Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) detection system is now widely used for nucleic acid detection and disease diagnosis. However, there are still fewer detections for single nucleotide polymorphisms (SNPs) and limited diversified detection systems for pathogen and SNP sites detection, which greatly limits their applications. Obviously, the development of a more diversified and convenient suite of detection tools is essential to unlock the full potential of CRISPR/Cas12a technology and to expand its applications across a wider range of scenarios. We have successfully developed an integrated CRISPR/Cas12a assay system. This system introduces crRNA during protein expression, reducing the number of steps and reaction time by adding only a fluorescent reporter gene and target DNA during subsequent detection. It enables on-site visualization of the assay in combination with a Recombinase polymerase amplification (RPA) reaction. Combined with the RPA reaction, we are able to rapidly detect African swine fever virus (ASFV) pathogens with high specificity. The system also enables genotyping of the SNP site of the porcine prolificacy-associated estrogen receptor (ESR) gene and the sheep prolificacy-associated Fecundity booroola (FecB) gene. Visualization is possible up to a final concentration of 3 nM, and effective differentiation of low concentrations within the concentration range of the assay. The integrated CRISPR/Cas12a assay system we developed has a robust design that ensures high-fidelity genotyping and pathogen detection are no longer restricted to the lab, allowing for rapid field analysis, which is crucial for timely interventions in agricultural and clinical settings. In addition, it has the advantages of low cost, easy operation and visualization of results.

The myometrium, composed of the inner circular muscle (CM) and outer longitudinal muscle (LM), is crucial in establishing and maintaining early pregnancy. However, the molecular mechanisms involved are not well understood. In this study, we identified the transcriptomic features of the CM and LM collected from the mesometrial (M) and anti-mesometrial (AM) sides of the pig uterus on day 18 of pregnancy during the placentation initiation phase. Some genes in the cellular zinc ion level regulatory pathways (MT-1A, MT-1D, MT-2B, SLC30A2, and SLC39A2) were spatially and highly enriched in uterine CM at the mesometrial side. In addition, the histone modification profiles of H3K27ac and H3K4me3 in uterine CM and LM collected from the mesometrial side were characterized. Genomic regions associated with the expression of genes regulating the cellular zinc ion level were detected. Moreover, six highly linked variants in the H3K27ac-enriched region of the pig SLC30A2 gene were identified and found to be significantly associated with the total number born at the second parity (P < 0.05). In conclusion, the genes in the pathways of cellular zinc homeostasis and their regulatory elements identified have implications for pig reproduction trait improvement and warrant further investigations.

Chromatin organization is important for gene transcription in pig genome. However, its three-dimensional (3D) structure and dynamics are much less investigated than those in human. Here, we applied the long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) method to map the whole-genome chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) in porcine macrophage cells before and after polyinosinic-polycytidylic acid [Poly(I:C)] induction. Our results reveal that Poly(I:C) induction impacts the 3D genome organization in the 3D4/21 cells at the fine-scale chromatin loop level rather than at the large-scale domain level. Furthermore, our findings underscore the pivotal role of CTCF-anchored chromatin interactions in reshaping chromatin architecture during immune responses. Knockout of the CTCF-binding locus further confirms that the CTCF-anchored enhancers are associated with the activation of immune genes via long-range interactions. Notably, the ChIA-PET data also support the spatial relationship between single nucleotide polymorphisms (SNPs) and related gene transcription in 3D genome aspect. Our findings in this study provide new clues and potential targets to explore key elements related to diseases in pigs and are also likely to shed light on elucidating chromatin organization and dynamics underlying the process of mammalian infectious diseases.

