B-box protein is a kind of zinc finger structure transcription factor, its function is universal and specific. In order to reveal the sequence and expression characteristics of the double B-box (DBB) gene family in Mangifera indica, in this study, based on the whole genome data of mango, using bioinformatics methods to analyze the sequence of mango DBB gene family, and the relative expression level of DBB gene family was studied by qRT-PCR during the infection of Colletotrichum gloeosporioides (Cg) and Xanthomonas campestris pv. mangiferaeindicae (Xcm). Nine DBB gene family members were identified from the mango genome and named MiDBB1~9. The 9 MiDBBs proteins were unstable hydrophilic acidic proteins, mainly located in the nucleus. Not only was the tertiary structures similar, but also the N-terminal were 2 conserved domains (B-box1 and B-box2) with the same site. According to protein structure prediction, phylogenetic relationship, conservative motifs and multiple sequence comparison analysis, it was inferred that the DBB gene family of mango was evolutionarily conserved in structure and similar in function. The phylogenetic tree constructed by mango with Arabidopsis thaliana, Nicotiana tabacum, Malus domestica, Ananas comosus and Populus trichocarpa showed that the DBB of mango were closely related to those of the P. trichocarpa, followed by apple. The results of qRT-PCR showed that during the infection of C. gloeosporioides, the relative expression of MiDBB1~9 continued to increase within 12~48 h. During the infection of X. campestris pv. mangiferaeindicae, the relative expression levels of MiDBB2 and MiDBB3 continued to increase within 12~72 h. It was preliminarily speculated that the expression of DBB gene in mangosteen was induced by C. gloeosporioides and X. campestris pv. mangiferaeindicae. The above findings provide reference for the follow-up study of the functions and mechanisms of mango DBB gene family members.

AQPs (aquaporins) play an important role in plant development and abiotic stress resistance. In this study, DeTIP2;1, the only up-regulated gene of AQPs family, was screened from drought transcriptome data of Xinjiang xerophyte Dontostemon elegans. Cloning the full-length sequence, bioinformatics analysis, expression analysis and subcellular localization were carried out. ：The results showed that the total length of CDS was 750 bp, encoding 249 amino acids, with a molecular mass of 24.92 kD and a theoretical isoelectric point of 5.30, and contained 2 highly conserved domain of asparagine-proline-alanine (NPA) motifs.. Both drought and salt stress could affect the expression of DeTIP2;1, and the expression was tissue specific. Subcellular localization showed that DeTIP2;1 was located in tonoplast. The above results showed that DeTIP2;1 may be involved in water absorption and transportation in the root of D. elegans. This study provides data supports for the mining and screening of functional genes of xerophytic resources in arid areas.

