Litter decomposition is a key process that can affect the forest ecosystem during plant invasion. The litter decomposition is an important part of the material cycle of the forest ecosystem, and it is of great significance to the improvement of soil fertility. The process of litter decomposition also affects the structure and quantity of soil microorganisms. Studies have shown that changes in the bacterial community structure are more sensitive to the response of litter decomposition. However, the characteristics of bacterial communities and their roles in litter decomposition during plant invasion in the forest are not well documented. An incubation experiment was conducted to investigate the effects of Moso bamboo (Phyllostachys edulis), evergreen broadleaf, and mixed bamboo-broadleaf litter additions on soil bacterial communities. The changes in bacterial communities before and after the litter addition were studied using terminal restriction fragment length polymorphism (T-RFLP), and quantitative real-time PCR (qPCR). The results showed that all 3 litter types significantly (P<0.05) increased the soil pH, organic matter, available potassium, and available nitrogen contents. At the end of the incubation, the number of soil bacteria increased with the addition of the 3 litter types. The bacterial abundance correlated positively with the addition rate of bamboo litter, while it didn't show correlation with the broadleaf forest litter. On the other hand, the bacterial abundance significantly (P<0.05) increased with only 2% addition rate of bamboo-broadleaf forests litter. The results of non-metric multidimensional scaling (NMDS) showed that the addition of the 3 litter types significantly (P<0.05) altered the composition of soil bacterial community as compared to the control. Redundant analysis (RDA) showed that the variation of the bacterial community composition was significantly affected by soil pH, organic matter, and available potassium contents. At the same time, the abundance of soil bacteria was positively correlated (P<0.05) with soil organic matter, total nitrogen, total potassium, and available potassium. In fact, there was no significant difference in the index of bacterial diversity among the treatments before the addition and incubation of 3 types of litter. At the end of incubation, both Shannon and evenness indices increased significantly (P<0.05) with the different litter treatments as compared to the control, while the Simpson index decreased significantly (P<0.05) with the different litter treatments as compared to the control. These results suggested that litter addition could increase soil nutrient content and promote bacterial proliferation. The magnitude of promoted bacterial proliferation varied with different types of forest litters. The abundance of soil bacteria increased with the increasing amount of litter addition of Moso bamboo, which was definitely different from the litter of evergreen broadleaf and mixed bamboo-broadleaf forests. Our results implied that the addition of Moso bamboo litter has positive effects on soil such as increasing nutrient availability and ameliorating soil acidity, as well as promoting the functional diversity of soil bacterial communities. This study revealed that the effects of litter addition on soil bacterial community varied with the type of forest stands, which might provide a reference for understanding the mechanism of the effect of litter decomposition on soil bacterial community during Moso bamboo invasion into broad-leaved forest.

Thaumatin-like proteins (TLPs), which is a kind of pathogenesis-related proteins possessing antifungal activity against many plant pathogens. In recent years, the antifungal function of TLPs has received more and more attention in plant pathology research. Our previous studies have reported that one wheat (Triticum aestivum) TLP gene named TaTLP1. It is related to wheat resistance to Puccinia triticina (Pt), and the expression of TaTLP1 was significantly up-regulated at 96 h after inoculation with Pt. In this study, the full sequence of the TaTLP1 gene was inserted into the expression vector pLGY-02 driven by the strong ubiquitin (Ubi) promoter, which can be highly expressed in monocotyledons. Then the recombinant vector pLGY-TaTLP1 was transformed into susceptible material 'JW' by Agrobacterium-mediated genetic transformation. Total 140 young embryos were infested, and 31 positive lines were obtained with a transformation rate of 22.1%. The transgenic TaTLP1 wheat from T₁~T₄ generations were verified by qRT-PCR, and the positive plants with stable genetic expression were screened out. qRT-PCR analysis confirmed the overexpression of TaTLP1 gene in transgenic positive plants. To further confirm the function of TaTLP1 gene in wheat resistance to Pt, the impact of TaTLP1 gene on transgenic wheat including 4 consecutive generations response to leaf rust pathogen infection was analyzed, the results showed that overexpression of TaTLP1 gene increased the resistance of transgenic plants to leaf rust. In addition, the determination of physiological and biochemical content of homozygous offspring further confirmed the phenotype observations, and proline, β-1,3-glucanase, hydrogen peroxide and peroxidase were also involved in wheat resistance to leaf rust. The results indicated that overexpression of TaTLP1 gene enhanced transgenic plants resistance to wheat leaf rust and played an important role in defense against wheat leaf rust, which provides a theoretical basis for further study on the function of TaTLP1 gene and the molecular mechanism between host-pathogen interaction.

