLC) method. The stability test proved that the test strips prepared in this study had an effective shelf life of at least 1 year. This research is suitable for the rapid detection of eugenol residues in aquatic products and provides a reference for the development of eugenol rapid detection technology.