Flavonoids are important secondary metabolites in plants, and they play an important role in plant growth and resistance to adversity and stress. Studies on the function and metabolic regulation of plant flavonoids (flavonoids, flavonols, etc.) have attracted more and more attention, especially in woody plants rich in flavonoid metabolites. The research on the synergistic interaction between key enzymes in flavonoid metabolic pathway will provide guidance for the better development and utilization of plant flavonoid functional components. Pigeon pea (Cajanus cajan) is a pioneer woody legume tree growing in karst soils with high acid aluminum content, which is rich in secondary metabolites such as flavonoids. In order to identify the key pathways of flavonoid biosynthesis, this study screened and identified 8 key enzyme gene families in pigeon pea, namely phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR). Bioinformatics software such as MEGA 7.0, MEME, TBtools v1.089 and PlantCare were used to analyze the phylogeny, gene structure, protein structure and cis-acting elements of the promoter, as well as the regulation of gene expression in the metabolic pathway under stress conditions. Phylogenetic analysis showed that some members of the PAL, C4H, 4CL and CHI gene families could cluster together with the genes in soybean (Glycine max), while the CHS, FLS and DFR gene families only showed sequence similarity among family members. Protein structure analysis showed that each gene family contained a special conserved domain. Analysis of promoter cis-acting elements showed that the genes containing methyl jasmonate (MeJA) response elements accounted for more than 50% of the total genes in 6 gene families. Meanwhile, in view of the habitat characteristics of pigeon pea, RNA-seq analysis was conducted on the 8 gene families under aluminum stress and methyl jasmonate treatment in the study. The results showed that CcPAL2, CcC4H1, CcCHS5~7 and CcCHS12 with the higher relative expression levels were all significantly up-regulated under aluminum stress, while CcCHI4~5 and CcFLS7 all showed a significant down-regulation trend, indicating that chalcone in pigeon pea might accumulate under aluminum stress. It was speculated that chalcone might play a role in aluminum resistance. Under MeJA treatment, CcC4H1, CcCHS5, CcCHS7, and CcCHI4 were significantly up-regulated, while CcF3H was down-regulated 0.6 times that of the control. At the same time, CcFLS1, CcFLS5, and CcFLS6 in the downstream pathway were significantly up-regulated. The above results indicated that MeJA may also promote the accumulation of naringin and flavonol, it was speculated that the substances might play an important role in the MeJA signaling pathway. The expression changes of the above flavonoid biosynthetic enzyme genes at different stages were verified by qPCR and consistent with the transcriptome sequencing results.This study reveals the expression patterns of key enzymes in flavonoid biosynthesis under different stresses and provides reference for further exploration of the regulatory role of flavonoid substances such as chalcones and flavonols in stress.

Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) can be obtained from linoleic acid by Propionibacterium acnes isomerase (PAI). Animals fed t10c12-CLA reduced fat deposition and milk fat content, and the dynamic balance of energy was affected. In this study, the codon optimized pai gene was transfected into mouse (Mus musculus) 3T3 cells. It was confirmed by gas chromatography that PAI could efficiently catalyze the substrate linoleic acid into t10c12-CLA. The content of t10c12-CLA was high, and reached 7.09% of the total amount of poly-unsaturated fatty acids in cells. When compared with the control cell line transfected with pIRES2-AcGFP1, the cell lines transfected with pai gene and synthesized t10c12-CLA showed linoleic acid accumulation (12.46% vs 14.38%~15.54%)(P<0.05) and arachidonic acid decrease (13.31% vs 9.03%~11.13%)(P<0.05), while stearic acid (14.07% vs 11.85%~12.54%)(P<0.05) and oleic acid (20.29% vs 14.89%~16.87%)(P<0.05) decreased simultaneously. T10c12-CLA produced in pai transfected cells could up-regulate the gene expression of stearoyl-CoA desaturase 1 (scd1) (P<0.05), which may be the reason for the decrease of stearic acid content in transgenic cells. However, exogenous t10c12-CLA did not down-regulate the expression of fatty acid desaturase 1 (fads1) and fads2 genes. The results of high pressure gas chromatography showed that the exogenous t10c12-CLA blocked the metabolism of linoleic acid to downstream arachidonic acid (P<0.05), that is, t10c12-CLA may competitively bind to FADS2/FADS1 enzyme system, so as to inhibit the normal metabolism of linoleic acid to arachidonic acid. This study confirmed that microbial pai gene could be normally expressed in mammalian cells and produce t10c12-CLA, which can provide a reference basis for the development of pai transgenic animals