The myometrium is the outer layer of the uterus. Its contraction and steroidogenic activities are required for embryo implantation. However, the molecular mechanisms underlying its functions remain unknown in pigs. The myometrium includes the inner circular muscle (CM) and the outer longitudinal muscle (LM) layers. In this study, we collected the CM and LM samples from the mesometrial side (named M) of the uterus on days 12 (pre-implantation stage) and 15 (implantation stage) of pregnancy and day 15 of the estrous cycle. The transcriptomic results revealed distinct differences between the uterine CM and LM layers in early pregnancy: the genes expressed in the LM layer were mainly related to contraction pathways, whereas the transcriptional signatures in the CM layer on day 15 of pregnancy were primarily involved in the immune response processes. Subsequent comparisons in the CM layer between pregnant and cyclic gilts show that the transcriptional signatures of the CM layer are implantation-dependent. Next, we investigated the genome-wide profiling of histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 trimethylation (H3K4me3) in pig uterine CM and LM layers. The genomic regions that had transcriptional activity and were associated with the expression of genes in the two layers were characterized. Taken together, the regulatory regions identified in the study may contribute to modulating the gene expression in pig uterine CM and LM layers during implantation.

Spatial chromatin structure is crucial for understanding the early growth and development of porcine skeletal muscle. However, its characteristic of 3D architecture and elaborate regulation of gene transcription remains unclear. In this study, ChIA-PET method is used to study the changes of early chromatin three-dimensional structure in skeletal muscle of lean type Yorkshire pig and fat type Meishan pig. Integrating the in situ Hi-C data revealed the 3D architecture and long-range interaction of the porcine muscle. The results showed the CTCF/RNAPII mediated long-range interaction shapes the different chromatin architecture and dominates the unique regulation of enhancers. In addition, the results revealed that key myogenic genes like ssc-mir-1 had a unique enhancer regulation function in myogenesis. Interestingly, the FGF6 gene is of breed-specific regulation, implying the difference between two breeds in skeletal muscle development. Our research thus may provide a clue for the porcine genetic improvement of skeletal muscle.

Expression of key transcription factors can induce transdifferentiation in somatic cells; however, this conversion is usually incomplete due to undefined intrinsic barriers. Here, we employ MyoD-induced transdifferentiation of fibroblasts as a model to illustrate the chromatin structures that impede the cell-fate transition. Focusing on the three-dimensional (3D) chromatin interactions, we show that MyoD directly establishes chromatin loops to activate myogenic transcriptional program. Similarly, dynamic changes of CTCF-mediated chromatin interactions are favorable for fibroblast-to-myoblast conversion. However, a substantial portion of CTCF-mediated chromatin interactions remain stable, and the associated genes are steady in expression and enriched for fibroblast function that may restrict cell-identity transformation. Temporal CTCF depletion can interrupt the resistant chromatin loops to enhance myogenic transdifferentiation in mice, pig, and chicken fibroblasts. Therefore, during induced transdifferentiation, the transcription factor can directly reorganize the 3D chromatin interactions, and perturbation of CTCF-mediated genome topology may resolve the limitations of cell fate transitions.

[目的]获得具有体外切割活性的来自毛螺旋菌属(Lachnospiraceae)ND2006的LbCas12a蛋白,以期为LbCas12a的应用研究提供重要的生物学工具.[方法]根据pMBP-LbCas12a质粒(Addgene,113431)的基因序列合成LbCas12a基因,并将其与pET-28a(+)线性化载体进行同源重组,构建重组质粒pET28a-LbCas12a,经测序和NheⅠ、SalⅠ双酶切鉴定后将阳性重组质粒转入大肠杆菌BL21(DE3)感受态细胞中进行IPTG诱导表达.通过12％SDS-PAGE凝胶电泳检测确定IPTG诱导的最佳浓度及温度,鉴定重组蛋白的表达形式,通过Ni-NTA树脂亲和层析的方法进行纯化、超滤法浓缩,BCA法检测蛋白浓度.将浓缩后的LbCas12a与猪细小病毒(PPV)靶标DNA、CRISPR RNA(crRNA)及偶联荧光基团的非特异性单链DNA(ssDNA)探针FQ共孵育,设置1个无LbCas12a蛋白的对照组、4个不同蛋白浓度(125、250、500、1 000 nmol/L)的试验组,检测不同组别ssDNA探针的荧光强度,检测测试位点PPV活性.[结果]测序和Nhe Ⅰ、Sal Ⅰ双酶切鉴定结果表明,重组质粒pET28a-LbCas12a构建成功且无移码突变.12％SDS-PAGE凝胶电泳检测结果表明,IPTG诱导表达的最佳浓度为0.5 mmol/L,最佳温度为37℃,表达形式主要为可溶性表达.蛋白浓度检测结果表明,浓缩后的蛋白浓度为485 ng/μL,质量为143 ku.活性检测结果表明,125、250、500、1 000 nmol/L LbCas12a蛋白组的荧光强度均极显著高于对照组(P＜0.01).[结论]本研究成功表达出高活性的LbCas12a蛋白,且LbCas12a蛋白具有体外切割ssDNA的反式活性,为后续基于CRISPR-LbCas12a系统的分子检测技术奠定了基础.

