Sugars will eventually be exported transporters (SWEETs), a novel family of membrane-transporters widely distributed in eukaryotes, which performed essential roles in plant growth and development processes, such as phloem loading for long distance sugar transport, hormone transport, pollen development, fruit development, seed filling and plant-pathogen interactions. To analyze the function of StSWEET16b in sugar transport and plant-pathogen interactions, in this study, potato (Solanum tuberosum 'Qingshu 9') was used as material, after treated with sucrose, fructose and glucose individually, the expression of StSWEET16b was analyzed by qRT-PCR in the leaves of 'Qingshu 9'. The expression pattern of StSWEET16b was changed under 3% sucrose, fructose and 3glucose treatments respective, suggesting that may be involved in the translocation of sucrose, fructose and glucose in potato. By analyzing the growth condition of the transgenic tobacco (Nicotiana benthamiana) on different sugar medium and measuring the contents of sucrose, fructose and glucose in the plants, it was found that the transgenic tobacco grew weaker than the wild-type plants on glucose and sucrose medium, and the contents of sucrose and glucose in the transgenic plants were lower than those in the wild-type plants. However, transgenic tobacco grew significantly better than wild-type plants on fructose medium (P<0.05), and the fructose content of transgenic plants was higher than that of wild-type plants, indicated that the heterologous expression of StSWEET16b in tobacco could promote fructose transport, and inhibit the transport of sucrose and glucose. Transient expression results showed that StSWEET16b gene was involved in the interaction between tobacco and Phytophthora infestans. Furthermore, transient expression of StSWEET16b enhanced the resistance to P. infestans in tobacco, resulting in inhibiting the expansion of late blight disease spots. It inhibited the expansion of the disease spot of P. infestans and improved the disease resistance of the plant. The results suggesed that StSWEET16b protein played an important role in sugar transport and plant-pathogen interactions for resistance to late blight. This study provides a theoretical basis for improving potato quality and breeding of disease-resistant varieties.

Firmness is an important indicator of fruit shelf-life, and cell wall metabolism is an important factor affecting the firmness and softening process of fruit. In order to understand the relationship among the softening process, cell wall metabolism genes and regulation of fruit ripening hormones in tomato (Solanum lycopersicum), the expression of cell wall metabolism genes during tomato fruit ripening was systematically analyzed, and the effects of the fruit ripening hormones, abscisic acid (ABA), ethylene (C₂H₄) and their inhibitors on the expression of these genes were investigated by applying exogenous hormones and their inhibitors and qRT-PCR technique. The results showed that the expression of cell wall metabolism related genes, SlCel1 (endo-β-1,4-glucanases), SlTBG7 (β-galactosidase precursor), SlXETB2-F2 (xyloglucanendotransglycosylase), SlXET4-F1, SlTBG3, SlCel2, SlExp1 (expansin), SlTBG1, SlPG (polygalacturonase) and SlTBG4 are increased with fruit maturation and softening, suggesting their close relation to the softening process during fruit ripening in tomato. The treatment results of fruit ripening hormones and their inhibitors indicated that exogenous ABA could promote the expressing of SlCel1, SlExp1, SlTBG3, SlTBG4 and SlXET4-F1 genes,while the expression of SlCel1, SlExp1 and SlTBG4 genes was inhibited by NDGA (nordihydroguaiaretic acid). The application of ethylene could up-regulate the expressing of SlExp1, SlPG and SlTBG4 genes, but their expressions were inhibited by 1-MCP (1-methylcyclopropene). These results implied that the regulation of cell wall metabolism related genes, SlCel1, SlExp1, SlTBG3, SlTBG4, SlPG and SlXET4-F1 require ABA and ethylene to regulate the softening process during fruit ripening. Therefore, appropriate application of NDGA or 1-MCP might be beneficial for inhibiting cell wall metabolism and would extend the fruit shelf-life by inhibiting the ABA and ethylene signaling. The cultivation of new tomato varieties with low expression levels of SlPG, SlCel1, SlExp1 and SlTBG4 genes may effectively realize a new way to keep tomatoes fresh and maintain good fruit quality after harvest. These results has guiding significance for realizing the ripening regulation of tomato fruit.