Black bone chickens (Gallus gallus domesticus) are excellent indigenous chicken resources in China and commonly believed to have medicinal properties. The study on the genetic diversity and breed identification of the black bone chickens is of great significance for genetic conservation. For this study, the complete mitochondrial DNA D-loop sequences of 197 chickens from 5 breeds (Silkie, Dongxiang Blue-eggshell, Jinhu Black-bone, Yugan Black-bone and Zhu Si) were analyzed. The size of full-length chicken mtDNA D-loop of 5 black bone breeds were 1 231 to 1 232 bp, with a single-base deletion from the 859 bp site in the 1 231 bp sequences. A total of 27 variable sites that defined 18 haplotypes were identified. The average haplotype diversity, nucleotide diversity and mean number of nucleotide differences were 0.862±0.013, 0.005 80±0.001 07 and 7.137, respectively. The median network showed that genetic structure of the mtDNA haplotypes of black bone chickens were distributed across 4 clades (haplogroups): Clades A, B, C, and E. However, haplogroups were dispersed in different breeds, and the distribution of morphology and geographical pattern was not obvious. This study data suggested that genetic distance between Zhu Si and Silkie was longer than the other breeds, which verified that Zhu Si chickens were hybridized from fast-growing broiler as maternal line. Chinese black bone chicken had relatively lower genetic diversity and likely shared 4 common maternal lineages. The results could be used as a reference for preservation, improvement and identification of Chinese black bone chickens.

Subgroup J of avian leukemia is a new subgroup of avian leukemia caused by Avian leukemia virus subgroup J (ALV-J) isolate from broilers (Gallus gallus). Clinically, it mainly causes the growth and immune suppression of diseased poultry, the prevalence of the disease has brought serious harm to the poultry industry. In this study, ELISA method was used to detect the infection of ALV-J in the local breed chicken in GuangDa Poultry Farm in Hangzhou, Zhejiang Province, and the virus was isolated and identified from positive samples. By amplifying the GP85 gene of the isolate, the genetic evolution of the isolated ALV was analyzed. The results showed that the positive rate of ALV-J among 10 local breeds was very different, with the lowest rate being only 4%, and the highest rate being 46%. 3 strains of ALV-J (named ZJ20-1, ZJ20-2 and ZJ20-3, respectively) were isolated from the positive samples using primary chicken embryo fibroblasts (CEFs). The GP85 specific fragment was amplified from the 3 isolates by PCR method, and the genetic evolution of GP85 gene was analyzed by DNA STAR software. The results showed that the ALV-J isolates isolated in this study had a certain genetic evolution relationship with the reference strains. The ALV-J isolates could be divided into 2 topological groups (G1 and G2), and the 3 isolated strains belonged to G1 group. The homology with other domestic isolates was 88%~98.0%. This study provides a reference for further study on the prevalence of ALV-J in chicken farms in China and the purification of avian leukemia.

Growth hormone (GH) is a polypeptide secreted by pituitary, which plays an important role in the development of teleost and the growth of somatic cells. In order to explore the role of GH in the growth dimorphism between male and female blotched snakehead (Channa maculata) and the mechanism of its interaction with sex hormones, in this study, GH gene (GenBank No. MW574005) of blotched snakehead was cloned and its expression pattern and response to exogenous steroid hormones were analyzed. The results showed that, the cDNA sequence of blotched snakehead GH gene was 882 bp in length, with an open reading frame (ORF) of 639 bp, encoding 212 amino acids. The length of GH sequence of blotched snakehead was 1 604 bp and contained 5 exons and 4 introns. A 1 199 bp flanking sequence was identified at the upstream of 5'-terminal, which contains pre-B-cell leukemia transcription factor 3 (PBX3), signal transducers and activators of transcription 1 (STA1), forkhead box O3 (FOXO3) and other transcription factors binding sites. Tissues expression analysis showed that GH was highly expressed in the pituitary of blotched snakehead, and the expression in male blotched snakehead was extremely significant higher than that in female (P<0.01); Expression analysis in different developmental stages showed that GH gene expression in the pituitary of male blotched snakehead was the highest at 365 d post hatching, which was extremely significant higher than that in female (P<0.01). The results of ELISA showed that the serum GH content in male blotched snakehead was higher than that in female in each developmental period. Hormone induction experiment showed that 17α-ethynylestradiol (EE₂) inhibited GH gene expression in female and male blotched snakehead; 17α-methyltestosterone (MT) promoted GH gene expression in male pituitary, but decreased GH gene expression in female pituitary. This study provides the reference for further exploring the growth dimorphism mechanism of male and female blotched snakehead and the molecular mechanism of growth regulation.

