As an important aquatic vegetable in China, Jiaobai was the swollen stem of Zizania latifolia induced by hyphal infection of Ustilago esculenta. Resistance to pseudomonas maculicola 1 (RPM1), as a disease resistance related gene, can recognize the invasion signal of pathogens and activate the defense response of plants. In this study, 2 genes, ZlRPM1.1 (GenBank No. KP 729626) and ZlRPM1.2 (GenBank No. KP 729627) in Jiaobai responding to hyphal infection of U. esculenta were cloned by the methods of rapid amplification of cDNA ends (RACE). Alignment analysis of amino acid sequence predicted that the 2 genes belonged to CC-NBS-LRR (coiled coil-nucleotide binding site-leucine rich repeat) genes related to plant disease resistance, and were similar to the genes in Oryza sativa as high as 61% (ZlRPM1.1) and 79% (ZlRPM1.2), respectively. After artificial infection of U. esculenta in the plantlets of Z. latifolia, qPCR analysis of the stem showed that hyphae infection of U. esculenta could significantly induce the enhanced expression of ZlRPM1.1 and ZlRPM1.2 in Z. latifolia. The expression of the 2 ZlRPM1 genes among different swollen stems showed that the grey Jiaobai had significantly higher expression than that in normal Jiaobai, which might be related to the ability of hyphae infection between MT and T type of U. esculenta. During the development of swollen stem in normal Jiaobai, the expression of the 2 ZlRPM1 genes were significantly induced at the stem of mid- and late development stages. The highest expression was found at the swollen stem which was expanded to the length of 15 cm. There was significantly increased expression of ZlRPM1.2 gene in the initial swollen stem at the stage of 8-leaf, following with the higher expression in the stem than that in the leaf. However, no significant increased expression of ZlRPM1.1 was detected at initial stage of stem swelling, and the expression in the leaf was significantly higher than that in the stem. Above results indicate that both the 2 ZlRPM1 genes were involved in the formation of swollen stem induced by the infection of U. esculenta, and the ZlRPM1.2 gene might be an important regulatory gene involved in the response of stem in Z. latifolia to hyphal infection. The present study provides a reference for further investigation on the regulation mechanism of Z. latifolia plants in response to hyphal infection and stem swelling.

Plant growth-promoting bacteria (PGPB) with low-temperature tolerance are beneficial for developing biofertilizers used in cold environments. To obtain cold-adapted PGPB resources, strains with nitrogen fixation and phosphorus solubilization abilities were screened from 2 leguminous species, Melissitus ruthenica and Oxytropis ochrocephala, which are the dominant leguminous plants grown in the alpine grasslands of Qilian Mountains. They were isolated from the root and rhizosphere of plants using selected medium. Other growth-promoting characteristics, including secretion of indole-3-acetic acid (IAA), production of siderophores and ACC deaminase, were further studied by qualitative and quantitative methods. 16S rRNA PCR-restriction fragment length polymorphism (PCR-RFLP) and 16S rRNA gene sequencing were used to identify the strains. Then the excellent strains were selected to verify their effects on Elymus nutans at low temperatures. Elymus nutans are the main reseeding forage specie of natural grassland in this area. As a result, a total of 87 isolates were screened, of which 45 strains still exert high activity at 4 ℃. The inorganic phosphate solubility was negatively correlated with pH. The lower the pH was, the greater the inorganic phosphate solubility, but there was no linear relationship. The genetic diversity of PGPB in the rhizosphere and root of the two legumes was not rich. They were divided into 5 genera by 16S rRNA gene sequencing and phylogenetic analysis, of which Pseudomonas were the dominant genera, and 63.22% of them had two or more plant growth-promoting characteristics. The inoculation of Elymus nutans. with cold-adapted PGPB possessing different functional characteristics had a significant promoting effect on both the development of root system (root length, root average diameter, root surface area, root dry weight, and the number of root tips) and aboveground parts (aboveground stem height, stem diameter, and stem dry weight), especially the nitrogen-fixing and IAA secreting strains. The cold-adapted PGPB have potential applications for agriculture stock production of cold areas, which is of great significance to the development of microbial fertilizer suitable for the near-natural restoration of degraded alpine grasslands on the Qinghai-Tibet Plateau.