Shikimate quinate hydroxycinnamoyl transferase (HCT) is one of the key enzyme in lignin synthesis pathway, also affected the quality of cotton (Gossypium spp.) fiber. The study on the expression characteristics of GbHCT13 in cotton fiber development can provide a reference for exploring the mechanism of cotton fiber quality development in the future. HCT gene involved in lignin metabolism named GbHCT13 was cloned from the fiber (10 d) in Gossypium barbadense 'XH21'. Bioinformatics analysis was carried out on its sequence.The expression patterns and characteristics were analyzed in G. barbadense and G. hirsutum by qRT-PCR, respectively. Effects of caffeoyl coenzyme A on fiber development were studied by in vitro feeding experiment of cotton ovule. These results showed that GbHCT13 gene (GenBank No. MW048849) was located on the Gossypium barbadense chromosome 10 of group D, the total length was 1 400 bp, CDS coding region was 1 311 bp, predicted that the proteins encoded by this gene were a class of hydrophilic non-membrane proteins, consisting of 1~432 amino acids, and the multi-sequence comparison from other species revealed that the amino acid sequence of GbHCT13 contained a BAHD acyltransferase family of HXXXD and DFGWG regions, which were closely related to Gossypium arboreum and Gossypium raimondii. Using qRT-PCR analysis GbHCT13 gene expressed in different cotton varieties , the results showed that the gene expression in different Gossypium barbadense varieties were similar trends. The expression level was higher at 5~15 d in the early stage of fiber development, especially at the 10 d of fiber development. In vitro feeding of ovule showed that certain concentration of caffeoyl coenzyme A promoted fiber elongation and the expression of GbHCT13 gene. This study provides the theoretical basis for the future study of the molecular mechanism of GbHCT13 gene involved in lignin synthesis.

9-cis-epoxycarotenoid dioxygenase (NCED) is the key rate-limiting enzyme in abscisic acid (ABA) biosynthesis pathway. In this study, bioinformatics method was used to identify 10 genes with complete NCED conserved domain RPE65 from the whole genome of Phyllostachys edulis. The length of the NCED proteins ranged from 240 to 654 aa with molecular mass of 28.03 to 70.04 kD. Phylogenetic analysis showed that the 10 NCED genes could be divided into 2 subgroups, and most NCED genes were closely related to rice (Oryza sativa) NCED genes, except for PH02Gene49300 and PH02Gene41427. The predictive analysis of cis-acting elements showed that NCED gene promoter contained a large number of abiotic stress response elements, indicating that they might play an important role in the stress resistance of Ph. edulis. Transcriptome data analysis showed that the expression of NCED genes in different tissues and developmental stages of Ph. edulis was specific, especially highly expressed in the roots and shoots (P<0.05). The qRT-PCR results showed that NCED genes had different expression patterns under ABA, salicylic acid (SA), drought and salt stress treatments, which suggested that NCEDs might play different roles in the regulation of stress and hormones response. The present work provides a basic data for further study of the function of NCED gene family in Phyllostachys edulis.

Histone acetylation is the first histone modification to be identified, which regulates gene expression by changing chromatin structure and accessibility, and is widely involved in the regulation of various biological events during early embryonic development of mammals. H3K64ac is a previously unidentified histone modification on the lateral surface of the histone octamer. It is known to be involved in regulating nucleosome stability and promoting gene expression in vivo. However, the role of H3K64ac in the early development of porcine (Sus scrofa domesticus) somatic nuclear transfer embryos is still unknown. In this study, H3K64ac in early embryos derived from parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) were examined by immunofluorescence staining. PA 4-cell embryos were treated with aphidicolin, α-amanitin and cycloheximide, respectively. The results showed that H3K64ac modification was always present in both PA and SCNT embryos, and H3K64ac levels were the maximum at the pronuclear stage, decreased at the 2-cell stage, slightly increased at the 4-cell stage, and remained low status from the 8-cell stage to the blastocyst stage. Comparison analyses of H3K64ac levels between PA and SCNT embryos revealed that H3K64ac levels in SCNT embryos at each stage were significantly higher than those in PA embryos (P<0.05). Inhibition of DNA replication, RNA and protein synthesis did not affect H3K64ac levels in porcine embryos. The results suggested that H3K64ac was abnormally reprogramming during early development of porcine cloned embryos, and the reduction of H3K64ac levels in early embryonic development was an active reactive process independent of DNA replication, RNA and protein synthesis. This study provides a theoretical basis for further study on the molecular mechanism of H3K64ac involved in regulating the development of porcine somatic nuclear transfer embryos.