Calcineurin B-like proteins (CBLs) are a set of small proteins that contain multiple Ca²⁺ binding domains and are highly conserved in amino acid sequence. In plant, CBLs and CBL-interacting protein kinases (CIPKs) are recruited to regulate responses to biotic and abiotic stresses. In this study, a pathogenic protein P3 encoded by Rice grassy stunt virus (RGSV) as a bait was used to screen a cDNA library of Nicotiana benthamiana. It was found that RGSV P3 interacts with NbCBL1. Thus, OsCBL1 coding sequence from the leaves of rice (Oryza sativa ssp. japonica cv. Nipponbare) plants was cloned. The interaction between RGSV P3 and OsCBL1 was further confirmed through conducting yeast two-hybrid and bimolecular fluorescence complementarity (BiFC) assays. In addition, the results of qPCR analysis showed that RGSV infection could significantly induce the expression of OsCBL1. Taken together, RGSV P3 may affect the OsCBL1-mediatedion signaling pathway to regulate virus infection through the association with OsCBL1. This study will provide more insights to reveal the RGSV pathogenicity.

MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factors are widely present in plant pathogenic fungi, and play a variety of roles in the growth, development and stress response of fungi. In order to understand the function of the MYB transcription factor family of Botrytis cinerea, this study used bioinformatics methods to conduct phylogenetic analysis, conserved domain analysis, and analysis of the MYB transcription factor family in B. cinerea. Analysis of the expression regularity during the infection period; and using qPCR technology to detect the expression of the MYB family genes of B. cinerea after NaCl and H₂O₂ treatment. The results showed that there were 22 MYB transcription factor family genes in Botrytis cinerea, and phylogenetic analysis divided them into 6 subfamilies, namely MYB, MYB-Hox, Zn-ZZ-MYB, DEXDc-HELICc-MYB, HAS- MYB and BAH-PND-MYB-Zn-GATA; MYB family genes showed different expression patterns at different developmental stages and infection stages of conidia, and different expression patterns after NaCl and H₂O₂ treatments, indicating that the genes of MYB family of B. cinerea might play an important role in the growth and development of the pathogen, the process of infection and responding to stress. The present study provides basic material for studying the gene function of the MYB transcription factor family and the control of B. cinerea.

With the continuous cultivation of high value-added vegetable crops in facility greenhouses, the problem of soil-borne diseases has gradually become prominent, and fumigants have also been more widely used. However, based on the broad spectrum of fumigants, while killing harmful organisms, it will inevitably have a certain impact on non-target organisms. In this experiment, qPCR technology, selective medium separation and counting methods were used to study the inhibitory effect of Dazomet, Calcium cyanamide and Metam sodium in lettuce (Lactuca sativa) planting soil on Fusarium and the influence of soil microbial community. During the harvest of lettuce, the agronomic traits and yield of the plant were measured, and the correlation analysis of the interaction between fumigant and soil microorganisms was carried out based on the physical and chemical properties of the soil. The results showed that the DNA content of Fusarium was 3.29×10⁹, 2.85×10⁹, and 5.85×10⁹ copies/g before the treatment of Dazomet, Calcium cyanamide and Metam sodium, respectively, and the DNA content of Fusarium decreased to 1.72×10⁹, 2.17×10⁹ and 5.25×10⁹ copies/g at 0 d after fumigation and unmasking. The number of microorganisms in the 3 fumigant treatments was significantly reduced at 0 d after the film was uncovered, and the number of actinomycetes recovered and was higher than the initial level after 60 d after the film was uncovered in the Calcium cyanamide treatment. The control effects of Dazomet, Calcium cyanamide and Metam sodium on the root rot of lettuce were 78.90%, 62.55% and 48.64%, respectively. The yield weights of Dazomet, Calcium cyanamide and Metam sodium after treatment were 0.72, 0.70 and 0.70 kg, respectively. Compared with CK, the yield increase rate of the lettuce in Dazomet treatment was 60.00%, and the growth promotion rate was more than 16%, followed by Calcium cyanamide and Metam sodium treatment. The pH before treatment with Calcium cyanamide and Dazomet was 6.89 and 6.88, respectively, and increased to 7.12 and 6.98 after 60 d of uncovering the film. The initial soil electrical conductivity of Dazomet, Calcium cyanamide and Metam sodium were 284.50, 324.00, and 330.00 μs/cm, respectively, and decreased by 22.38%, 13.88%, and 37.18%, respectively, after 60 d of uncovering the film. Therefore, the 3 fumigant treatments can inhibit Fusarium to varying degrees, increase the agronomic characteristics and yield of lettuce, restore the number of microorganisms in the later stage, and will not significantly disturb the soil microbial community, which was safe for the environment. This research lays a technical support for the restoration and reconstruction technology of lettuce planting soil micro-ecological.

