Abscisic acid (ABA) responsive element binding factors/abscisic acid responsive element binding proteins/abscisic acid insensitive 5 (ABF/AREB/ABI5) belongs to the basic leucine zipper (bZIP) transcription factors A subfamily, which participate in ABA signaling pathway by regulating the expression of downstream genes, and thereby affect the plant growth and development, as well as in response to abiotic stress. In this study, the members of the ABF/AREB/ABI5 subfamily were identified in the whole genome of wheat (Triticum aestivum), and the phylogenetic relationship, physical and chemical properties, gene structure, gene replication, cis-acting elements of promoters and the expression pattern of wheat TaABFs were analyzed. The results showed that a total of 36 TaABFs were identified in wheat, which were distributed on all chromosomes except 7B and there were tandem duplication and fragment duplication events on the Ⅲ homologous group. The coding region of wheat TaABF proteins were 672~1 191 bp in length, and their isoelectric point were 5.22~9.93, all of which were hydrophilic proteins. According to phylogenetic analysis, TaABFs were divided into 3 groups, and in each group, the TaABFs showed high similarity in structure, conserved motifs and physicochemical properties. The promoter analysis of TaABFs indicated that there were a large number of cis-acting elements in response to hormones and abiotic stresses in their promoters. Transcriptome expression pattern analysis showed that the TaABF genes expressed differentially in different tissues and developmental stages, most of which were highly expressed during seed development. The TaABF genes also showed different responses to drought, salt and low temperature stress, and the TaABF genes in group Ⅰ homologous to the Arabidopsis AREB/ABF subfamily were up-regulated under both drought and salt stress. Further expression analysis of TaABF genes in Group Ⅰ by qPCR showed that, under ABA treatment, TaABF18 was up-regulated, while TaABF17 and TaABF19 were significantly down-regulated. TaABF17, TaABF18 and TaABF19 were all significantly up-regulated under drought and salt stress. These results provide basic information for further study on the resistance function of TaABFs in wheat.

BNS (Bai-nong sterility) is a nuclear thermo-sensitive male sterile line of common wheat (Triticum aestivum), which plays an important role for the study of hybrid wheat. In order to explore the physiological mechanism of BNS sterility, Affymetrix wheat microarray was used to detect the transcriptomes of BNS male sterility and male fertile in anthers and spike-stalks in the developmental stage of tetrad and mononuclear, and analyzed metabolic networks generated by differential probe genes. The results found that an acetyl-CoA metabolic sub-network could be formed in the profiles of up-regulated genes in fertile anthers, but the sub- network was incompletely in partly sterile anthers, and it was not formed in sterile anthers. The acetyl-CoA sub-metabolic network consisted of 6 nodes, which were acetyl-CoA synthesis and metabolism, acyl-CoA synthesis and metabolism, and thioester synthesis and metabolism, respectively. The nodes of sub-network of acetyl-CoA first linked to that of hormone metabolism, waxy metabolism, amino acid metabolism and linked to ATP synthesis, mitochondrial electron transfer, and so on, then connected with stigma and anther development. Acetyl-CoA was a pivotal substance in cell energy metabolism, so the deficiency of this metabolic pathway directly affected the production of energy carriers such as ATP, thus affecting microspore development. ATP content in anther and flag leaf tissue of BNS sterile and fertile plants were detected, respectively. The results showed the content of ATP in the sterile anther decreased by about 10% compared with fertile anther (P<0.01), and the content of ATP in the sterile flag leaf decreased by 50% compared with fertile flag leaf (P<0.01). The results indicated that there was a shortage of ATP supply in the critical period of microspore development. Thus it was believed that the metabolic defect of acetyl-CoA was an important physiological mechanism for BNS male sterility. The mechanism provides an important clue for revealing the occurrence of BNS infertility.