Based on early study, the high-throughput sequencing found that ssc-miR-204 was significantly differentially expressed in piglets infected with Clostridium perfringens type C in the resistance and susceptibility group. This study aims to verify the targeted regulatory relationship between miR-204 and discs large homolog 5 gene (DLG5). In the present study, the TargetScan software was used to predict the targeted binding sites and conservation between miR-204 and DLG5 gene. The wild-type and mutant-type pmirGLO dual-luciferase reporter vector in the 3'UTR region of DLG5 gene was constructed and the targeting relationship between miR-204 and DLG5 confirmed by dual luciferase activity experiment. Further, the regulation of miR-204 on DLG5 expression was detected by qRT-PCR, Western blot and cell immunofluorescence experiments after transfected with miR-204 mimic and inhibitor in intestine porcine epithelial cells, respectively. The results showed that the miR-204 mature seed region sequence was complementary to the nucleotide sequence of DLG5 at 3'UTR 1 333~1 339, and was highly conserved among multiple species (such as human (Homo sapiens), pig, chimpanzee (Pan troglodytes), rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), cattle (Bos taurus), etc). Through double enzyme digestion and sequencing identification, this experiment successfully constructed pmirGLO-DLG5-3'UTR dual luciferase vector; the results of dual luciferase reporter gene experiment showed that miR-204 mimic could significantly reduce the luciferase activity of DLG5 gene (P<0.01), the two had a target relationship. Then, the results of qRT-PCR and Western blot indicated that after transfected with miR-204 mimic, the expression level of DLG5 was significantly inhibited (P<0.01). On the contrary, after transfected with miR-204 inhibitor, the expression level of DLG5 was significantly upregulated (P<0.01). The immunofluorescence results showed that compared with the mimic NC group (89.90±3.818), DLG5 fluorescence intensity was significantly reduced (62.65±3.790, P<0.01) after transfected with miR-204 mimic. While, comparing with the inhibitor NC group (85.52±6.220), the fluorescence intensity of DLG5 was significantly enhanced (102.80±3.588, P<0.05) after transfected with miR-204 inhibitor. From the above results, it could be seen that miR-204 could target the DLG5 gene, and miR-204 had a negative regulatory effect on the expression of DLG5. The results of this study can provide a basis for the further verification of the functions of miR-204 and DLG5 genes at the cellular level, and provide a basis for the role of miRNAs in the regulation of C. perfringens type C diarrhea of piglets.

Apoptosis of ovarian granulosa cells affects follicle development and ovulation in animals. Resveratrol plays an important role in the apoptosis of ovarian granulosa cells. This research was aimed to provide data support for the application of resveratrol in pig (Sus scrofa) production by analyzing the mechanism of resveratrol and miR-222 regulate the apoptosis of porcine ovarian granulosa cells (pGCs). The primary pGCs were treated with different concentrations of resveratrol (0~100 μmol/L), the morphological changes of pGCs were observed under an inverted microscope, the apoptosis rate and cell cycle distribution were detected by flow cytometry, and the expression of CyclinD, CyclinB, p21 and miR-222 were detected by the qRT-PCR method. The pGCs apoptosis change was also detected in the miR-222 overexpression condition. The results showed that when the concentration of resveratrol reached 100 μmol/L, the morphology of pGCs was changed, the apoptosis of pGCs was decreased and the cell cycle of pGCs was inhibited. The expression level of miR-222 was affected by different concentrations of resveratrol and reached the lowest level at 75 μmol/L. The pGCs apoptosis was significantly decreased when miR-222 overexpressed in 75 μmol/L resveratrol. These results indicated that resveratrol (100 μmol/L) could inhibit pGCs proliferation, and regulate miR-222 expression depend on the concentration. Overexpression of miR-222 could decrease pGCs apoptosis with 75 μmol/L resveratrol treatment. The results of the study provide more reference data for resveratrol combined miRNA to regulate the biological functions of pGCs.