Corn (Zea mays) stalk is the main source of feed for cattle, sheep and other livestock, and contains a large amount of cellulose. Exogenous cellobiohydrolase (CBH) can promote the degradation of cellulose during silage. As probiotics, lactic acid bacteria (Lactococcus lactis) can improve the quality of silage. In present study, mixed DNA of the cattle (Bos taurus domestica) rumen microbes was obtained by crude extraction, and the cbh gene was amplified and cloned into food-grade vector pMG36e, which was used to construct a secreted expression vector pMG36e::CBH. The expression vector was transformed into L. lactis NZ9000, and the corresponding enzyme activity was determined by 3,5-dinitrosalicylic acid (DNS) method, and the enzymatic properties of the recombinant enzyme were analyzed. The results showed that a sequence with 1 600 bp was cloned from cattle rumen, the molecular weight of the recombinant enzyme was about 56 kD, the filter paper enzyme activity was (2.218 1±0.834 3) U/mL, and the exoglucanase activity was (10.499 2±1.113 5) U/mL. The recombinant enzyme had the highest specificity for regenerated amorphous cellulose (RAC), followed by microcrystalline cellulose, lower specificity for filter paper and absorbent cotton, but almost no activity for sodium carboxymethyl cellulose (CMC-Na); the optimal pH and temperature were pH 6 and 60 ℃; 1 mmol/L Mn²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Co²⁺, and 1% Tween-20 promoted the enzyme activity of recombinant CBH, while 1 mmol/L Ba²⁺ and Hg²⁺ had almost no effect on the enzyme activity, but 1 mmol/L K⁺, Mg²⁺, and EDTA inhibited the enzyme activity, 5 mmol/L Mn²⁺, Fe²⁺, Ca²⁺, Cu²⁺, and Co²⁺ promoted the enzyme activity, 10% Tween-20 and 5 mmol/L Zn²⁺, Ba²⁺, Hg²⁺, K⁺, Mg²⁺ and EDTA inhibited the enzyme activity. The recombinant lactobacillus with cbh gene from cattle rumen microbes constructed in this study is conducive to promoting the enzymatic hydrolysis of straw during silage and improving the nutritional value of silage.