The porcine monomyeloid cell line, or 3D4/21 cells, is an effective tool to study the immune characteristics and virus infection mechanism of pigs. Due to the introduction of the neomycin resistance gene and the SV40 large T antigen gene, its genome has undergone essential changes, which are still unknown. Studying the variation in genome structure, especially the large fragments of insertions and deletions (InDels), is one of the proper ways to reveal these issues. In this study, an All-seq method was established by combining Mate-pair and Shotgun sequencing methods, and the detection and verification of large fragments of InDels were performed on 3D4/21 cells. The results showed that there were 844 InDels with a length of more than 1 kb, of which 12 regions were deletions of more than 100 kb in the 3D4/21 cell genome. In addition, compared with porcine primary alveolar macrophages, 82 genes including the CD163 had lost transcription in 3D4/21 cells, and 72 genes gained transcription as well. Further referring to the Hi-C structure, it was found that the fusion of the topologically associated domains (TADs) caused by the deletion may lead to abnormal gene function. The results of this study provide a basis for elaborating the genome structure and functional variation in 3D4/21 cells, provide a method for rapid and convenient detection of large-scale InDels, and provide useful clues for the study of the porcine immune function genome and the molecular mechanism of virus infection.

The differences in reproductive processes at the molecular level between viviparous and oviparous animals are evident, and the site in the ovary that synthesizes sex hormones (androgens and oestrogens) and the trends for enriching sex hormones during follicle development in chickens are different from those in mammals, suggesting that the effect of sex hormones on follicle development in chickens is probably different from that in viviparous animals. To explore the specific role of androgen receptors (ARs) on chicken follicular development, we matched the correspondence of follicular development stages among chickens, humans, cows and identified chicken-specific genes related to follicle development (GAL-SPGs) by comparing follicle development-related genes and their biological functions among species (chickens, humans, and cows). A comparison of the core transcription factor regulatory network of granulosa cells (or ovaries) based on super-enhancers among species (chicken, human, and mouse) revealed that AR is a core transcriptional regulator specific to chickens. In vivo experiments showed that inhibition of AR significantly reduced the number of syf (selected stage follicles) in chickens and decreased the expression of GAL-SPGs in F5 follicles, while in vitro experiments showed that inhibition of AR expression in chicken granulosa cells (GCs) significantly down-regulated the expression levels of GAL-SPGs, indicating that AR could regulate follicle selection through chicken-specific genes related to follicle development. A comparison among species (77 vertebrates) of the conserved genomic regions, where chicken super-enhancers are located, revealed that the chicken AR super-enhancer region is conserved in birds, suggesting that the role of AR in follicle selection maybe widespread in birds. In summary, we found that AR can regulate follicle selection through chicken-specific genes related to follicle development, which also emphasizes the important role of AR in follicle selection in chickens and provides a new perspective for understanding the unique process of follicle development in chickens. Our study will contribute to the application of androgens to the control of egg production in chickens and suggests that researchers can delve into the mechanisms of follicle development in birds based on androgen/androgen receptors.