Epididymis serves as a main site for sperm transportation, maturation and storage, and its structure and function maintenance as well as gene expression extremely depend on the regulation of androgen and androgen receptor (AR). AR is highly expressed in epididymis of many animals and regulates the function of epididymal epithelium and lumen fluid, which is important for sperm maturation. The purpose of this study was to explore the expression characteristics of AR in postnatal epididymis of Congjiang Xiang pig (Sus scrofa). The qRT-PCR, immunohistochemistry and Western blot were used to detect the expression of AR in the epididymis of Congjiang Xiang pigs at 4 development stages of pre-puberty (15 d), in-puberty (30 d), post-puberty (60 d), and sexual maturity (180 d). qRT-PCR results showed that AR was expressed in the epididymis of Congjiang Xiang pigs from postnatal 15 d to 180 d with the increase of age, and reached to the highest expression level at 180 d. In different regions, the expression of AR gene was observed highest in region Ⅳ(epididymal cauda) and the lowest in regionⅠ(epididymal caput) (P<0.05). Although there was no significant difference of AR expression between region Ⅰ and Ⅲ(epididymal corpus), the statistical distinction among other regions was found (P<0.05). Western blot results showed that AR protein expression abundance had the same trend with that of mRNA level in different stages and 5 epididymis regions. Immunohistochemical staining showed that AR was mainly located in the epididymal epithelial cells (nucleus) and spermatozoa, and its expression displayed segment-specific. In conclusion, this study found that the expression of epididymal AR increased from 15 d to 60 d, and then maintained a high level to 180 d; AR mainly distributed in the nucleus of epididymal epithelial cells and spermatozoa. These data indicated that the epididymal AR expression was in a stage- and segment-specific manner. In addition, it was speculated that the high expression of AR at age of 180 d might be related to the existence of a large number of spermatozoa in this period. This study provides reference for further exploring the mechanism of sperm maturation in the epididymis of Congjiang Xiang pigs.

miR-133a is the microRNA (miRNA) specifically expressed in skeletal muscle and myocardium, but its role in the proliferation and differentiation of skeletal muscle-derived satellite cells (MDSCs) in Bos taurus is unknown. In order to further understand the effect of miR-133a on the proliferation and differentiation in bovine MDSCs, and enrich the molecular genetic mechanism affecting the growth and development of bovine muscle, in this study, based on the established in vitro differentiation model of Bos taurus MDSCs, qRT-PCR method was used to detect the temporal expression profiles of miR-133a, myogenin (MyoG), myogenic differentiation (MyoD) and myosin heavy chain (MHC) in MDSCs. Then mimic and inhibitors of miR-133a were used to transfect BMSCs to construct a model of miR-133a overexpressing or inhibiting, and transfection effects on MDSCs proliferation were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining for proliferating cells. The techniques of qRT-PCR, Western blot and immunofluorescence were used to detect the mRNA and protein levels of MyoG, MyoD and MHC. The results showed that the expression level of miR-133a increased significantly at the differentiation stage of MDSCs (P<0.01). After overexpression of miR-133a, the positive ratio of proliferating cells significantly decreased (P<0.01), while the gene expression of MyoG (P<0.05) and MHC (P<0.01) significantly increased; The protein expression level of MHC significantly increased (P<0.01) and the number of fused myotubes increased. After miR-133a expression was inhibited, the positive ratio of proliferating cells significantly increased (P<0.01), while the gene expression of MyoG (P<0.01), MyoD (P<0.05) and MHC (P<0.01) significantly decreased; the expression of MHC protein significantly decreased (P<0.01) and the number of fused myotubes decreased. The above results confirmed that miR-133a played a role in inhibiting proliferation and promoting differentiation of MDSCs. The present study enriches the molecular genetic research of bovine muscle growth and development, and provides an important reference for further research on the function of miRNA in regulating animal muscle growth and development.

Circular RNA (circle RNA, circRNA) is a covalently enclosed endogenous biomolecule, which plays an important role in the hypoxia adaptation of plateau animals. High-throughput sequencing technology has been widely used in mammalian and human disease research due to its advantages of low cost, high throughput, and fast speed. However, there are few reports on the circRNAs related hypoxia adaptation in Tibetan cattle (Bos taurus). The heart tissue is sensitive to the high-altitude hypoxia environment. In this study, the circRNAs in the heart tissues of Tibetan cattle and Sanjiang cattle were sequenced using the Illumina HiSeq^TM 4000 platform. Then analyzed the differential expression profiling of circRNAs and predicted the targeting relationship between circRNA-miRNA-mRNA, constructed a circRNA-miRNA-mRNA visualization regulatory network. The results showed that a total of 283 differentially expressed circRNAs were filtered between the two species, which included 278 up-regulated and 5 down-regulated. Gene Ontology functional enrichment analysis showed that the source genes of differentially expressed circRNA were annotated to 45 functional terms, and the KEGG pathway analysis showed that they mainly significantly enriched in ubiquitin mediated proteolysis, cGMP-PKG signaling pathway and ect.. In addition, novel_circ_018959 was found had a targeting relationship with miR-142-x, miR-144-y, miR-1908-x, miR- 2302-y, and miR-1273-y, and the targets of these miRNAs including ECE1, NFATC1, CAST, and COX7C were reported as hypoxia adaptation candidate genes, indicating that novel_circ_018959 was likely to be involved in the hypoxia adaptive regulation in Tibetan cattle, Its function and regulation mechanism need to be further studied. The results provide the data support for further revealing the molecular mechanism of circRNA in Tibetan cattle hypoxia adaptation process.