In the glucose-specific phosphotransferase (PTS^Glc) system, glucose-specific enzyme ⅡBC (EⅡBC^Glc) has a comprehensive effect on the metabolism of bacteria, but of which the function is still unclear in Glaesserella parasuis. In this study, EⅡBC^Glc protein coding gene (ptsG) mutant and comlementary strain were constructed by natural transformation method. The growth status of wild strain SC1401 and ptsG mutant strains in the trypton soy broth (TSB)+glucose medium were compared in this study. Moreover, the bacterial morphology, biofilm formation, tolerance to oxygen pressure and virulence to mice (Mus musculus) of the SC1401 and ptsG mutant strain were measured. The results showed that the ptsG mutant strain could take in more reducing sugars for its own growth. Through transmission electron microscopy that compared with the wild strain, ptsG mutant strain had more high electron density areas inside the bacteria. In addition, the formation of biofilm and the tolerance to hydrogen peroxide were decreased in ptsG mutant strain. But there was no significant difference between SC1401 and ptsG mutant strains on mice. This study found that the ptsG gene played an important role in the formation of biofilm and oxidative stress tolerance, which will lay a foundation for the study of pathogenic mechanism in Glaesserella parasuis.

Novel duck reovirus (NDRV) is a new pathogen discovered in recent years, which can cause diseases in multiple species of ducks and geese and huge economic losses to the duck industry in China. σC protein is one of the outer capsid proteins of NDRV, which is able to induce type-specific antibodies against the virus and functions in the identification and attachment of the virus to the target cell, probably via a receptor-mediated event. For the preparation of monoclonal antibody (McAb) against protein σC of NDRV and identification of antigen epitope, SP2/0 myeloma cells were fused with spleen from BALB/c mice (Mus musculus) immunized with purified recombinant σC expressed in Escherichia coli. Four hybidoma cell lines (A5-A1, A5-B6, A9-D4 and B9-6) stably secreting McAbs against σC of NDRV were identified by indirect ELISA. Western blot results showed that all 4 McAbs could recognize whole virus of NDRV and recombinant σ C protein from prokaryotic expression. Indirect immunofluorescence assay (IFA) indicated that 4 McAbs reacted positively with NDRV, but did not show cross reaction with Classical duck reovirus (CDRV) and Avian reovirus (ARV). The antigenic epitopes recognized by 4 McAbs were identified by synthetic overlapping peptide library and a series of truncated peptides, and the results showed that the core antigenic epitope of ¹⁷⁷PILSGPADA¹⁸⁵ was recognized by 3 McAbs (A5-A1, A5-B6, and B9-6). The antigenic epitopes recognized by A9-D4 might be spatial conformational epitopes. Conservative analysis showed that the antigenic epitope was highly conservative in the NDRV isolates from different regions and different hosts. The results suggested that ¹⁷⁷PILSGPADA¹⁸⁵ was a type-specific linear B-cell epitope of NDRV. This study could provide a basis for further research on the structure of σC protein and the establishment of NDRV clinical detection methods.

Mycoplasma synoviae (MS) is an important pathogenic mycoplasma in poultry, which can cause exudative synovitis, arthritis, tarsometatarsal swelling and respiratory tract inflammation in chickens (Gallus domesticus) and turkeys (Meleagris gallopavo), causing great economic losses in chicken industry. Methylenetetrahydrofolate dehydrogenase (MTHFD) is a key enzyme in folic acid metabolism, catalyzing 5,10-methylenetrahydrofolate to 5,10-methoxytetrahydrofolate and participating in the synthesis of purine nucleotides. To study the immunogenicity of MTHFD protein of MS and its distribution in MS, primers were designed according to the folD (bifunctional 5, 10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase) gene of MS WVU1853 strain in GenBank. On the basis of sequence analysis and gene optimization, the full-length folD gene was successfully obtained through PCR, which the length of CDS of folD is 837 bp and the sequence similarity was as high as 99.6% with other MS strains. The prokaryotic expression vector pET-folD was constructed and transformed into E.coli BL21 (DE3). After induction, the recombinant protein (r) MS MTHFD was expressed through SDS-PAGE and molecular weight of rMS MTHFD was approximately 36 kD. The antiserum was prepared by immunizing New Zealand rabbits (Oryctolagus cuniculus) , Western blot, ELISA and immunofluorescence test were used to analyze the distribution of MTHFD in MS. The results showed that MTHFD protein was distributed in MS cell membrane and cytoplasm, but more in cytoplasm. The study build up a basis for in-deepth research of biological characteristics of MS MTHFD.