In the process of plant regeneration mediated by developmental regulators, overexpression of developmental regulators could lead to pleiotropic effects such as developmental malformation and pollen abortion. Therefore, finding a suitable promoter to control the suitable expression of developmental regulators in plants may be one of the methods to obtain normal regenerated plants. LEAFY COTYLEDON2 (LEC2), a specific B3 transcription factor in plant embryogenesis, plays a crucial regulatory role in seed maturation. In the study, the results of semi-quantitative PCR showed that GbLEC2 was not or weakly expressed in roots, stems, leaves or embryogenic callus in Gossypium barbadense, but significantly highly expressed in globular embryo, torpedo embryo and cotyledon embryo. Bioinformatics analysis displayed that there were 2 seed-specific cis-elements Skn-1 element and RY repeat element in GbLEC2 promoter sequence. To further identify the transcriptional characters of GbLEC2 promoter, 4 different GbLEC2 promoter fragments including ProGbLEC2-1 (2000 bp), ProGbLEC2-2 (1500 bp), ProGbLEC2-3 (1000 bp) and ProGbLEC2-4 (500 bp) were constructed and then transformed into the different cotton tissues and cells by Agrobacterium-mediated transient transformation method. And the GUS activity assay demonstrated that ProGbLEC2-3 (1000 bp) and ProGbLEC2-4 (500 bp) promoter fragments displayed a specific transcriptional activity in somatic embryogenesis, which preliminarily met the Lowe's requirements for the promoters to drive suitable expression of developmental regulators in plant. Therefore, the GbLEC2 promoter fragment cloned in the study is expected to be a new candidate promoter to precisely regulate the expression of developmental regulators, and thus be applied to a novel molecular breeding technolody platform mediated by developmental regulators-promoting plant regenesis in G. barbadense.

Raffinose plays an important role in plant response to abiotic stress, and galactinol synthase (Gols) is an important enzyme involved in the synthesis of raffinose. In this study, Gols gene families were identified and analyzed in the whole genome, 8, 7, 14 and 14 Gols genes were identified in Gossypium arboreum, G. raimondi, G. hirsutum and G. barbadense, respectively. Evolutionary analysis showed that the Gols gene family could be divided into 7 subgroups. The resules of conservative motifs and gene structure analysis showed that gene structure and composition of conserved motifs were also similar between closely related family members. A lot of cis-elements relevant to plant response to stresses were distributed on promoter regions of GhGols. Tissue expression pattern analysis showed that some GhGols genes were predominantly expressed in specific tissues. Expression analysis showed that the expression of some GhGols was induced by PEG treatment, among which the expression of GhGols6 was significantly up-regulated under PEG treatment. The results of qPCR analysis showed that the transcription level of GhGols6 was also significantly induced under drought stress. Further the GhGols6 gene was silenced using virus induced gene silencing (VIGS) technology, the result showed that silencing GhGols6 decreased the tolerance of cotton to drought stress, indicating that GhGols6 was a positive regulator of cotton drought response. This study provides a reference for further study on the function of Gols gene in cotton drought response.

Fruit flesh of Citrus is rich in citrate and other nutrients. To better understand the effects of differential expression of genes (DEGs) on citrate and other important metabolites in Citrus, both fruit qualities and RNA-seq analysis were performed between 'Sweet lemon' and 'Eureka', respectively. The comparative analysis between 2 cultivars had been implemented using transcriptomics technology to identify the DEGs, which caused citrate accumulating in pulp. After transcriptomic sequencing for each fruit in 3 replicates, 73 678 unigenes had been obtained, out of which the total 4 044 unigenes were differentially expressed between 2 cultivars. Subsequently, DEGs were further enriched into different pathways by KEGG. The upregulating DGEs in 'Sweet lemon' with lower citrate contents were enriched into 24 pathways, including GABA shunt. Besides, upregulating DEGs in 'Eureka' with higher citrate contents were enriched into 13 pathways, including citrate cycle. At fruit ripening stage, the significantly differential expression of citrate synthase was not found between 2 cultivars. In addition, proton pump gene in 'Eureka' had higher transcription levels than 'Sweet lemon' at fruit ripening stage, so that it was benefit for vacuole acidification in fruit. It could be concluded that up-regulating expression of genes involved into citrate metabolism and proton transporting were correlated with organic acidity accumulations in Citrus fruits. This study provides an insight into organic acids metabolism in Citrus and is useful for breeding program in future.