Ethylene responsive factors (ERFs) belong to the AP2/ERF (APETALA2/ethylene responsive factor) superfamily, which are one of the largest transcription factor families in plants. Previous studies have shown that ERFs are involved in a variety of biological processes in plants, such as growth and development, signal transduction, biotic, abiotic stresses and so on. In this study, in order to study the function of potato (Solanum tuberosum) StERF109 gene (GenBank No. XM_006355301.2), bioinformatics methods were used to analyze the StERF109 protein, and the subcellular localization fusion expression vector StERF109-EGFP was constructed to locate the StERF109 gene. The tissue-specific expression of StERF109 was analyzed using qPCR technology. The results showed that StERF109 contained a typical AP2 domain, which was a hydrophilic protein without transmembrane structure. StERF109 was located on the nucleus and cell membrane. StERF109 gene had the highest expression level in potato roots, but there were differences in the expression level in potato stems and leaves of different potato varieties. StERF109 gene was up-regulated under drought and salt stress, which indicated that StERF109 gene might played an important role in drought and salt stress. The results of the study provide a reference for further research on the function of potato StERF109 gene.

Flavonoid-3'-hydroxylase (F3'H) is the key enzyme in the second stage of the anthocyanin synthesis pathway, it is very important in the anthocyanin synthesis pathway. In order to explore the formation mechanism of F3'H in the formation of anthocyanins in Vitis amurensis, the F3'H gene and its promoter sequence were cloned from the 'shuanghong' variety of V. amurensis, and the bioinformatics and prokaryotic expression analysis were performed on it, and the different length deletion fragment promoters of F3'H gene were transformed into tobacco (Nicotiana tabacum) leaves for transient expression. The results showed that the ORF of the F3'H gene was 1 530 bp in length and the promoter sequence was 1 051 bp in length. Bioinformatics analysis showed that the composition of the 'Shuanghong' variety F3'H was different from that of the 'Shuangfeng' variety in isoelectric point, molecular weight and secondary structure. The prediction of promoter cis-acting elements showed that in addition to the core promoter elements CAAT-box and TATA-box, there were a series of light-responsive elements, stress elements, hormone response elements in the promoter sequence. The transient expression results of different length deletion fragment promoters transformed into tobacco leaves showed that as the length of the deletion fragment promoter increases, the GUS enzyme activity of the F3'H promoter showed an upward trend. Through the construction of prokaryotic expression vector, it was found that the F3'H recombinant protein had a relative molecular weight of the predicted recombinant protein in the supernatant sample under the conditions of 30 ℃, culture time 11 h, and isopropyl β-D-thiogalactoside (IPTG) concentration 0.8 mmol/L, expression bands with consistent mass and high concentration. This study provides basic data for further study on the regulation mechanism of Vitis amurensis F3'H gene in the process of anthocyanin synthesis and the transcription mechanism of promoter.

Genomic imprinting is an important epigenetic modification mechanism in mammals, which is closely related to the growth and development of organisms. The ATP10A (ATPase phospholipid transporting 10A) located at one end of the PWS/AS imprinted region, which is related to the development of human (Homo sapiens) PWS (Prader-Willi syndrome) and AS (Angelman syndrome) syndromes. ATP10A is a brain-specific imprinted gene in human, but its imprinting status in cattle (Bos taurus) is still unknown. In this study, genomic DNA from 32 bovine heart tissues and 15 placentas were amplified by PCR first, and the single nucleotide polymorphic sites were found on ATP10A gene by direct sequencing of PCR products, the A/G heterozygous SNP locus (rs136134264) was identified on exon 20 of bovine ATP10A gene. Then total RNA in tissues and placenta of heterozygous cattle were used for RT-PCR to determine the allele expression status of ATP10A gene. The sequencing results of PCR products were compared with above heterozygous SNP locus, it was determined that ATP10A gene was monoallelic expression in bovine brain tissues and biallelic expression in other tissues and placenta. The DNA methylation status of the promoter region of ATP10A gene in bovine placenta and adult bovine tissues was further analyzed by bisulfite sequencing. It was found that the 46 CpG loci in the detected region showed hypomethylation in bovine tissues and placenta, indicating that the DNA methylation modification in the promoter region of ATP10A gene might not be involved in regulating imprinted gene expression of ATP10A. The present study provides a reference for further exploring of bovine ATP10A gene function and imprinting mechanism.