Polymerase chain reaction (PCR) is widely used in many fields because of its unique advantages. Traditional PCR technology takes a long time, which is generally more than 60 min, so a method of fast PCR is proposed. This paper focuses on the fast PCR reaction device. According to the heat transfer mode, the contact (continuous flow, fixed chamber, oscillating swing) and non-contact (infrared, laser, metal nanostructure, microwave, electromagnetic induction) thermal cycling instruments are introduced and analyzed respectively. Finally, the operation integration of rapid PCR technology, the further research and development of metal nanostructures and the commercial production of rapid thermal cycling instrument are discussed and prospected. This review provides a reference for the research and development of low-cost, miniaturized, automatic and high-throughput heat cycle instrument in the future.

Iron is one of the most important trace elements for plant growth and development. As an enzyme cofactor or part of the electron transport chain, it plays a role in various important metabolic processes such as plant photosynthesis, respiration and amino acid biosynthesis. To cope with iron deficiency, plants have evolved complex transcriptional regulatory networks to maintain iron homeostasis and strictly control iron uptake, transport, assimilation and storage. These regulatory networks are composed of multiple transcription factors, among which the basic helix-loop helix (bHLH) family is one of the most critical transcription factor families. In this paper, two strategies of plant response to iron deficiency were briefly summarized. This paper introduced the structure, classification and action form of bHLH transcription factors, and discusses the latest progress of bHLH transcription factors such as FER-like iron deficiency-induced transcription factor (FIT), PYE (POPEYE), upsteam regulator of iron-regulated transporter 1 (URI) and the iron deficiency regulatory protein E3 ubiquitin ligase BRUTUS (BTS) in the regulation of iron homeostasis cascade. This paper provides theoretical basis for the study of the mechanism of bHLH transcription factors in plant iron deficiency regulation network.

Goat (Capra hircus) and sheep (Ovis aries) are main domestic animals raised by human beings. Goat and sheep can be divided into four categories including milk, meat, fleece and both according to its economic use. A clear understanding of the genetic evolution and selection progress of population can be provided through exploring the characteristics of selection signatures among different population. Moreover, the genetic progress can be accelerated and the breeding process can be shortened by applying positive selection. The functional genes detected by the selection signature analyses can be further verified as the causal genes, so as to realize molecular design breeding and the improvement of important economic traits. In this review, the differences and application scopes of five commonly used selection signature detection methods are summarized, and the research progress of functional genes determined using selection signature analyses for important economic traits in goat and sheep are also summarized. This study lays a theoretical foundation for further analysis of the molecular mechanism of functional genes regulating the formation of important economic traits in goat and sheep, and provides genetic sources and molecular materials for population genetic improvement or new varieties (lines) cultivation through molecular design breeding.