The yak (Bos grunniens) with yellow body fat showed obvious ability to enrich carotenoids. β- carotene (β-C) is a kind of carotenoids, which plays an important role in carotenoid metabolism of yak. In this study, 15 yaks aged 2~3 years were randomly divided into 5 groups: The control group was fed basic diet; The experimental group included low, medium and high-dose groups, in which the amount of added β-C was 720, 1 440 and 1 620 mg/d, respectively; Positive control was added β-C with 3 440 mg/d. After β-C feeding for 90 d, the expression differences of carotenoid related regulatory gene β-carotene-15,15'-momoxygenase (BCMO1), β-carotene-9', 10'-dioxygenase (BCDO2) and 4 related metabolic regulatory factors - intestine specific homeobox (ISX), peroxisome proliferator-activated receptor γ (PPARγ), scavenger receptor class B type Ⅰ (SR-B1) and retionic acid receptor (RAR) were measured by qRT-PCR, and correlation between BCMO1, BCDO2 and related metabolic regulators was analyzed by Pearson method. The results showed that, in yak duodenum, the expression of BCMO1 in middle dose group was significantly higher than that of other groups; Low, medium and high dosage had no significant effect on BCDO2 expression; Low dosage of β-C was more favorable for ISX expression; There was significant positive correlation between BCDO2 and PPARγ, SR-B1. In the jejunum, the expression of BCMO1 in low and middle dose groups was significantly higher than that of other groups; BCDO2 expression was not affected with β-C supplementation except for the positive group; There was significant positive correlation between BCDO2 and ISX, PPARγ, RAR. In the ileum, the expression change of BCMO1 and BCDO2 were smooth, and the medium dose group was beneficial to the expression of BCMO1 and BCDO2; The expression trends of the 4 metabolic regulatory factors were different, and low dose was beneficial to the expression of ISX, and medium dose was beneficial to the expression of SR-B1; There was significant positive correlation between BCMO1 and PPARγ, SR-B1. In the cecum, the expression of BCMO1 and BCDO2 was not affected by the supplementation. In the colon, the expression of BCMO1 increased with the supplementation; Except for the positive group, the BCDO2 expression was not affected by β-C supplementation; The expression of ISX and RAR showed the maximum in low dose group. The present study provides basic materials for mechanism study of carotenoid metabolism in ruminants, scientific supplementary feeding of yak and the production of high quality yak meat.

Tong sheep is a well-known local sheep (Ovis aries) with fat tail and semi-fine wool for both meat and wool. Adipose tissue is an organ that stores energy and supplies energy to the body. Adipose tissue could secrete leptin (LEP), leptin receptor (LEPR), inflammatory factors and so on.. The LEP and LEPR genes in the adipocytokine signaling pathway can coordinately regulate fat deposition and energy metabolism. In this study, in order to verify the insertion/deletion (InDels) variation of the LEPR gene and its influence on the fat traits and growth traits of the sheep tail, InDel locus of the LEPR gene was screened through the Ensemble database, and primers were designed according to the sequence of Indels locus screened in the database. 166 healthy individuals of Tong sheep were identified and analyzed. Association analysis showed that the 17 bp InDel polymorphism of LEPR gene had no significant effect on tail width and tail length traits of the same sheep (P>0.05), indicating that this locus had little effect on tail fat traits; The breast depth of DD genotype at this locus was significantly higher than that of ID genotype (P>0.05). The DD genotype was significantly higher than that of the II genotype in the growth traits of ram body height, back height and recommended height (P<0.05). The ewe waist width of the II genotype at InDel locus was the significantly higher than that of the DD genotype (P<0.05), indicating that this locus had a significant impact on some growth traits. These results provide a theoretical basis for the selection of the LEPR gene in tail type of Tong sheep and the selection and breeding of the Tong sheep, and provide an important reference for the genetic breeding and improvement of the Tong sheep.