In brief Transforming the endometrial luminal epithelium (LE) into a receptive state is a requisite event for successful embryo implantation. This study suggests the role of a transcription factor in regulating endometrial LE receptivity. Abstract The endometrial luminal epithelium (LE) undergoes extensive remodeling during implantation to establish receptivity of the uterus in response to the conceptus signals, such as interleukin 1β (IL1B). But the mechanisms remain to be fully understood. This study investigated the role of CCAAT/enhancer-binding protein β (C/EBP-β) in regulating pig endometrial LE receptivity. Our results showed that C/EBP-β was expressed and activated only in the endometrial LE in an implantation-dependent manner. In addition, C/EBP-β was highly activated at the pre-attachment stage compared to the attachment stage, and its activation was correlated with the expression of IL1B-dependent extracellular signal-regulated kinases1/2-p90 ribosomal S6 kinase signaling axis. Subsequent chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the binding of C/EBP-β within the promoter was positively associated with the transcription of genes related to cell remodeling. One such gene is matrix metalloproteinase 8 ( MMP8 ), which is responsible for extracellular matrix degradation. The expression of MMP8 was abundant at the pre-attachment stage but dramatically declined at the attachment stage in the endometrial LE. Consistent with C/EBP-β, the expression and activation of MMP8 were limited to the endometrial LE in an implantation-dependent manner. Using ChIP-qPCR and electrophoresis mobility shift assay approaches, we demonstrated that C/EBP-β regulated the expression of the MMP8 gene during implantation. Furthermore, we detected that MMP8 and one of its substrates, type II collagen, showed a mutually exclusive expression pattern in pig endometrial LE during implantation. Our findings indicate that C/EBP-β plays a role in pig endometrial LE receptivity by regulating cell remodeling-related genes, such as MMP8 , in response to conceptus signals during implantation.

生物统计学是学习开展现代生物学研究所必需学习的基础核心课程,其理论相对比较复杂,细节要求严格.新冠肺炎疫情暴发以来,线上教学由之前的辅助教学方式转变为主要教学方式,生物统计学课程的特点使其线上教学的效果受到影响.文章试以课程内容设计、教学方式革新、考核方式改革以及对学生综合能力的培养为切入点,探讨疫情期间生物统计学课程教学模式的创新变化与教学实践思考,力争在新冠肺炎疫情防控常态化的背景下,能够实现生物统计学线上教学的基本目标,从而提高教学效果.

旨在制备猪RNA聚合酶Ⅱ的单克隆抗体并进行初步应用.本研究采用生物信息方法预测免疫原,将其化学合成后免疫5只4～8周龄Bal b/c雌性小鼠,对免疫后呈现阳性的小鼠进行细胞融合试验,取其脾细胞与骨髓瘤细胞进行融合并获得能稳定分泌抗RNA PolⅡ的杂交瘤细胞.鉴定结果显示,RNA PolⅡ单克隆抗体的重链为IgG 2A型,轻链为Kappa型.利用间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了 8株稳定分泌RNA PolⅡ单克隆抗体的杂交瘤细胞株.将其初步应用到染色质免疫共沉淀(ChIP-seq)技术,并与商业化抗体的富集性进行比较,结果表明,本研究得到的单克隆抗体富集性更强.本研究获得的猪RNA PolⅡ单克隆抗体可为表观遗传学的研究提供理论基础,并且提供重要的生物学材料.

miRNAs are short noncoding RNAs that post-transcriptionally regulate gene expression by binding to complementary regions of the target mRNA. The miRNAs associated with the deposition of intramuscular fat (IMF) content in pigs, which is an important meat quality trait, still remain to be investigated. In this study, the longissimus dorsi muscles (LDMs) from 234 individuals were collected from Yorkshire pigs at 90 kg body weight and the miRNA deep sequencing was conducted by using two tailed groups which were taken five individuals each from high (2.94 +/- 0.04%) and low (1.62 +/- 0.02%) IMF samples. The results showed that total 268 mature miRNAs were identified, of which 70 were previously known, 162 were conserved among species and 36 were identified specifically in pigs. Moreover, 28 miRNAs involved in adipogenesis were differentially expressed in the two groups, and five out of 16 miRNAs were validated by quantitative PCR (qPCR) using stem loop primers. Our results may serve as a fundamental basis for understanding the roles of miRNA in IMF development in pigs. The miRNAs identified in our study can be utilized for research IMF trait in pig population and will provide further clues to the study of meat quality regulatory mechanisms.