Lycoris sprengeri is a kind of non-model perennial bulbous plant of Lycoris, whose flower color is bicolor with red and blue, and which is great ornamental value. It is of great significance to explore the mechanism of flower color formation by using genetic engineering technology for the breeding of new varieties of L. sprengeri. Virus-induced gene silencing (VIGS) is a new reverse genetics technology, which could be used to study the gene function of non-model plants. In this study, LsMYBs VIGS vectors were constructed by seamless cloning technology , the buds of L. sprengeri were infected using vacuum permeation method, and the expression of LsMYBs and anthocyanin contents of the petals in L. sprengeri after infection were detected, which would establish the VIGS technology system in L. sprengeri and analyzed the gene function of LsMYBs. The results showed that the expression of LsMYBs genes decreased significantly and the total contents of anthocyanin increased significantly after LsMYBs genes silencing. The relative expression of LsMYBs gene was relatively stable by 400 bp insertion fragment in VIGS vector, while the relative expression of LsMYBs was unstable after inserting a full length fragment of cDNA. The relative expression of structural genes ((chalcone synthase (LsCHS), chalcone isomerase (LsCHI), flavanone 3-hydroxylase (LsF3H), flavonoid 3'-hydroxylase (LsF3'H), dihydroflavonol-4-reductase (LsDFR), anthocyanidin synthaes (LsANS), flavonoid 3-o-glycosyltransferase (LsUFGT)) related to anthocyanin formation was affected after LsMYB4 gene silencing, the relative expression of all of the structural genes increased significantly during the whole anthocyanin biosynthesis process (early biosynthesis genes (EBGs) and late biosynthesis genes (LBGs)) and the degree of change was varying; while the expression of LBGs genes (LsANS, LsDFR3 and LsUFGT) increased significantly when LsMYB5 silencing, however, the expression of other genes did not change significantly. It could be inferred that LsMYB4 and LsMYB5 might play a negative regulatory role in the regulation of anthocyanin formation, while the target genes may be different. The results in this study provide a technical and theoretical basis for further analysis of the regulatory mechanism of LsMYBs transcription factors on the biosynthesis of anthocyanin in L. sprengeri.

Acetyl-CoA carboxylases alpha (ACACA) is a key rate-limiting enzyme in the de novo synthesis of fatty acids, which plays an important role in the regulation of fatty acid biosynthesis. In this study, 0.5-year-old and 4.5-year-old Tianzhu white yaks (Bos grunniens) were studied using quantitative real-time PCR (qRT-PCR) and heavy sulfite sequencing (BSP) methods. The expression of ACACA gene in 13 tissues such as heart, liver, and subcutaneous fat, as well as the methylation level of CpG island in PⅠ promoter region in subcutaneous adipose tissue were detected, and the gene expression regular and epigenetic mechanism in subcutaneous adipose tissue were studied. The results showed that the expression of ACACA gene in yaks were different in tissues and ages.The liver, brain, dorsal longest muscle and kidney tissues of 0.5-year-old yaks were highly expressed and the liver expression was significantly higher than other tissues (P<0.05);The expression of subcutaneous adipose and liver in 4.5-year-old yaks was extremely significantly higher than other tissues (P<0.01); The expression in the kidney of 0.5-year-old yaks were extremely significantly higher than that of 4.5-year-old yaks (P<0.01), but the expression of subcutaneous adipose, liver and testicular was significantly or extremely significantly less than that 4.5-year-old yaks (P<0.05 or P<0.01).In the subcutaneous adipose tissue, the ACACA gene PⅠ promoter 1st CpG island area (g.-795 bp~-193 bp) was highly demethylated and there were age differences. The average methylation level of BSP-1 primer amplification zone in 0.5-year-old yaks was extremely significantly higher than that in 4.5-year-old yaks (P<0.01). 0.5-year-old yaks BSP-1 primer amplification zone CpG11, CpG31, CpG35, CpG37 site and BSP-2 primer amplification zone CpG5', CpG13', CpG15', CpG27', CpG28' site methylation level significantly or extremely significantly higher than 4.5-year-old yaks (P<0.05 or P< 0.01); The level of methylation in the 1st CpG island region of the PⅠ promoter was negatively correlated with the expression of the gene in subcutaneous adipose tissue. This study revealed the differences of ACACA gene expression and PⅠ promoter methylation in yaks of different ages, which provide theoretical basis for molecular genetic research of yaks.