Insulin like growth factor 1 (IGF1) is a type of single-chain polypeptide hormone, which can promote embryo development and cell proliferation and differentiation in the animal body and plays an important role in improving animal reproduction performance. This study was based on the previous research of the prior research group found that the birth rate of Nubian goats (Capra hircus) can be improved by feeding 0.07% N-acetylcysteine (NAC), which also has the best impacts. Meanwhile, filtering out IGF1 which is a significantly different gene based on the transcriptome sequencing of goat uterine horn. This experimental studied and collected the uterus, fallopian tube, pituitary, hypothalamus, ovary tissues of a Nubian goat and extracted total RNA from each tissue, reverse transcribed into cDNA and used qRT-PCR to verify the result of transcriptome sequencing. At the same time, bioinformatics analysis was performed on the CDS region of the cloned Nubian goat IGF1 gene; the eukaryotic expression vector of IGF1 gene was constructed and then transfected into uterine epithelial cells and detected the expression level of the homeobox genes related to reproductive traits of Nubian goats by the qRT-PCR test, which were homeobox A10 (HOXA10), leukemia inhibitory factor interleukin 6 family cytokine (LIF), C-X-C motif chemokine ligand 14 (CXCL14). The results showed that IGF1 gene was expressed in all 5 tested tissues. In contrast, the IGF1 gene in the uterus, fallopian tubes, hypothalamus and ovary of the experimental group was significantly higher than that of the blank group (P<0.01). The IGF1 gene CDS region of the cloned Nubian goat was 471 bp and encoding 153 amino acids. The protein prediction results showed that the molecular formula of Nubian goat IGF1 protein was C₇₃₉H₁₁₇₈N₂₁₂O₂₁₅S₁₅, the relative molecular mass was 16.953 67 kD and the isoelectric point was 9.36. The phylogenetic tree analysis showed that the Nubian goat IGF1 gene and sheep (Ovis aries) had the closest genetic distance. The result of qRT-PCR showed that the expression level of IGF1 gene mRNA in the cells of the successfully transfected recombinant plasmid group was significantly higher than that of the blank control group. The mRNA expression levels of HOXA10, LIF, and CXCL14 genes of the successfully transfected recombinant plasmid group (P<0.01) were significantly higher than those in the blank control group (P<0.05). The result showed that overexpression of the IGF1 gene would promote the expression of lambing-related genes which were HOXA10, LIF, and CXCL14. This study successfully constructed the eukaryotic expression vector of pEGFP-N3-IGF1 and detected the effect of the IGF1 gene on the expression of Nubian goat reproduction-related genes. It provides more information for further research on the effect of the IGF1 gene on Nubian goat reproduction traits.

Absent, small, or homeotic-like (ASH2L) is an important histone methylation modification enzyme involved in a variety of cell development. However, its function and molecular mechanism in the process of male germ cell development are not clear. The purpose of this study was to study the expression of Ash2l in the formation of chicken (Gallus gallus) male reproductive stem cells and to construct Ash2l eukaryotic fusion expression vector NCBI conserved domain analysis, UniPort, TMHMM Serverv.2.0 and SignalP online websites were used to analyze the conserved domain, hydrophilic and hydrophobic domain, transmembrane domain and signal peptide of ASH2L. qRT-PCR was used to detect the expression of Ash2l in different cells (embryonic stem cells (ESC), primordial germ cell (PGC) and spermatogonial stem cell (SSC)) and tissues of 18.5 d chicken embryo. The recombinant vector pcDNA3.1-Ash2l-Flag was identified by double restriction endonuclease digestion and sequencing. The expression of recombinant protein in PGC was detected by qRT-PCR and Western blot.The binding of recombinant protein and interaction protein was verified by Co-immunoprecipitation (Co-IP). The results showed that ASH2L protein contained 2 conserved domains, SPIa and ryanodine receptor (SPRY) and plant homeodomain (PHD) had histone methylase characteristic domain, and had high hydrophilicity, no transmembrane domain and no signal peptide expression. The results of qRT-PCR showed that the expression of Ash2l in testis was the highest, which was significantly higher than that in other tissues (P<0.01), and that in PGC was significantly higher than that in other cells (P<0.01). The results of double restriction endonuclease digestion showed that Ash2l was correctly inserted into pcDNA3.1 vector without frameshift and mutation. qRT-PCR showed that the recombinant vector in PGC could overexpress Ash2l gene, and Western blot indicated that the recombinant protein was successfully expressed in PGC. Co-IP results showed that the recombinant protein could bind to the interacting proteins Dpy-30 histone methyltransferase complex regulatory subunit (DPY30) and WD repeat containing protein 5 (WDR5) to form a complex. These results provide a reference basis and technical means for the study of the function and mechanism of Ash2l in the formation of male germ cells in chickens.