As an important gene regulatory element, microRNA (miRNA) is expressed in various mammalian skin and hair follicles, regulating the growth and development of skin and hair at post-transcriptional level. The aim of present study was to analyze and identify miRNAs in the skin tissues of Tibetan cashmere goats, and explore the role and regulatory mechanisms of miRNAs in fiber diameter traits of Tibetan cashmere goats (Capra hircus). Four Tibetan cashmere goats with fineness cashmere (average fiber diameter (12.04±0.03) μm) and 4 goats with coarse cashmere (average fiber diameter (14.88±0.05) μm) were selected, respectively. Shoulder skin tissues were collected to extract RNA and established libraries for high-throughput sequencing. After the raw sequences were filtered and blasted with reference genome (ARS1), the miRNAs and other small RNAs were identified based on the known miRNAs in miRBase and the location information of other non-coding RNAs (ncRNAs) in the reference genome. The differentially expressed miRNAs in the skin tissues of Tibetan cashmere goats with different fiber diameters were screened out, and target-gene prediction and function analysis were performed. qPCR was used to detect the consistency of 5 miRNAs with the sequencing results. The results showed that each sample obtained at least 20.09 M raw reads and at least 19.46 M clean reads after filtering. A total of 426 known miRNAs and 119 novel miRNAs were identified. Twelve differentially expressed miRNAs were screened, of which 5 miRNAs were significantly up-regulated in the skin tissues of the goats with fineness cashmere when compared with that of coarse group, and 7 miRNAs were significantly down-regulated. Differentially expressed miRNAs were predicted to obtain a total of 621 target genes. KEGG enrichment analysis showed that the target genes were enriched in biological pathways such as B cell receptor signaling pathway, FcεRI signaling pathway, and T cell receptor signaling pathway. Meanwhile, qPCR results showed that the expression trends of selected 5 miRNAs were consistent with that of the sequencing results. The above results suggests the fineness of Tibetan cashmere might be regulated by different miRNAs, which further affecting the expression of target genes and simultaneously participating in the regulation of multiple signaling pathways. The present study provides a reference for deep researches on miRNAs and related signaling pathways regulating the fineness of cashmere.

Neuregulin-4 (NRG4) is a cytokine highly expressed in mammalian brown adipose tissue. NRG4 is involved in the regulation of energy balance and glucolipid metabolism through the activation of ErbB4 signaling pathway. To date, due to the lack of brown adipose tissue in chicken (Gallus domesticus), the tissue expression and function of NRG4 gene in chickens have not been investigated. In this study, the full-length coding sequence of chicken NRG4 gene was amplified by PCR from the cDNA of chicken adipose tissue, and cloned to yield the NRG4 expression vector. Expression profiling of NRG4 gene was performed using qPCR on Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Four promoter fragments of NRG4 gene were amplified by PCR from the chicken genomic DNA, cloned, sequenced and analyzed by bioinformatics. A series of NRG4 promoter reporters were constructed and their promoter reporter activities were determined and compared. The results showed that chicken NRG4 gene encoded a protein of 112 amino acids and shared low amino acid identity with human (Homo sapiens) and mouse (Mus musculus) NRG4, 57.76% and 52.59%, respectively. Tissue expression profiling analysis showed that NRG4 gene was widely expressed in various chicken tissues, with the highest expression in the kidney and the lowest expression in the spleen. Expression analysis of NRG4 gene in NEAUHLF showed that NRG4 gene expression in crop fat, abdominal fat and gizzard fat was significantly higher in the fat line than that in the lean line (P<0.01 or P<0.05). Reporter gene assays showed that compared with the empty vector pGL3-basic, pGL3-NRG4 (-2854/-2076) and pGL3-NRG4 (-1451/+117) had high promoter activity (P<0.01 or P<0.05). This study paves the way for further studies on the role and mechanism of chicken NRG4 in adipogenesis and adipose tissue development.