Terpenoids are the main floral components of tree peony (Paeonia suffruticosa). 1-deoxyxylulose-5- phosphate reductoisomerase (DXR) and 2-C-methylerythritol-2, 4-cyclodiphosphate synthase (MCS) play important roles as key enzymes in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in plant terpene synthesis. To investigate the role of PsDXR and PsMCS genes in the terpene synthesis pathway of tree peony, the PsDXR (GenBank No. ON092613) and PsMCS genes (GenBank No. ON092614) were cloned using the 'Huang Guan' cDNA of tree peony in this study, and their bioinformatics analysis, subcellular localization and qRT-PCR were performed to analyze the expression in different flower developmental stages and tissues of tree peony 'Huang Guan'. The results showed that the cDNAs of PsDXR and PsMCS genes were 1 401 and 702 bp in length, encoding 466 and 233 amino acids, respectively. The proteins encoded by the 2 genes were stable hydrophilic proteins. The PsDXR protein had the conserved domains of DXP_reductoisom and DXP_redisom_C, and the PsMCS protein had the MECDP-specific domain. The results of subcellular localization showed that both proteins were localized in the chloroplast. The expression patterns of PsDXR and PsMCS genes both increased and then decreased in flower development stages; both 2 genes were differentially expressed in different tissues (P<0.05). The results indicated that PsDXR and PsMCS genes might play important roles in the MEP metabolic pathway of tree peony, provides a molecular basis for the biosynthesis of terpenes in tree peony.

Iridoid oxidase (IO) belonging to the CYP76 family A26 subfamily and is the step 4 rate-limiting enzyme of the iridodial pathway in the upstream synthetic pathway of terpenoid indole alkaloids, catalyzing the synthesis of 7-deoxyloganetic acid from iridoid. In this study, UrIO (GenBank No. ON227515) was cloned based from Uncaria rhynchophylla transcriptome data, which encoded 516 amino acids. Phylogenetic relationship showed that the relationship between UrIO of U. rhynchophylla and CcIO of Cinchona calisaya was the closest, followed by CrIO of Catharanthus roseus. Multiple sequence alignment analysis showed that UrIO had 3 typical domains of cytochrome P450 proteins (CYP) family. The expression level of UrIO in stem was the highest, followed by fruit, and the expression of UrIO was significantly increased under methyl jasmonate (MeJA) treatment. However, it was not induced by abscisic acid (ABA). The promoter of UrIO was cloned by fusion primer and nested integrated PCR (FPNI-PCR) method as well, which contained stress response and hormone response elements by plant cis-acting regulatory elements (PlantCARE) analysis. Transient transformation experiments in tobacco (Nicotiana tabacum) showed that the promoter could drive GUS reporter gene expression. Furthermore, yeast one hybrid (Y1H) confirmed the interaction between UrMYC2 and UrIO promoter. This study provides a reference for further analysis of the synthetic pathway of indole alkaloids from U. rhynchophylla.

Hypericum perforatum is an important perennial medicinal plant in the form of herb in China. APETALA2 (AP2) is a special transcription factor family in plants, which plays a key role in plant growth and development. H. perforatum, as a sequenced species in Malpighiales, its AP2 gene family has not been analyzed. In order to reveal the characteristics and functions of HpAP2 gene family, a total of 21 AP2 transcription factors were identified using bioinformatics methods based on the transcriptome sequencing data in different tissues of H. perforatum, and their physicochemical properties, gene structure, conserved motifs, phylogenetic relationship, cis-acting elements and expression pattern were analyzed. In addition, the expression of selected 5 genes in H. perforatum under the treatment of 3 kinds of hormones were detected by qRT-PCR. Results showed that physicochemical properties of HpAP2 family were different, but they all belonged to hydrophilic protein. In the secondary structure of AP2 protein, the proportion of random coil and α-helix was large, while that of extended strand and β-turn was small. The HpAP2 family was subdivided into the eu-AINTEGUMENTA (euANT), euAP2, and basalANT groups according to the phylogenetic relationship. The same subfamily members shared similar gene structure and conserved motifs. Cis-acting elements in the promoter region of HpAP2 genes included 4 categories responding to transcription, development, abiotic or biological stress and hormone. Transcriptome data showed that most AP2 family genes (57.14%) were expressed in 4 tissues of root, stem, leaf and flower, but there were significant differences in expression levels. qRT-PCR analyses demonstrated that the 5 HpAP2 genes were differentially expressed under the treatment of 3 kinds of hormones. Among them, the gene expression of HpAP2-5, HpAP2-11, and HpAP2-18 were significantly upregulated when treated by abscisic acid (ABA), salicylic acid (SA), and gibberellin (GA), respectively, suggesting that these genes might play roles in the response to stress-related phytohormones. This study laid a foundation for further study on the function of HpAP2 genes and genetic improvement of H. perforatum. This study provides reference for further exploring the function of HpAP2 gene family and analyzing its regulatory mechanism in stress responses.