Wumeng black-bone chicken is a kind of local chicken breed which has been bred for more than 20 years in Bijie area of Guizhou province. Its eggs are rich in a variety of trace elements and have high research value. To explore the effect of SNP of estrogen receptor α (ESR1) and β (ESR2) genes on egg quality of Wumeng black-bone chicken, two strains of Wumeng black-bone chicken, white-shell layer hens and green-shell layer hens, were used as materials to screen SNPs of ESR1 and ESR2 genes by direct sequencing of PCR products, and their correlation with egg quality were analyzed. The results showed that SNPs were not found in the ESR1 gene. There were 4 SNPs on exon 8 and its near introns of ESR2 gene, which were g.53223010 A>T mutation in intron 7, g.53223142 G>A mutation, g.5322338 C>T mutation in exon 8, causing threonine to be a methionine, which belonged to missense mutation, and g.53223404 G>A in intron 8. The mutation of g.53223338 C>T in white-shell layer hens was low polymorphism, and the other sites were moderate polymorphism, and all sites didn't deviate from Hardy-Weinberg equilibrium. The results of linkage disequilibrium analysis showed that the SNPs g.53223010 A>T and g.53223404 G>A of ESR2 gene in 2 chicken strains were completely balanced; There were linkage disequilibrium among SNPs sites g.53223142 G>A, g.53223404 G>A and g.53223010 A>T in white-shell layer hens, but there was no linkage disequilibrium in green-shell layer hens. There was no linkage imbalance among other sites. Five identical haplotypes were detected in both strains, 8 haplotypes were detected in white-shell layer hens, and 11 haplotypes were detected in green-shell layer hens. The results of egg quality correlation analysis showed that SNPs g.53223010 A>T, g.53223404 G>A and g.5322338 C>T might be the marker sites affecting egg-type index and yolk relative quality of white-shell eggs, while SNPs g.53223142 G>A might be the marker site affecting yolk weight of green-shell laying hens. White-shell layer hens H5H5 and H1H4 diploid had significant advantages in the egg-type index and eggshell thickness (P<0.05), respectively, while green-shell layer hens H3H4 diploid had significant advantages in egg yolk weight (P<0.05). When other diploids had significant advantages in one egg quality index, this diploid had significant disadvantages in another, but they could all be used as potential genetic markers affecting egg quality. This experiment provides a new genetic marker for molecular breeding of Wumeng black bone chicken.

Rhodeus ocellatus is a kind of ornamental fish with a fair market prospect due to its bright color and beautiful body. The sequencing and analysis of the mitochondrial genome (mtGenome) of R. ocellatus, and the exploration of mtGenome differences of R. ocellatus from different geographical populations and the phylogenetic position of R. ocellatus in Acheilognathinae subfamily at the mtGenome level are of great significance for the phylogenetic study of R. ocellatus. In this study, PCR amplification, sequencing, and software splicing were used to obtain the complete mtGenome of R. ocellatus from Ou River. The full-length sequence (GenBank No. MW007386) was 16 675 bp, and the base composition was A (28.98%), G (17.09%), C (26.51%), and T (27.42%). There were 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs. The (A+T) content of the mtGenome and PCGs in the mtGenome of R. ocellatus from Ou River was 56.41% and 56.27%, respectively. Both mtGenome and PCGs had noticeable AT preference. The 22 tRNAs, except tRNA-Ser^(AGY), had a typical clover structure. Based on BLAST alignment, the sequence identity of R. ocellatus from Ou River was 83.84% with that of R. ocellatus from Dong River, 95.07% with that of R. ocellatus from Japan, 93.11% with that of R. ocellatus with GenBank No. DQ026430, 99.03% with that of Acanthorhodeus chankaensis from Tumen River, 83.72% with that of A. chankaensis from Heilong River, and 83.84% with that of A. chankaensis from Yuan River. In addition, the genetic distance between R. ocellatus from Ou River and A. chankaensis from Tumen River was the closest, followed by R. ocellatus from Japan, and R. ocellatus from Ou River was farther away. Phylogenetic tree was constructed based on complete mtGenomes of 25 species of Acheilognathinae subfamily belonging to 3 genera. R. ocellatus from Ou River was closely related to A. chankaensis from Tumen River, but was relatively distant to R. ocellatus from Dong River. The present study provides reference for the genetic diversity protection and breeding of R. ocellatus from Ou River.