Early Pregnancy Factor (EPF) is a bioactive protein that firstly separated from gestational mammal serum. EPF prevents maternal rejection of the fetus in the manner of regulating immunosuppressive action. To analyze the biological characteristics and variation pattern of EPF in yak (Bos grunniens) blood, verify the correlation between blood EPF concentration and pregnancy, yak ovarian EPF gene sequencing was conducted after RT-PCR. The physicochemical properties and protein structure of yak EPF were predicted using bioinformatics related software. Blood samples were collected from 69 yaks during 20~40 d after artificial insemination (AI). The EPF level was measured by enzyme-linked immuno sorbent assay (ELISA). The complete coding sequence (CDS) of yak EPF gene was cloned and submitted to GenBank (No. MK992914). The open reading frame was 207 bp in length and encoded 69 amino acids. The ELISA results showed that the serum EPF level was closely related to the pregnancy status of female yak. In pregnancy group, the EPF concentration in serum gradually increased after the 20 d of AI, and reached the highest value on the 28 d and subsequently decreased slightly. In the non-pregnant group, the EPF concentration in blood maintained a stable and low concentration during 20~40 d after AI, and was significantly lower than that in pregnancy group (P<0.01). These results implied that blood EPF, as a candidate factor, could be used in early pregnancy diagnosis. It shortened the confirming time to 20 days by using blood EPF inspection in pregnancy diagnosis of yak. This study provides basic data for the research and development of products related to early pregnancy diagnosis of yaks and the establishment of technical systems.

Endometritis of cows (Bos taurus) has a significant impact on the fertility of dairy cows and restricts the development of dairy industry. Postpartum bacterial infection is the leading cause of endometritis. The main pathogenic bacteria isolated clinically are Escherichia coli and Staphylococcus aureus. In this study, bovine endometrial epithelial cells (BEECs) were cultured in vitro by two-step enzymatic digestion, and were infected with E.coli and S.aureus at different concentrations with corresponding multiple of infection (MOI). The TUNEL kit was used to detect apoptosis induced by the infection, and the mRNA levels of apoptosis-related genes Bax, Caspase-3 and Bcl-2 were detected by qRT-PCR after 12 h of the infection. The immunocytochemical identification showed that marker proteins (vimentin and keratin 18) of endometrial epithelial cells were positively stained. In E.coli and S.aureus infection groups, the apoptosis rate was the highest at 10⁷ CFU/mL; E.coli and S.aureus infection induced increased expression of Bax and Caspase-3 (highest at 10⁶ and 10⁷ CFU/mL, respectively), and Bcl-2 expression decreased (lowest at 10⁷ CFU/mL). This study indicated that E. coli and S. aureus could cause endometrial epithelial cell apoptosis, which might provide a reference for related mechanism researches on cow endometritis.

Sertoli cells can provide energy substrate for the development of male germ cells through glycolysis. Triosephosphate isomerase 1 (TPI1) is a key enzyme in glycolysis. In order to explore the biological function of TPI1 gene in the development of reproductive organs of male Tibetan sheep (Ovis aries), the cDNA sequence of TPI1 gene (GenBank No. MN847717) was cloned, and its expression and localization patterns in developmental testis and epididymis were detected by real time quantitative PCR (qRT-PCR), Western blot and immunohistochemistry. The results showed that the length of CDS region of Tibetan sheep TPI1 gene was 861 bp and encoded 286 amino acids, showing high degrees of homology with other mammals. TPI1 gene and its protein were expressed in testis and epididymis in all developmental stages, but mainly in testes and epididymides from 1-year-old and 3-year-old sheep. TPI1 protein was mainly distributed in Sertoli cells within testes at all stages of development, and sperm tails within epididymides from post-puberty (1-year-old and 3-year-old). In addition, weak TPI1 protein signals were present in Leydig cells, spermatogonia and spermatocytes from testes, and pseudostratified columnar ciliated epitheliums and loose connective tissues from epididymides. These results taken together demonstrated that TPI1 gene might be involved in the regulation of glycolysis in Sertoli cells and sperm flagella, as well as the secretory function of pseudostratified columnar ciliated epithelium of epididymis, which in turn might promote the formation and maturation of sperms. This study provides basic materials for further investigation on the regulatory mechanism of TPI1 gene in ovine and other mammalian spermatogenesis.