Inosine monophosphate (IMP), as an important material basis of meat flavor, has become an important index to evaluate the umami taste and freshness of meat. In order to explore the role of key regulatory genes in the process of inosine monophosphate deposition in muscle tissue of Jingyuan chicken (Gallus gallus) and Pudong chicken, and to provide theoretical basis for the study of muscle quality among breeds, in this study, based on IMP, inosine, and some physical indexes of Jingyuan chicken and Pudong chicken, the differentially expressed genes related to meat quality among different breeds were screened by transcriptome sequencing, and the expression differences of cyclin A1 interacting protein (protein interacting with cyclin A1, PROCA1) and chicken bile toxin 1 (axis inhibition protein 1, AXIN1) gene in breast muscle tissues of Jingyuan chicken and Pudong chicken were detected by the qPCR method. The results showed that the IMP content of Jingyuan chicken was higher than that of Pudong chicken, and the IMP content of breast muscle of Jingyuan chicken and Pudong chicken was significantly higher than that of leg muscle (P<0.05). The results of transcriptome sequencing showed that the differential genes PROCA1 and AXIN1 among different varieties were screened in glycerol phospholipid metabolism pathway, arachidonic acid metabolism, and Wnt signal pathway. By comparing the gene expression of different breeds, it was found that the expression of PROCA1 and AXIN1 genes in breast muscle of Jingyuan chicken was higher than that of Pudong chicken, and the trend of differential expression was consistent with that of transcriptome sequencing. In summary, PROCA1 and AXIN1 genes may be candidate genes that affect the specific deposition of inosinic acid in the muscle tissue of Jingyuan chicken and Pudong chicken. The results of this study provide a reference for further exploring the regulation mechanism of IMP-specific deposition of PROCA1 and AXIN1 genes.

Chemerin is an adipocytokine secreted by adipose tissue, which can regulate biological processes such as adipocyte generation and metabolism. In this study, the total RNA of chicken (Gallus gallus) liver tissue was used as the template, and the full-length Chemerin cDNA sequence of chicken was amplified by reverse transcription PCR (RT-PCR) and rapid amplified cDNA ends (RACE). qPCR was used to detect the expression of Chemerin gene in different tissues. The eukaryotic expression vector of Chemerin-Myc fusion protein was constructed. The transcription efficiency of chicken Chemerin gene promoter was assessed by dual luciferase reporter vectors with various promoter fragments. The results showed that the full length of the cDNA was 733 bp, encoding 162 amino acids, including a 55 bp 5'UTR and a 189 bp 3'UTR. Chemerin gene was mainly expressed in liver, kidney and adipose tissue. The eukaryotic expression vector pCMV-3Tag-9-gga-chemerin-Myc was successfully constructed. After transfection into chicken Leghorn male hepatoma (LMH) cells, fusion protein of Myc and Chemerin was detected by Western blot. The 4 constructed pGL3b-chemerin-F1/F2/F3/F4 vectors all showed that the luciferase activity was very significantly increased compared with the control group (P<0.01), and the promoter transcription efficiency of F1 and F3 was the highest. The luciferase activity of pGL3b-chemerin-mutF1 was very extremely significantly decreased after the site-directed mutation of Kruppel-like factor 5 (KLF5) binding site in pGL3b-chemerin-F1 (P<0.001). After the interference of KLF5 gene expression in LMH cells, the mRNA expression of Chemerin gene significantly decreased (P<0.01), which implied that there was a cis-element with positive regulator KLF5 binding in F1 fragment. These results provide basic data for further study on the function of Chemerin gene in chicken.

Growth differentiation factor 9 (GDF9), a growth factor secreted by oocytes, is involved in the regulation of ovarian development in mammals. In order to study the SNPs of GDF9 gene and find out the molecular markers related to laying performance of Jinding ducks (Anas platyrhynchos domestica), in this experiment, the direct sequencing method was used to detect GDF9 SNPs in 458 female Jinding ducks, and the correlations between different genotypes of each polymorphic site and the age at first egg as well as egg number at 300 d old were analyzed. The results showed that a total of 10 mutation sites were found in exon 1 and exon 2 of GDF9 of Jinding ducks. Among the mutation sites, 6 sites were missense mutation sites, and all sites had 3 genotypes; genetic analysis showed that 7 loci were moderately polymorphic (0.25<PIC<0.5), Hardy-Weinberg balance test showed that loci C344T and G557A were in equilibrium (P>0.05); Linkage disequilibrium analysis showed that there was no complete linkage disequilibrium among the 10 loci. Analysis on the correlation between polymorphic loci and laying performance indicated that sites C2111T and G2261T were significantly correlated with egg numbers at 300 d old (P<0.05), and sites T2345C and C2390T were significantly correlated with the age at first egg (P<0.05). In summary, these loci of GDF9 gene could be used as genetic markers for laying performance selection in Jinding ducks. The results may provide a reference for marker-assisted selection.