Wool fiber is one of the important economic products of sheep. The development of the wool market has stimulated people's demand for different characteristics of wool. Peratin-associated proteins (KAPs)genes--KRTAPs and keratin-associated proteins (KIFs) genes and their respective variant genes are candidate genes for identifying the genetic basis of wool fiber differences. Using the homologous genes of human or goat and other mammalian keratins, we can identify the sheep genome and explore new potential functional KRTAPs or KIFs genes. In this review, the morphological structure of hair follicles is introduced, and the development of keratin-related protein genes is reviewed. The expression and location of KAP protein in hair follicles are also described. Finally, the effect of KRTAPs gene polymorphism on wool traits is summarized. This review provides a theoretical basis for the molecular breeding of sheep hair follicle growth and development and hair fiber traits.

The commonly used CRISPR/Cas9 system only recognizes targets with a NGG protospacer-adjacent motif (PAM) sequence, while the evolved xCas9 and SpCas9-NG systems can recognize targets with a NG PAM, which broadens the recognition range of CRISPR/Cas9. However, the off-target effects have always hindered the development and application of the CRISPR/Cas9 system. Previous studies showed that the double-nicking system could increase the specificity of DNA targeting. Four nicking mutants, named xCas9-D10A, SpCas9-NG-D10A, xCas9-H840A, and SpCas9-NG-H840A, and the corresponding single guide RNA (sgRNA) expression vectors were designed and constructed in this study to improve the specificity of gene editing of the CRISPR/Cas9 system. These vectors, with SSA-DKK1, SSA-DKK2 reporter plasmid, were co-transfected into sheep (Ovis aries) fibroblast cells through a specific plasmid combination, verifying cutting mutants activity with luciferase expression. The results showed that the SpCas9-NG-D10A and SpCas9-NG-H840A mutants had significantly greater reporter gene activity against most target vectors that recognize PAM as NG than the control group (P<0.05), and the xCas9-H840A variant had no activity at all.However, xCas9-D10A only showed cleavage activity in DKK1 gene. In contrast, the SpCas9-NG-D10A variant had stable activity and high efficiency in cutting DNA single strands, so this vector can be used for further research applications. The results of this study provides important reference data for the further research and application of NG PAM's Cas9 double-nicking system.

Fusarium wilt and Anthracnose are the most common and major diseases in the strawberry (Fragaria ananassa), and the pathogens are Fusarium oxysporum and Colletotrichum spp., respectively. At present, the main detection methods are traditional pathogen culture method and conventional PCR method, but loop-mediated isothermal amplification (LAMP) can be used for rapid detection of Fusarium wilt and Anthracnose in strawberry. In this study, a set of LAMP amplification primers was designed for the conserved region of intergenic spacer (IGS) in rDNA of Fusarium oxysporum, and the LAMP specific primers were designed to detect the specific sequence of internal transcribed spacer (ITS) in Colletotrichum spp. The LAMP technique was used to identify and detect strawberry Fusarium oxysporum and Colletotrichum spp. rapidly and accurately. The results showed that the established LAMP method was able to specifically detect F. oxysporum and Colletotrichum spp., and the detection sensitivity was 10×10^-5 ng/uL, which was 1 000 times that of conventional PCR. As a result, the rapid LAMP detection method of Fusarium wilt and Anthracnose in strawberry developed in this study had the advantages of high sensitivity, rapidity and simplicity, which could be popularized to grass-roots units and play an important role in real-time monitoring and early warning of strawberry pathogens.

Genetically modified crops (GMC) bring huge agricultural, socio-economic and environmental benefits with their excellent traits and quality, which are considered to be one of the potential methods to alleviate the global food crisis. Based on patent data, this study attempts to mine and analyze the global research situation, technology hotspots, major countries, and major patent applicants of genetically modified crops. It was revealed that the patents of genetically modified crops had been booming in the past two decades, and genetically modified soybeans and genetically modified corn were the hotspots. The United States led the patents research of genetically modified crops, and Monsanto, Pioneer Seed, Stine Seed Farm and Syngenta were the major applicants, they focused on germplasm resources with multiple combinations of traits. Chinese researchers paid more attention to gene mining of important agronomic traits. Finally, this study proposed to integrate superior resources to seize market opportunities; promote cooperation between academies and enterprises to accelerate the process of industrialization, pay attention to the international layout of patents and improve the intellectual property defense system. This study presents a panoramic view of the global patent research on genetically modified crops, and provides references for the research layout and innovation decision-making in the field of genetically modified crops.