Rab5a molecule is an intracellular transporter, while the invariant chain (Ii) is an immune molecule that helps MHC (major histocompatibility complex) to assemble, mature and present antigen peptide. For a long time, the intracellular pathway mechanism of Ii is not clear. The purpose of this study was to explore the structural characteristics of Rab5a in chicken (Gallus gallus) and its role in the intracellular transport of Ii. First, chicken Rab5a gene was cloned with self-designed primer, and the eukaryotic expression recombinant vectors containing chicken cIi and its mutants (Ii_D81-87aa and Ii_D91-99aa) were constructed. Secondly, the protein structure of Rab5a in chicken and mouse (Mus musculus) were compared by software. Finally, Rab5a and Ii were transfected into mouse dendritic cell line DC2.4 cells, respectively. The localization and co-localization of chicken Rab5a and Ii, cIi_D81-87aa and cIi_D91-99aa in eukaryotic cells were observed by immunofluorescence and laser confocal microscope. The results showed that the Rab5a gene was 648 bp, which was consistent with the expected size. The molecular structural analysis showed that Rab5a of chicken was highly similar to that of mouse, and the homology was as high as 95.4%. The results of homologous modeling analysis showed that Rab5a of chicken and mouse contained 5 alpha helixes and 6 beta helixes, indicating that Rab5a of mammal and poultry were homologous. Laser confocal microscope observation showed that chicken Rab5a and Ii could be localized in the early endocytosis of cells, but the mutants Ii_D81-87aa and Ii_D91-99aa could not be localized in the early endocytosis. The results of GST pull-down and Western blot showed that Rab5a could bind Ii molecule. In conclusion, the results of co-localization and binding of Rab5a and Ii in the early endocytosis suggest that Rab5a is involved in the transport of Ii in cells, which provides a new basis for further study of its mechanism.

Animal mitochondrial cytochrome b (Cytb) gene is an important functional protein-coding gene. Its evolutionary rate is moderate, a small gene segment contains genetic evolutionary information from within species to between species and even between families, which was considered as an ideal molecular marker for studying animal genetic polymorphism and systematic evolution. The aim of the study was to prove up the heredity polymorphism of Cytb gene on some indigenous chicken (Gallus domesticus) breeds. Four Jiangxi chicken breeds were used as materials, and the Cytb gene was amplified by using sequencing and the heredity polymorphism was analyzed. The results revealed that the complete sequence of Cytb was 1 143 bp, which had 12 SNPs and 9 haplotypes. The hap4 was found in four chicken breeds. Average number of nucleotide differences (K), haplotype diversity (Hd) and nucleotide diversity (Pi) of four chicken breeds were 1.971, 0.724 and 0.001 72, respectively; the K and Pi of Anyi Gray chicken were biggest, which were 3.105 and 0.002 72, respectively; the Hd of Baier Yellow chicken was biggest, which was 0.752; The K, Pi and Hd values of Silkies were all the lowest. The phylogenetic relationship and origin analysis of the four chicken species in the formation process showed that Silkies may originate from Gallus gallus spadiceus. Dongxiang Blue-eggshell chicken, Anyi Gray chicken and Baier Yellow chicken probably originated from Gallus gallus spadiceus and Gallus gallus murghi. The results of this study provide a theoretical basis for the protection, development and utilization of genetic resources of chicken breeds.