Cinnamomum camphora var. linaloolifera is a kind of camphor trees with linalool-rich essential oils in their branches and leaves. Due to its high economic value, this variety has become one of the important industrial material tree species in South China. In this study, a linalool synthase gene (CcTPS14) was cloned from C. camphora var. linaloolifera, and was analyzed based on bioinformatics, phylogeny and tissue-specific expression patterns. The results showed that the full length of CcTPS14 was 725 bp, with a 684 bp CDS and encoding 227 amino acids. The relative molecular weight of CcTPS14 protein was 25.69 kD, the theoretical isoelectric point was 5.39. The fat coefficient was 87.19 and the instability coefficient was 51.40. The CcTPS14 protein was unstable and hydrophilic. Based on subcellular localization predicts, CcTPS14 was mostly located in chloroplasts. Its secondary structure was mainly irregular crimping (65.86%) and α -helix (22.52%), with a small amount of extended chains (12.60%) and β-folding (9.01%). Phylogenetic relationship analysis showed that CcTPS14 was closely related to TPS from C. micranthum and C. osmophloeum, indicating that the presence of similar evolutionary process and biological function of those TPS genes in different species. And it was distantly related to TPS genes from Tetracentron sinensis and Rhododendron griersonianum. The relative expression of CcTPS14 in different tissues of C. camphora was significantly different (P<0.05), among which the expression was higher in mature tissues (such as stems and old leaves) and lower in young tissues (such as young leaves, young fruits and flowers). This study provides the molecular basis and theoretical reference for further understanding of the functional of CcTPS14.

In mammals, imprinted genes play key role in placental development and fetal growth. Changes in the expression of imprinted genes can lead to abnormal growth and development in mammals and cause diseases through various physiological responses and signaling pathways. KLF14 (Kruppel like factor 14) and phosphodiesterase 10a (PDE10A) genes were identified as imprinted genes in human (Homo sapiens) and mouse (Mus musculus). At present, the imprinting studies on KLF14 and PDE10A in cattle (Bos taurus) have not been reported. In this study, the expression of KLF14 and PDE10A genes in bovine heart, liver, spleen, lung, kidney, brain and placenta were firstly analyzed by RT-PCR. The results showed that KLF14 gene was only detected in the placenta; PDE10A gene was expressed in all tested tissues. The allelic expression status of KLF14 and PDE10A genes were further analyzed by direct sequencing of RT-PCR products based on SNP genomic DNA, KLF14 gene was identifiied to be a maternally imprinted gene in bovine placenta. PDE10A gene showed biallelic expression in placenta ant other tissues. PDE10A gene was non-imprinted in bovine. The methylation status of the promoter region of KLF14 gene was analyzed by sulfite sequencing method, hypomethylation were found in both bovine placenta and sperm, and there was no differentially methylated region, which indicated that the methylation modification of the promoter region of KLF14 gene might not be involved in regulating the imprinted expression of KLF14 gene. The above results suggest that bovine KLF14 gene is a placenta-specific maternally imprinted gene and bovine PDE10A gene is non-imprinted gene. This study provides a reference for further study of the function of the bovine KLF14 and PED10A.

The infection of bovine viral diarrhea is becoming more and more serious, which has caused serious losses to the breeding industry. At present, the lack of effective treatment leads to the complicated infection pattern in China, and the research on the pathogenesis and virus-host interaction is still unclear. To investigate the effect of vesicle transport protein (SEC22B) deletion on Bovine viral diarrhea virus (BVDV) replication, according to the SEC22B gene sequence in GenBank database, small guide RNA (sgRNA) was designed using Benchling platform and cloned into LentiCRISPR V2 vector for lentivirus packaging. The SEC22B gene in madin-darby bovine kidney cells (MDBK) was knocked down (KD) by CRISPR/Cas9 gene editing technique, and identified by Western blot. After BVDV infection with SEC22B KD cells at different times, the changes of 5' UTR mRNA level and accumulation of double-stranded RNA (dsRNA) were detected by qPCR and immunofluorescence staining, respectively. Reed-Muench method was used to measure the titer change of progeny virus after BVDV infection at different time. The results showed that compared with scramble cells, the protein expression level of SEC22B in SEC22B KD cells was significantly decreased by Western blot. Compared with scramble cells infected with BVDV, the level of 5' UTR mRNA and dsRNA of SEC22B KD cells were extremely significantly decreased (P<0.01) after BVDV infection, and the titer of progeny virus was extremely significantly decreased (P<0.01). The results showed that knockdown SEC22B gene significantly inhibited the replication of BVDV. This study provides a target for the establishment of new methods for the prevention and control of BVDV.