Estradiol (E2) plays an important role in regulating the growth and development of granulosa cells.To explore the effect of estradiol (E2) at different concentrations on Goose (Anser cygnoides domestica) hierarchical follicular granulosa cells (HFGCs) and its molecular mechanism, in this study, the grade granulosa cells of 'Tianfu' geese were randomly divided into experimental group (E5, E7, E9) and control group (NC), the granulosa cells of experimental group were treated with different concentrations of estradiol (1×10^-5, 1×10^-7 and 1×10^-9 mol/L). The samples were collected for MTT assay, oil staining assay and transcriptome sequencing analysis. MTT results showed that the cell activity of the experimental group was significantly higher than that of the control group (P<0.05); Oil red staining results showed that with the decrease of E2 concentration, the lipid deposition of granulosa cells decreased, and the difference was significant when E2 concentration was 1×10^-9 mol/L (P<0.05); By transcriptome sequencing analysis, E5, E7, E9 were compared with NC under the condition of fold change (FC)>1.2 or <0.83 and P<0.05, 267, 79 and 162 differentially expressed genes were screened, respectively; KEGG enrichment analysis showed that the differentially expressed genes of E5, E7, E9 and NC were significantly enriched in sphingolipid metabolism, insulin signaling and Wnt signaling, ABC transporter and other pathways, respectively. 3-ketodihydrosphingosine reductase (KDSR), ceramide synthase 6 (CERS6) and ceramide synthase 4 (CERS4) in sphingolipid metabolism pathway, mitogen-activated protein kinase 8 (MAPK8), inositol polyphosphate phosphatase like 1 (INPPL) and sclerostin (SOST) in insulin and Wnt signaling pathway were involved in regulating granulocyte activity; Galactose-3-O-sulfotransferase 1 (GAL3ST1), sphingosine-1-phosphate lyase 1 (SGPL1), ATP binding cassette subfamily A member 2 (ABCA2) and ATP binding cassette subfamily A member 5 (ABCA5) in sphingolipid metabolism and ABC transporter pathway were involved in regulating granulosa cells lipid deposition when E2 concentration was 1×10^-9 mol/L. The results showed that exogenous E2 treatment could improve the activity of goose granulosa cells and inhibit lipid deposition by affecting sphingolipid metabolism, insulin signaling, Wnt signaling and the expression levels of related genes in ABC transporter pathway. The results lay a foundation for exploring the molecular mechanism of estradiol regulating the growth and development of goose granulosa cells.

As key immune protein in innate immune mechanism of crucian carp (Carassius auratus), c-type lysozyme is a good candidate for the development of green feed additives or natural antimicrobial agents. In present study, the cDNA encoding crucian carp c-type lysozyme was cloned by reverse transcription PCR (RT-PCR). Subsequently, 3 times of PCR were performed to add the XhoⅠrestriction endonuclease site, Kex2 signal peptidase cleavage site and a 6×His tag to its 5' end, and the XbaⅠrestriction endonuclease site and stop codon to its 3' end; the obtained target gene was named CrCLm (crucian carp c-type lysozyme mature peptide). Using pPICZɑA as expression vector and Pichia pastoris X-33 as engineering strain, the recombinant P. pastoris strain X-33/pPICZɑA-CrCLm was constructed. After the recombinant strain was induced at initial pH 6.0, 28 ℃ and 250 r/min for 96 h with 1.0% methanol, the obtained recombinant protein was purified by a nickel ion affinity chromatography column. The purified protein was used for Western blot and LC-MS/MS analysis. On the other hand, the antibacterial activity and stability for the recombinant protein were determined. The results indicated that under above fermentation conditions, the recombinant protein with a yield of 18.3 mg/L was obtained, and it was identified by LC-MS/MS analysis as expected recombinant CrCLm with a molecular weight of 14.5 kD. The results of antibacterial test showed that the recombinant CrCLm had obvious activity against Gram-positive Staphylococcus aureus, Bacillus subtilis, B. cereus and Listeria monocytogenes, and Gram-negative Escherichia coli, Salmonella, Vibrio parahaemolyticus and Pseudomonas aeruginosa. In addition, different acidic environments, high temperature treatment and hydrolysis of proteinase (such as trypsin, pepsin and proteinase K) did not affect its antibacterial activity. The recombinant P. pastoris strain constructed in the present study can effectively synthesize CrCLm with obvious antibacterial activity and good stability, which could provide technical support for its large-scale preparation and application.