As an adaptor in Toll-like receptor (TLR) signaling pathway, TIR domain-containing adaptor inducing interferon-β receptor (TRIF) mediates downstream signaling cascades and plays an important role in host innate immune responses. In order to investigate the potential role of TRIF in anti-disease immune response of Nile tilapia (Oreochromis niloticus), the cDNA sequence (GenBank No. MT199561) of TRIF gene was obtained by reverse transcription PCR (RT-PCR) and fragment amplification, and bioinformatic analysis was also performed. Real time quantitative PCR (qRT-PCR) was used to detect TRIF expression in healthy individuals and those with intraperitoneal injection of Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C). The recombinant eukaryotic expression vector was constructed to analyze the subcellular localization of TRIF in 293T human (Homo sapiens) embryonic kidney cells and the activation of nuclear factor κ B (NF-κB). The results showed that TRIF cDNA was 3 135 bp in length which contained an ORF of 1 653 bp encoding a polypeptide with 550 amino acid residues. The deduced amino acid sequence contained Toll/IL-1 receptor (TIR) domain. In the phylogenetic tree, Nile tilapia was clustered with other fish and then clustered with zebrafish (Danio rerio) and mammals. The TRIF expression were widespread in all the tested tissues and organs (liver, skin, heart, kindey, stomach, gill, brain, spleen, intestine, blood and muscle), with the highest expression in the muscle and blood. After challenged with pathogenic bacteria S. agalactiae, the TRIF gene expression increased in the spleen and kidney, and then reached the peak at 24 h post infecton (hpi). After infection with LPS, TRIF was up-regulated in the liver and spleen, while down-regulated in the intestine and kidney. TRIF was up-regulated in the intestine and liver, and down-regulated in the kidney after infection with Poly I:C. The result of subcellular localization showed that TRIF protein distributed in the cytoplasm and could significantly increase NF-кB activity. Taken together, the above findings suggested that TRIF gene might play an important role in immune responses of Nile tilapia. This study could provide a reference for further studying on the function of TRIF gene in immunity evolution of teleost.

Alkaline phosphatase (AKP) can catalyze a variety of phosphorus-related hydrolysis reactions, and is a reference indicator for immune function and health status of organisms. Leuciscus waleckii is a kind of fish that is extremely resistant to the environment (especially high carbonate). In order to clarify various physiological reactions and health conditions in high alkali environments, two geographical groups of L. waleckii, which were Dalinuoer Lake L. waleckii (short for alkaline L. waleckii) from alkaline water and Songhua River Leuciscus waleckii (short for freshwater L. waleckii) from fresh water, were set to 30 and 50 mmol/L NaHCO₃ for 20 d. Then the blood was taken out and placed in the Automatic microplate reader to determine the concentration of AKP in serum. Under the condition of 30 mmol/L carbonate alkalinity, the AKP activity of freshwater L. waleckii was significantly higher than that of alkaline L. waleckii (P<0.05). At the same time, based on the AKP gene sequence of the closely related species Cyprinus carpio, the AKP gene in L. waleckii was cloned, and AKP gene expression in the muscle, sputum, liver, brain and kidney was detected by real-time quantitative PCR. The results showed that the AKP ORF of L. waleckii was 876 bp in length (GenBank No. MN473375) and encoded 291 amino acids. The expression of AKP gene in different tissues of the two squids were different. The AKP gene expression in alkaline water L. waleckii increased in the liver with the increasing of alkalinity. The AKP gene expression in freshwater species increased in the kidney with the increasing of alkalinity. The AKP gene expression at different alkalinity levels in multiple tissues showed significant differences (P<0.05). In summary, the AKP gene might play a role in the alkali-resistant response in L. waleckii. This study could provide basic data for further investigation on the involvement of AKP in the alkali-resistance in L. waleckii.

Gene editing is a new genetic engineering technology that modify the specific target genes of organism genomes. It has been widely used to edit plant genomes accurately at present to obtain excellent crop varieties. This paper attempts to mine and analyze patent data related to the global plant gene editing technology. On the basis of sorting out the development path of gene editing technology, the focus was on analyzing the development trend and competition situation from the perspective of the main patent layout areas and rights holders, R&D (Research & Development) and industrialization. The analysis found that China's plant gene editing technology patent lacked global patent deployment, and the overall quality and economic value of the patent were relatively low. The final achievements still remained in model crop creation, lack of industrialization mechanism based on intellectual property cooperation. Finally, countermeasures and suggestions were proposed such as strengthening the original innovation of editing system, strengthening the intellectual property layout of the global and whole industrial chain, and promoting intellectual property cooperation to make up for shortcomings.