Alternative splicing is an important way for eukaryotes to precisely regulate cell types and devel‐ opment. Peroxisome proliferator activated receptor γ (PPARγ) is a key regulator of adipocyte differentiation. Its pre-mRNA could produce a subtype of PPARγΔ5 through alternative splicing and affecting adipocyte dif‐ ferentiation. In this study, the preadipocytes were isolated from subcutaneous adipose tissue of three-day-old piglets (Sus scrofa) and induced to adipogenic differentiation. The mRNA expression of related genes was de‐tected by qPCR. it was observed that porcine adipocytes expressed PPARγΔ5 mRNA, and the expression in‐creased significantly in the early stage of differentiation, peaked in the middle and decreased in the later stage (P<0.01), which was consistent with the expressions of PPARγ and serine/arginine-rich splicing factor 1 (SRSF1). The expressions of PPARγΔ5 and SRSF1 in the adipocytes were extremely significant promoted by rosiglitazone treatment (P<0.01), and reduced by GW9662 (P<0.01). Furthermore, the PPARγΔ5-siRNA re‐ combinant lentivirus was constructed, and after transfected with porcine preadipocytes, the PPARγΔ5 expres‐ sion significantly decreased about 75% (P<0.01). Compared with the cells transfected with negative control lentivirus or non-transfected, PPARγΔ5 expression silencing significantly stimulated the differentiation of pre‐ adipocytes and the mRNA expressions of PPARγ, fatty acid binding protein 4 (FABP4) and stearoyl-CoA de‐ saturase (SCD) (P<0.05), and decreased the expression of lipoprotein lipase (LPL) (P<0.05). These results suggested that the porcine adipocytes expressed PPARγΔ5 through selective splicing depending on SRSF1 and the expression and activity of PPARγ. PPARγΔ5 was an inhibitor of adipocyte differentiation, which could at‐ tenuated the differentiation of porcine preadipocytes via negatively regulating PPARγ expression. This study provides a new clue for understanding the regulation of porcine adipogenesis.

The 17β-hydroxysteroid dehydrogenase (17β-HSD) eventually oxidizes androstenedione converted from cholesterol to testosterone, which is essential for testosterone synthesis and male reproductive traits. 17β- HSD3 was found to be involved in the pre-embryonic gonadal development of the Mulard duck (Cairina moschata) through the steroid hormone synthesis pathway. In this study, the full-length sequence of 17β-HSD3 gene was obtained by rapid amplification of cDNA ends (RACE) cloning method, sequence characteristics and physicochemical properties of proteins were predicted using bioinformatics analysis, and phylogenetic trees were constructed. The qPCR was utilized to analyze the tissue-specific expression of 17β -HSD3 gene in Mulard duck (brain, heart, liver, lung, muscle, gonad, kidney) and differential expression in the gonads of Mulard ducks, male parent Muscovy and female parent Pekin ducks. The results showed that the full-length sequence of 17β-HSD3 gene was 1 332 bp in Mulard duck, containing a 963 bp of open reading frame, and the length of 5' and 3' non-coding regions were 26 bp and 343 bp, respectively. The encoded protein contained 320 amino acids, which was an unstable protein. Homology and phylogenetic tree phylogenetic analysis showed that the 17β-HSD3 gene in Mulard duck have the closest relationship with swans (Cygnus olor). Tissue-specific expression analysis revealed that the 17β-HSD3 gene was highly significantly more expressed in the gonads of Mulard ducks than in other tissues (P<0.01), suggesting that 17β -HSD3 gene might be involved in gonadal development in Mulard duck. The expression of the 17β -HSD3 gene was significantly higher in Muscovy ducks and Pekin ducks with normal reproductive traits than in Mulard ducks (P<0.01), revealing that 17β - HSD3 gene might be associated with sterility in Mulard ducks. The study provides a theoretical basis for the function of the 17β -HSD3 gene in gonadal development, germ cell differentiation and other reproductive processes in the Mulard duck.