Pigs, as one of the most common livestock species worldwide, are expected to have a fast growth rate and lower subcutaneous fatness but higher intramuscular fat ("marbling meat"). Nowadays, it is believed that not only host genetics but also its gut microbiomes can modulate farm animal phenotypes, however, many of the mechanisms remain elusive. We measured the body weight (BW), average daily gain (ADG), backfat thickness (BFT), and intramuscular fatness (IMF) of 91 Enshi pigs at 260 days of age, then genotyped each one individually using a 50K single nucleotide polymorphism array and performed 16S ribosomal RNA gene sequencing on 455 microbial samples from the jejunum, ileum, cecum, colon, and rectum. The microbial diversity showed notable spatial variation across the entire intestinal tract, with the cecum and colon having the highest alpha-diversity. The cecal and colonic microbiotas made greater contributions to BW and ADG and accounted for 22-37% of the phenotypic variance. The jejunal and cecal microbiotas contributed more (13-31%) to the BFT and IMF than the other segments. Finally, from cecum, colon, and jejunum, we identified eight microbial taxa that were significantly correlated with the target traits. The genera Alloprevotella and Ruminococcaceae UCG-005 were highly positively correlated with BW and ADG. The genera Prevotellaceae UCG-001 and Alistipes in the cecum and Clostridium sensu stricto 1 in the jejunum were highly positively correlated with BFT and IMF. The genera Stenotrophomonas, Sphaerochaeta, and Desulfovibrio were negatively associated with the mentioned traits. These findings could aid in developing strategies for manipulating the gut microbiota to alter production performance in pigs.

Skeletal muscle satellite cells (SCs) play an important role in the repairment and regeneration of damaged muscle. The activation, proliferation, migration, and differentiation of SCs are essential to the response to muscle injury. Up-frameshift 1 (UPF1) is involved in the regulation of many developmental processes. However, the role of UPF1 and its associated regulatory mechanism in SCs are still unclear. Here, we analyzed changes in the transcriptome of porcine SCs with UPF1 knockdown. The results showed that focal adhesion and actin cytoskeleton processes were regulated by UPF1. We also confirmed experimentally that UPF1 promoted SC migration and adhesion by regulating the expression of F-Actin, Vinculin, and several adhesion-related genes. Furthermore, we found that phosphorylated focal adhesion kinase (p-FAK) was down-regulated by UPF1 knockdown. This study identifies the role of UPF1 in regulating SC migration and adhesion and therefore provides new insight into the regulatory mechanism of UPF1 in the process of repairing damaged muscle.

The development of the placental fold, which increases the maternal–fetal interacting surface area, is of primary importance for the growth of the fetus throughout the whole pregnancy. However, the mechanisms involved remain to be fully elucidated. Increasing evidence has revealed that long non-coding RNAs (lncRNAs) are a new class of RNAs with regulatory functions and could be epigenetically regulated by histone modifications. In this study, 141 lncRNAs (including 73 up-regulated and 68 down-regulated lncRNAs) were identified to be differentially expressed in the placentas of pigs during the establishment and expanding stages of placental fold development. The differentially expressed lncRNAs and genes (DElncRNA-DEgene) co-expression network analysis revealed that these differentially expressed lncRNAs (DElncRNAs) were mainly enriched in pathways of cell adhesion, cytoskeleton organization, epithelial cell differentiation and angiogenesis, indicating that the DElncRNAs are related to the major events that occur during placental fold development. In addition, we integrated the RNA-seq (RNA sequencing) data with the ChIP-seq (chromatin immunoprecipitation sequencing) data of H3K4me3/H3K27ac produced from the placental samples of pigs from the two stages (gestational days 50 and 95). The analysis revealed that the changes in H3K4me3 and/or H3K27ac levels were significantly associated with the changes in the expression levels of 37 DElncRNAs. Furthermore, several H3K4me3/H3K27ac-lncRNAs were characterized to be significantly correlated with genes functionally related to placental development. Thus, this study provides new insights into understanding the mechanisms for the placental development of pigs.