Long non-coding RNA (lncRNA) is an important class of functional non-coding RNA, which can regulate physiological processes such as cell proliferation, differentiation and metabolism, and participate in the regulation of various pathological processes in the body. In the rapid development of the intensive breeding industry, the intestinal diseases of livestock and poultry have a high incidence and mortality that seriously affect the health of livestock and poultry and cause significant economic losses to animal production industry. In recent years, many lncRNAs associated with intestinal inflammation and diseases have been identified by transcriptome sequencing technology. Researches on human (Homo sapiens) and mouse (Mus musculus) model organisms demonstrated that multiple lncRNAs are abnormally expressed in intestinal diseases, which regulate the expression and distribution of tight junction proteins, and also participate in the production of various inflammatory molecules and the activation of signaling pathways. In this paper, the regulatory function of lncRNA in intestinal barrier and immune-homeostasis, and the recent research progresses in livestock and poultry were reviewed. This review offers references for the follow-up research on resistance to intestinal diseases in livestock and poultry and the breeding of resistant varieties.

The reference materials (RMs) for genetically modified organisms (GMOs) detection are essential for ensuring the accuracy, reliability and traceability of test results. Among different types of RMs, the pure powder RMs has the characteristics of easy-preparation and reliable property value, therefore, to become the most commonly used RMs for GMO quantification. In this study, the homozygous seeds of transgenic soybean (Glycine max) MON89788 were used as raw materials to develop pure matrix RMs through the steps of raw material identification, grinding, particle size measurement, water content measurement and initial homogeneity test. Particals less than 200 μm in this batch of RMs accounted for more than 80% of total powders, and the water content was measured to be (4.53±0.03)%, less than the acceptable level of 10%. A total of 400 bottles of RMs were developed by filling 1 g powder in bottles. 15 bottles were randomly selected from the whole batch of RMs to evaluate the homogeneity by MON89788 and Lectin qRT-PCR methods, the F-test results showed that the RMs powder was well-homogenized among and within bottles. The minimum sampling amount was determined to be as low as 100 mg according to the standard of DNA extraction together with the homogeneity test results. For short-term stability study, the RMs were evaluated by analyzing 3 bottles stored at 4, 25, 37 and 60 ℃ for 1, 2 and 4 weeks. For long-term stability, the RMs were evaluated by analyzing 3 bottles stored at 4 ℃ and -20 ℃ for 1, 2, 4 and 6 months. The property value of RMs did not show obvious changes with the extension of transportation and storage time. A t test results indicated that the slopes of regression curves (β₁) were not significantly different from zero under the transportation and storage conditions at the 95% confidence level. The stability test indicated that the RMs could be transported at room temperature for one month and the long-term stability was up to 6 months. The property value of this batch of RMs was defined as the copy number ratio of transgenic DNA to total DNA (copy/copy), which was collaboratively characterized by 9 laboratories using MON89788/Lectin duplex digital PCR. Statistical analysis of the characterization data revealed no outlier and a nominal distribution of the data. The calculated average of all independent measurement results was assigned as the certified value. The certified value of DNA copy number ratio was calculated to be 1.00. The uncertainty of the RMs consisted of uncertainty components from characterization, homogeneity, and stabiltiy during short-term and long-term storage. The expanded uncertainty was estmiated to be 0.07 by combining the 3 components with a coverage factor k (k=2 at 95% confidence level). The property value was certified to be 1.00 with an extended uncertainty of 0.07. This batch of RMs can be used for the qualitative and quantitative detection of GM soybean MON89788, as well as the evaluation of MON89788-specific methods and laboratory quality control.