O157: H7, an enterohemorrhagic Escherichia coli (EHEC) strain, is a foodborne pathogen that can cause disease in livestock, poultry and humans (Homo sapiens). Magainins is a cationic peptide with antibacterial and antitumor properties, and its antibacterial activity against Gram-negative and Gram-positive bacteria has been reported in previous studies. The purpose of this study was to explore the effect of frog (Xenopus laevis) derived Magainin Ⅱ on intestinal inflammation and microflora in mice (Mus musculus) induced by EHEC O157: H7. Forty C57/BL6 male mice with similar weigh were randomly divided into 4 groups with 10 replicates in each group, CK group, O157 group, Mag Ⅱ group, O157+Mag Ⅱ group, respectively. All the groups received the same basal diet. The O157 group and O157+MagⅡgroup were orally administered 0.1 mL O157 bacterial solution with a concentration of 106 CFU/mL on 1, 5 and 9 d; CK group and MagⅡgroup were orally administered 0.1 mL PBS in the same time. After 1 h, MagⅡ and O157+MagⅡ group were intraperitoneally injected with 0.25 mL Magainin Ⅱ solution according to 5 mg/kg body weight, while CK and O157 group were intraperitoneally injected 0.25 mL sterilized saline. On the 10 d, mice were treatmented by euthanasia, and corresponding tissue samples were collected for testing. The results showed that Magainin Ⅱ treatment alone had no effect on the serum inflammatory cytokine concentration and the expression of jejunal inflammatory cytokines and barrier function related genes. EHEC O157: H7 treatment resulted in significant increase in the jejunum gene expression levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) and serum concentration of IL-1β, IL-6, TNF-α in mice (P<0.05). The serum IL-10 levels significantly decreased (P<0.05), as well as relative gene expression of jejunal tight junction proteins claudin-1, mucin-1 (MUC-1), and MUC-2 (P<0.05); Magainin Ⅱ treatment significantly improved EHEC O157: H7-induced relative gene expression of IL-1β, IL-6, TNF- α, claudin-1, MUC-1, MUC-2 in mouse jejunum and abnormal serum concentration of IL-1β, IL-6, IL-10 (P<0.05). EHEC O157: H7 infection could cause changes in intestinal microorganisms. The structure of intestinal microorganisms could be analyzed by 16S rDNA sequencing, and then the changes of intestinal microorganisms before and after Magainin Ⅱ intervention were analyzed. Sequencing results showed that at the genus level, compared with CK group, the relative abundance of Prevotella and Bacteroides in O157 group significantly increased (P<0.05). After MagaininⅡtreatment, the relative abundance of Bacteroides in O157+MagⅡ group decreased (P<0.05). The results showed that the abundance of Prevotella and Bacteroides related to the development of inflammation significantly increased after EHEC O157:H7 challenge, while the abundance of the unfavorable bacteria genus Bacteroides decreased significantly in the Magainin Ⅱ treatment group. This study showed that Magainin Ⅱ could maintain the intestinal barrier and alleviate the level of inflammation, thus playing a protective role in the intestinal injury induced by EHEC O157:H7 in mice, and the change of intestinal flora might be the related factor for Magainin Ⅱ to protect the intestinal injury. In conclusion, Magainin Ⅱ could be used as a potential drug for the treatment of intestinal diseases caused by EHEC O157:H7.