This paper aims to investigate the bending, buckling and free vibration problems of an axially loaded Timoshenko beam in a systematic manner. A recently-developed unified model is adopted, where a transition parameter characterizing the direction of the axial force during the deformation is introduced. For beams with various boundary conditions, closed-form expressions of deflections and critical buckling loadings are obtained in an analytical fashion and frequency equations are derived. It is highlighted that the inherent relations of the buckling loadings for the Timoshenko and Euler-Bernoulli beams are explicitly extracted. Numerical calculations are carried out to check the validity of the present solutions and to quantify the effects of the transition parameter. Significant effects of the transition parameter are revealed on the bending, buckling and free vibration of the axially loaded beam. The present analysis is of significance to axially-loaded beam-like mechanical system, especially for determination of the direction of axially force.

真核生物的基因转录受多种元件共同调控,其中增强子是重要的顺式作用元件,能够极大促进基因的转录.增强子的功能与细胞、组织、个体的特异性功能或表型密切相关,其异常功能往往导致性状改变和疾病发生.因此,研究增强子的功能对于揭示表型背后的分子机理具有十分重要的生物学意义,对于农业动物科学显得尤为重要.本文就增强子的特性、鉴定方法、活性检测以及在农业动物研究中的进展进行综述,以期能够对增强子的相关研究提供依据和参考.

BACKGROUND:Porcine skeletal muscle satellite cells play important roles in myogenesis and muscle regeneration. Integrated analysis of transcriptome and histone modifications would reveal epigenomic roles in promoting myogenic differentiation in swine.METHODS:Porcine satellite cells (PSCs) were isolated and in-vitro cultured from newborn piglets. RNA Sequencing (RNA-Seq) and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiments were performed using proliferating cells and terminal myotubes in order to interrogate the transcriptomic profiles, as well as the distribution of histone markers-H3K4me3, H3K27me3, and H3K27ac-and RNA polymerase II.RESULTS:The study identified 917 differentially expressed genes during cell differentiation. The landscape of epigenetic marks was displayed on a genome-wide scale, which had globally shrunken. H3K27me3 reinforcement participated in obstructing the transcription of proliferation-related genes, while its depletion was closely related to the up-regulation of myogenic genes. Furthermore, the degree of H3K27me3 modification was dramatically reduced by 50%, and 139 myogenic genes were upregulated to promote cell differentiation.CONCLUSIONS:The depletion of H3K27me3 was shown to promote porcine satellite cell differentiation through upregulating the transcription level of myogenic genes. Our findings in this study provide new insights of the epigenomic mechanisms occurring during myogenic differentiation, and shed light on chromatin states and the dynamics underlying myogenesis.

Development of placental folds is a critical event affecting placental function in pigs because it can increase surface area for improvement in capillary density as gestation advances. However, the molecular mechanisms of the event are not well defined. Histone modifications have important roles in gene regulation. To investigate their effects on regulation of genes controlling porcine placental development, RNA-seq and ChIP-seq of porcine placental tissues from gestational days 50 (establishment stage of placental folds) and 95 (expanding stage of placental folds) were carried out in this study. The differentially expressed genes were identified and of which the down- and up-regulated genes are related to endoplasmic reticulum (ER) stress and angiogenesis, respectively. In addition, we mapped the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are associated with transcriptional activation. A number of differential modification regions between the 2 gestational stages were identified and majority of them are those with increased signals of H3K4me3 (14,576 out of 16,931). Furthermore, we observed that the increase of H3K4me3 is significantly correlated with the elevated expression levels of the neighboring genes, and notably, these genes were enriched in pathways related to blood vessel formation and microvascular permeability. Taken together, the findings suggest important roles of histone modifications on placental remolding in response to developmental changes.