Taxus sp. can produce a natural diterpenoid anticancer compound-taxol, which has good curative effect on cancers of breast, lung, ovarian, endometrial, and cervical carcinoma. However, both the growth of yew trees and the content of taxol are very low. The conflict between the protection of yew trees and utilization of taxol is serious. Previous studies reported that AP2/ERF (APETALA2/ethylene-responsive factor) transcription factors involved in regulating the biosynthesis of many secondary metabolites, such as tanshiones. AP2/ERF transcription factors also play an important role in the regulation of taxol biosynthesis in Taxus. To better study the function of TcERF gene that had been isolated by our group previously, this study established 3 stable and suitable real-time fluorescent quantitative PCR (qRT-PCR) experiment systems. With the sequence of TcERF gene, three primer pairs were designed and synthesized with the TBC41 (3,5-epimerase-4-reductase) gene as the housekeeping gene. According to the relationship between the upstream and downstream of primers, three primer combinations were obtained. Then the best primer combination F1R2 was screened by PCR and qRT-PCR. The orthogonal test L₉(3⁴) method was used to design the test scheme. Under the 3 test schemes with the highest amplification efficiency (A1B3C3, A2B2C3 and A3B1C3), the amplification efficiency of the housekeeping gene were 70%~80%, and the amplification efficiency of F1R2 was 102% and 91% in A1B3C3 and A2B2C3, respectively, but only 72% in A3B1C3. The amplification efficiency of housekeeping gene and target gene did not reach 90%~110% at the same time. Therefore, the qRT-PCR experiment system had to be further optimized. Finally, the best combination of 5 µL small reaction system, 10 and 20 µL common system were screened out. The results indicated that in the optimized 5 µL system which include 2.5 µL Master Mix, 1.7 µL cDNA template, and 0.8 µL primer, the amplification efficiency of TBC41 and TcERF were 94% and 90%; in the optimized 10 µL system which include 5 µL Master Mix, 1.2 µL cDNA template, 1.3 µL primer and 2.5 µL ddH₂O, the amplification efficiency of TBC41 and TcERF were 95% and 94%, respectively; in the optimized 20 µL system which include 10 µL Master Mix, 0.5 µL cDNA template, 1.5 µL primer and 8 µL ddH₂O, the amplification efficiency of TBC41 and TcERF was 93% and 102%, respectively. All the mentioned amplification system regression coefficient R² were greater than 0.980. The expression level of TcERF in Tm3 cells that treated with methyl jasmonate for 24 h was detected by 10 µL reaction systems of before and after optimization. A significant difference was existed between the values of TcERF expression obtained from the optimized and unoptimized qRT-PCR composition, which indicated that unreliable results may be obtained by using the unoptimized qRT-PCR system. All the above described 3 reaction systems showed that the amplification efficiency of TBC41 and TcERF closed to 100%, which indicated that these detection programs were suitable to investigate the TcERF gene expression by qRT-PCR method. It provides experimental conditions and technical support for the subsequent transcriptional expression detection and functional research of TcERF gene.

qRT-PCR is one of the important methods for investigating mRNA expression levels in the cells and tissues. However, the selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. This study examined the expression of 9 internal genes (topoisomerase (DNA)Ⅱβ (Top2b), β-actin (Actb), ribosomal protein S18 (Rps18), peptidylprolyl isomerase A (Ppia), 18S ribosomal RNA (18S), TATA-box binding protein (Tbp), hydroxymethylbilane synthase (Hmbs), ribosomal protein L4 (Rpl4) and β-2-microglobulin (B2m) for heart, liver, spleen, lung, kidney, muscle, subcutaneous fat from three 15 d Bama minipigs (Sus scrofa) using qRT-PCR technique, and 3 well-known software packages: geNorm, NormFinder and BestKeeper were used to analyze the results. Rps18, Rpl4, Ppia and Tbp were found to own the highest stability across tissues. Only 2 internal reference genes could standardize the expression of target genes in the same tissue. And in the different tissue, the optimal gene combination was different. The results of this study provide a reference information on Bama minipig to select the most suitable internal reference gene, and it could provide a theoretical basis for the research of gene function about meat quality, litter size, development, reproduction, disease and so on of Bama minipig.

Rubber tree (Hevea brasiliensis) brown root disease caused by Phellinus noxius is a root disease that seriously harms rubber. Loop-mediated isothermal amplification (LAMP) has been widely used in many fields such as plant protection because of its high specificity, high sensitivity, rapid response, and low cost. Therefore, an acturate, sensitive and rapid detection method is a powerful tool to timely effective control measures. In this study, SYBR Green Ⅰ as an indicator, a fast visual detection method of LAMP for the detection of P. noxius was established. Four primers (2 inner primers FIP/BIP, 2 outer primers F3/B3) were designed for LAMP amplification based on the ribosome transcriptional spacer sequence specific to P. noxius. The reaction conditions were optimized and the specificity and sensitivity of LAMP were assayed. The suspected brown root disease samples in the field were verified. The study determined the optimal reaction system (100 ng/μL for template in 25 μL, 0.2 μmol/L for outer primer F3/B3, 1.2 μmol/L for inner primer FIP/BIP, 1.4 mmol/L for dNTPs, and 6 mmol/L for Mg²⁺, betaine is 0.8 mol/L, Bst DNA polymerase is 8 U/μL and optimal reaction temperature 63 °C, reaction 1 h). Specificity assays showed that a positive color (green) was only observed in the presence of P. noxius by SYBR GreenⅠas an indicator reaction prior to amplification, however none of other pathogens changed color (still orange). The lower limit of detection of the LAMP assay was about 1 pg/μL of genomic DNA, which was 1 000 times more sensitive than PCR method. Twenty strains of suspected samples in the field were tested by the LAMP system with the pathogen detection rate was 85%, while thenormal PCR detection rate was only 70% or 75%. The LAMP detection system established in this study can be used for the rapid detection of suspected samples of root rot of rubber tree, which was of great significance for effectively controlling the spread of the disease.