In recent years, deterioration of water environment, elevation of stocking density, occurrence of disease and abiotic stress have seriously affected growth and development of Eriocheir sinensis. Peroxiredoxin (Prx) plays an important role in oxidative stress response. Prx4 is an important member of Prx family. To discuss the role of Prx4 in the anti-bacterial response of E. sinensis, EsPrx4 (GenBank No. ON685195) was cloned from hepatopancreas with homologous cloning method. Sequence analysis showed that the coding region of EsPrx4 was 738 bp, encoding 245 amino acids. The physical and chemical propertiese analysis showed that the molecular weight of EsPrx4 protein was 27.31 kD, the theoretical isoelectric point was 5.55, and the total average hydrophilicity was -0.181, indicating that EsPrx4 protein was hydrophilic. Phylogenetic analysis showed that the EsPrx4 has high homology to that of Scylla paramamosain, Penaeus monodon and Litopenaeus vannamei, which indicated high conservation in Prx4 of shrimp and crab. Results of qPCR indicated that EsPrx4 was widely expressed in E. sinensis, it had the highest expression in hepatopancreas and the lowest expression in intestine. Under the stimulation of Listonella anguillarum, the expression of EsPrx4 reached the highest level at 24 h and recovered to the normal level at 72 h, indicating that EsPrx4 plays an important role in the immune response of E. sinensis. This study provides theoretical support for further research on innate immunity and Prx gene family of E. sinensis.

Starch content and its metabolism in potato (Solanum tuberosum) tubers have great impact on starch processing. Deciphering the functions and expression regulation of key genes involving in starch metabolism will promote to improve potato starch content and quality through genetic engineering. In this review, recent advances on genes encoding key enzymes in potato starch metabolism and their application in starch improvement were summarised. In particular, the identification of cis-regulatory elements for the starch- related genes and their potential advantages in improving starch content and quality were discussed. The application of cis-regulatory elements in potato breeding may provide a new feasible strategy to create cultivars required for both food and energy alcohol industry.

Drought is one of the most important environmental factors affecting crop productivity in many regions. Although sunflower (Helianthus annuus) is tolerant to drought conditions, its tolerance mechanisms at the molecular level are less studied. Elucidating the complex mechanisms of drought tolerance in sunflower will accelerate the selection of new drought-resistant varieties. In this paper, functional genes and transcription factors (AP/ERF, bZIP, WRKY, HB, NAC, MYB, bHLH, etc.) involved in drought stress response in sunflower are summarized. Finally, the current research is summarized and the future development trend is given in the hope that these candidate genes and transcription factors can be used as a potential biotechnological tool for future resolution and improvement of drought resistance and yield of sunflower.

5' rapid amplification of cDNA ends (5' RACE) is usually used to identify transcription initiation sites (TIS) of target gene for gene expression regulation research. However, it usually suffers unexpected failure when processing RNA from adipose tissue because high content of impurities and low content of RNA disturbs RNA extraction in fat, leading to evident declines in yield, quality and purity of extracted RNA compared to other tissues. Kruppel-like factor 4 (KLF4) is a key regulatory gene of bovine adipose deposition. And it is of great significance to determine KLF4 gene TIS in bovine adipose tissue for study of molecular mechanism of fat accumulation.“Classical 5' RACE”technique was considered to have great improvement potential for complex samples by means of comparison and analysis of various 5' RACE techniques. And it was adopted to identify KLF4 gene TIS with RNA extracted from subcutaneous fat tissue of Jiaxian Red Cattle (Bovine) as samples, of which some processes were modified according to sample characteristics. Firstly, specific reverse transcription primers were employed to produce cDNA of special gene, which could significantly increase the cDNA concentration of target gene compared to reverse transcription of total RNA; Then, cDNA purification before tailing was simplified to reduce cDNA losses, which was particularly necessary when adipose tissue with low amount of initial mRNA was confronted; Finally, appropriate GSP primers close to 5'end of mRNA ensured successful amplification of RACE fragment, reducing as many uncertainties as possible in PCR. In addition, in view of the controversy surrounding the best size of RACE fragment, the effects of amplified fragments on PCR in 5' RACE were also analyzed with different lengths. In this study, the RACE fragment of KLF4 from bovine adipose tissue was successfully obtained by the above improvement measures. By sequencing 5'-terminal nucleotide sequence of the amplified KLF4 fragment was confirmed to extend additional 8 bp of CGTATCGA to 5' end of KLF4 mRNA of NCBI (NM_001105385). Among the 8 extended nucleotides, two adjoining ones of GA at 3' end exactly matched with counterpart sequence in 5' end region of KLF4 gene of bovine genome, which indicated a new TIS of KLF4 gene to be found in Chinese native breeds. Under the same experimental conditions, short RACE fragment (<500 bp) was well achieved by PCR, while long RACE fragment (>1000 bp) suffered failed amplification in both kit method and optimized method. In summary, the optimized RACE method was proved to apply to adipose tissue, by which, a new TIS of KLF4 was successfully identified at 2 bp upstream of the 5’end of published KLF4 sequence of NCBI in Jiaxian Red Cattle, implying the genetic specificity of Chinese cattle breeds, and provides a reference for further studying KLF4 gene function in fat deposition.