Garlic (Allium sativum) is an important vegetable crop for asexual reproduction, and the molecular mechanism research of somatic embryogenesis is still relatively weak. Selection of the stable reference genes will help clarify the differential expression of genes and miRNAs in somatic cell formation, help lay the foundation for the research to reveal the mechanism of garlic somatic embryogenesis. To select the most stable reference genes for mRNA and miRNA qPCR detection system during garlic somatic embryogenesis, this study chose 7 mRNA candidate reference genes, including the actin (AsACTIN), glyceraldehyde-3-phosphate dehydrogenase (AsGAPDH), 18S ribosomal RNA (As18S rRNA), polyubiquitinase (AsUBQ), α-tubulin (AsTUA), transcription factors LATE ELONGATED HYPOCOTYL (AsLHY) and Brassinosteroid resistant 1 (AsBES1). 5 miRNA candidate reference genes, including AsU6snRNA, As5.8S rRNA, AsmiR159a-1, AsmiR168a and AsmiR168a-5p. qPCR technology was used to detect their Ct value at different stages of somatic embryo development in different genotypes, explants, and 2,4-D concentrations. Delta CT, BestKeeper, NormFinder, and GeNorm were applied to analyze the expression stability of candidate reference genes. The results were combined with RefFinder to evaluate the expression stability of candidate reference genes comprehensively. The results showed that for mRNA qPCR expression analysis, AsACTIN was the most suitable reference gene for different genotypes. AsBES1 was the most stable reference gene under different explants and different 2,4-D concentrations. The most stable reference gene in all samples was AsBES1. For miRNA, the most stable reference gene in different genotypes was AsmiR159a-1, AsmiR168a-5p was the most stable reference gene in different explants, and As5.8S rRNA was the most stable reference gene under different 2,4-D concentration treatments. The ideal reference gene in all samples was AsU6 snRNA. Using the 7 mRNA candidate reference genes and 5 miRNA candidate reference genes, plant hormone related transcription factors myelocytomatosis protein 2 (AsMYC2) and AsmiR167 were analyzed by qPCR, which were differentially expressed during garlic somatic embryogenesis. The results showed that AsBES1 and AsmiR168a-5p, which were the most stable reference gene during garlic somatic embryogenesis of different explants, were used as reference gene, the expression trends of AsMYC2 and AsmiR167d-1 were consistent with the sequencing data. When other genes (miRNA) were used as controls, the gene expression trends (miRNA) differed, further confirming the reliability of reference genes. This study provides supports for accurately detecting genes and miRNAs during garlic somatic embryogenesis, it is helpful to further study the mechanism of somatic embryogenesis in garlic.

Duck hepacivirus (DuHCV) is a newly identified Hepatitis C virus in ducks (Anas) with abnormal egg production syndrome. In order to establish a molecular biological detection method of DuHCV for the epidemiological investigation of the disease and further explore its pathogenic mechanism. In this study, based on the NS5B gene characteristics of the DuHCV reference strain (HCL-2 strain, GenBank No. MK737640.1), specific primers were designed to establish a TB Green real-time fluorescent quantitative PCR method for the detection of DuHCV. The established TB Green real-time fluorescent quantitative PCR method of DuHCV had high sensitivity, with the limit of detection was 64 copies/μL. It had good specificity, with no cross-reaction with common duck origin infectious disease pathogens (such as Duck hepatitis A virus type 1, Duck hepatitis A virus type 3, Avian influenza virus, Avian paramyxovirus type 1, Avian tembusu virus, Muscovy duck reovirus and novel Muscovy duck reovirus), and the melting curve appeared a specific peak, with Tm value was (82.22±0.21) ℃. It had good reproducibility, and the coefficient of variation of repeated experiments within and between groups was 0.13%~0.54% and 0.53%~1.12%, respectively. 103 samples came from three species of ducks, Muscovy ducks (Cairna moschata), Jinding ducks and Putian black ducks were collected, and then were tested with the established TB Green real-time fluorescent quantitative PCR method and reverse transcription PCR (RT-PCR) method simultaneously for DuHCV infection. The results showed that 8 positive (positive rate was 7.8%) samples were detected by the RT-PCR method, while the TB Green real-time fluorescent quantitative PCR method had 11 positive (positive rate was 10.68%) samples. Moreover, 8 RT-PCR positive samples were all positive by TB Green real-time fluorescent quantitative PCR method, with the coincidence rate was 100%. DuHCV positive was not detected in Muscovy ducks, but both DuHCV positive were found in Jinding ducks and Putian black ducks. Previously data showed no reports of Putian black ducks infected with DuHCV, which means this study confirmed the existence of DuHCV firstly. In conclusion, TB Green real-time fluorescent quantitative PCR method for the detection of DuHCV was successfully established, with high specificity, sensibility and well repetitiveness, which provides a useful candidate tool for epidemiological investigation and pathogenic mechanism research of DuHCV.