Porcine epidemic diarrhea (PED) caused by Porcine epidemic diarrhea virus (PEDV) is a highly contagious and devastating enteric infectious disease to pig (Sus scrofa) production. Serological techniques are absent for distinguishing PEDV variants from classical strains. Monoclonal antibody (McAb) is a kind of important and basic experimental materials for serological testing or diagnosing technique researches. In order to prepare McAb for distinguishing PEDV variants from classical strains, this study compared and analyzed the nucleotide and amino acid sequences of spike (S) protein of PEDV virulent variants and classical strains by the software of DNAMAN and DANStar7.1. The polypeptide, ⁵⁵IGENQGVNSTWYCAGRHPTAS⁷⁵ being different between the PEDV variants and the classical ones, was screened out and synthesized. BALB/c mice (Mus musculus) were immunized 3 times at an interval of 2 weeks with the polypeptide conjugated keyhole limpet haemocyanin (KLH), and then one strain of hybridoma (named 3^#) which secreted IgG2bκ monoclonal antibody (named McAb 3^#) was obtained through cell fusion technique, 3 rounds of clone selecting as well as enzyme-linked immunosorbent assay (ELISA) detection. The McAb could specifically react with PEDV-S protein whereas it did not combine with PEDV nucleocapsid (N) protein by ELISA and Western blot, meanwhile, PEDV variants could specifically bind to the McAb 3^# in Vero-81 cells using the indirect immunofluorescence assay (IFA). The specific ELISA antibody titer reached to 1∶10⁶ in mouse ascitic fluid induced by 3^# hybridoma cells. Moreover, IFA based on the McAb 3^# was performed in antigen detection of PEDV variant HBQY2016 and the classical attenuated strain CV777 in Vero-81 cells, respectively. Green fluorescence signals were observed only in the cytoplasm of Vero-81 cells infected with PEDV variant HBQY2016, but not in the cells infected with the classical attenuated strain CV777, which indicated the McAb 3^# could distinguish the PEDV variant from the classical strain. The results showed that the McAb prepared in this study specifically combined with PEDV S protein and could distinguish the PEDV variant from classical strain. Its preparation could be used for studying PEDV infection immunity and developing antigen or antibody detection methods to differentiate PEDV variants from classical strains.

Bluetongue virus (BTV) is an arbovirus that seriously endangers ruminants. Since 2008, 3 new serotypes of BTV-25, -26 and -27 have been discovered in the worldwide, and China is also threatened by the invasion of BTV-25, -26 and -27. The DNA sequences of BTV-25, -26 and -27 genomic segment 2 (Seg-2) with lengths of about 500 bp were synthesized and transcribed into single strand RNA (ssRNA) in vitro using as the reference nucleic acids. Serotype specific amplification primers and TaqMan probes were designed based on Seg-2 sequences of BTV-25, -26 and -27 and the one step qRT-PCR detection assays were established. The results showed that the reference nucleic acids of BTV-25, -26 and -27 were successfully obtained; The established BTV-25, -26 and -27 serotype specific one step qRT-PCR detection assays possessed strong specificity, high sensitivity and good repeatability, which did not cross-react with other BTV serotype virus, Epizootic haemorrhagic disease virus, Akabane virus and African horse sickness virus inactivated vaccine virus;and that showed the minimum ssRNA template detection limits of 4.43×10¹, 5.61×10¹ and 4.88×10¹ copies/μL for BTV-25, -26 and BTV-27 ssRNA, respectively; and displayed the intra- and inter-group coefficient of variation varying between 0.48% and 1.79%. To determine the presence of BTV-25, -26 and -27 infection, 120 BTV nucleic acid positive animal blood samples collected from southern China were tested, and the results were all negative, representing none of the animals were infected by BTV-25, -26 and -27. This study successfully established the one step qRT-PCR detection assays of BTV-25, -26 and -27, and provides an effective technical means for the monitoring of new serotype BTV intrusion into China.