Northern leaf blight caused by the heterothallic ascomycete Exserohilum turcicum, it is an important foliar fungal disease that affects corn (Zea mays) and sorghum (Sorghum bicolor) production in China. To select suitable reference genes for quantitative real-time PCR (qRT-PCR) in E. turcicum, the elongation factor-1α (EF-1α), nascent-polypeptide-associated complex alpha polypeptide (NACA), 60S ribosomal protein L2 (RPL2), 60S ribosomal protein L37a (RPL37A), ubiquitin-conjugating enzyme (UBC), α-tubulin (TUBA), actin (ACT) and β-tubulin (TUBB) genes were served as candidate reference genes, and the samples from 4 different isolates of E. turcicum, three different stages of development, four different periods of infection in vivo, and 4 different times under difenoconazole stress were used as experimental materials in this study. The qRT-PCR technique was used to measure the expressions of candidate reference genes of E. turcicum under different conditions as mentioned above, and the analytical methods of geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), Delta-CT, and RefFinder (http://blooge. cn/RefFinder/) were used for comprehensive assessment the expression stability of candidate reference genes. Meanwhile, the expression pattern of lanosterol 14α -demethylase (CYP51) gene of E. turcicum under difenoconazole stress was analyzed using the screened most stable and least stable reference gene, respectively. The results showed that the amplification efficiencies of the 8 candidate reference genes ranged from 90.3% to 97.8% (r>0.99), with the melting curve peak being a single peak, which met the requirements of amplification efficiency in qRT-PCR. TUBA and TUBB were suitable for reference genes among different isolates of E. turcicum. RPL2 and TUBA were steadier reference genes at different stages of development. NACA and TUBA were more ideal reference genes for different stages of infection in vivo. ACT was appropriate for reference gene at different times under difenoconazole stress. qRT-PCR analysis indicated that the expression of CYP51 gene of E. turcicum significantly increased over time under difenoconazole stress, with the highest expression at 24 h, while CYP51 was significantly down expressed at 36 h (P<0.05). When a more stabile gene of ACT was used as the internal reference gene, the expression of CYP51 gene of E. turcicum was significantly up-regulated under difenoconazole stress compared with those of the non-stress group (P<0.05). However, an opposite result of the CYP51 gene expression level of the 2 isolates was obtained when the unstable TUBB gene was served as the reference gene. This study affords an effective correction tool for quantitative analysis of gene expression in E. turcicum, ensures the accuracy of quantitative analysis of gene expression, and provides a reliable reference for further study on the overexpression of fungicide resistant genes in E. turcicum.

TypeⅢsecretion system (T3SS) is ubiquitous transport machinery in Gram-negative bacteria and an important way for bacterial virulence factors to produce pathogenic effects. Ara-C type DNA binding protein ExsA is one of the main activators of T3SS and can regulate the expression of T3SS-related proteins. In this study, ExsA gene of Pseudomonas plecoglossicida PQLYC4 strain was cloned into the prokaryotic expression vector pET28a (+). The recombinant expression plasmid pET-28a-ExsA was transformed into competent cells of Escherichia coli BL21 (DE3) for induced expression. The results showed that the molecular mass of the expressed recombinant ExsA protein was slightly less than 30 kD, which was consistent with the expected molecular mass (29.7 kD). The purified recombinant ExsA protein was used as an antigen to immunize rabbits (Oryctolagus cuniculus) to prepare polyclonal antibodies, and the titer was above 1∶256 000. The prepared antibody were used to detect ExsA protein in P. plecoglossicida and the over expressed ExsA protein in the epithelioma papulosum cyprini (EPC) of carp (Cyprinus carpio) by Western blot and indirect immunofluorescence methods, the results showed specific reactions. This study provides a tool for further exploring the structure of ExsA protein and its function in regulating the virulence factor T3SS system of P. plecoglossicida.