Phenoloxidases (POs) are important immune effector molecules of insect and belong to the type 3 copper protein familywith oxidoreductase activities. Generally, PO is present as a zymogen, prophenoloxidase (PPO), which is activated by a serine protease cascade upon recognition of foreign invaders, and then catalyzes both melanin biosynthesis and the oxidation of phenols to quinones. Gomphocerus sibiricus is dominant pest in alpine and subalpine grassland areas in Xinjiang. The study of immune related gene of G. sibiricus can lay a foundation for the future study of its interaction with pathogenic organisms to carry out biological control. The full length cDNA of PPO2 were cloned by RACE technology in this study, and which consisted of 2 439 bp (GenBank No. KY981766). Bioinformatic analysis indicated that PPO2 is highly similar to Locusta migratoria and Diabolocatantops pinguis. It contained 2 common copper-binding regions, 2 potential proteolytic activation sites, a plausible thiolester site, and a conserved C-terminal region. Genetic expression analysis showed that the gene expression level was the highest inhindgut of G. sibiricus. The expression level in posterior intestine was significantly higher than midgut, and there were no difference between salivary glands and malpighian tubules, the expression was least in fat body. This study could provide a theoretical basis for further study of the function of PPO2 gene in G. sibiricus.

Southern corn leaf blight (SCLB) caused by Cochlibolus heterostrophus is an important foliar fungal disease that significantly affects corn (Zea mays) yield and quality. To determine the population genetic structure and pathogenicity of C. heterostrophus populations from sweet corn in different geographical locations, inter-simple sequence repeat (ISSR) molecular markers and pathogenicity assays were used for analysis the population genetic structure and pathogenicity of 7 C. heterostrophus populations (Nanping, Ningde, Fuzhou, Sanming, Putian, Longyan, Zhangzhou) from sweet corn in Fujian province. A total of 198 loci were detected using optimized 13 ISSR primers, and the percentage of polymorphic DNA loci (P_L) were 100.0%, with the highest level of DNA polymorphism being detected from Fuzhou population (P_L=73.7%) and the lowest level of DNA polymorphism being detected from those in Longyan (LY) and Zhangzhou (ZZ) populations (58.6% and 56.6%). ISSR marker results indicated that a total of 126 multilocus haplotypes were detected among C. heterostrophus populations from the 7 geographical locations in Fujian province, with no shared multilocus haplotypes being detected from these populations. The most abundant multilocus haplotype diversity (H_S) was detected from Nanping (NP) population (H_S=0.323), whereas the lowest diversity was detected from those in LY (H_S=0.157). A low genetic differentiation was detected between C. heterostrophus populations from NP and Ningde, Putian and Sanming, and LY and ZZ (Φ_PT<0.088), with the frequent gene flow being detected between these populations (Nm>5). However, moderate to high genetic differentiation was detected among populations in the other geographical locations. One hundred and twenty-six multilocus haplotype isolates from sweet corn in Fujian province were divided into 8 genetic clusters at genetic similarity coefficient of 0.768, with different isolates originated from the same location clustering together. The analysis of molecular variance (AMOVA) of 7 C. heterostrophus populations indicated that approximately 81.9% and 18.1% of the total variation occurred within and among populations, respectively. This result revealed that the major source of genetic variation in Fujian C. heterostrophus population was derived from within populations. The principal coordinates (PCoA) and population structure analysis showed that Fujian C. heterostrophus population could be divided into 2 genetic groups. Pathogenicity assays indicated that the 7 regional populations of C. heterostrophus had higher pathogenicity to 4 resistant and susceptible sweet corn cultivars, with the average disease index ranging from 36.42 to 59.23. Analysis of variance showed highly significant differences in pathogenicity among isolate, location, and cultivar, respectively (P<0.001). Nevertheless, no significant differences in pathogenicity among isolate×cultivar (P=0.999) and location×cultivar (P=0.361) interactions were detected at the level of 99%. This study provides a theoretical reference for the further investigation of genetic variation of C. heterostrophus and the resistance breeding efforts.

Cotton verticillium wilt caused by Verticillium dahliae is an important soil-borne fungal vascular disease threatening cotton production in China. In previous studies, the transcriptional expression of a gene encoding hypothetical protein in the virulent defoliating V. dahliae V592 strain isolated from cotton (Gossypium hirsutum) was dramatically down-regulated when V592 strain was cultured with cotton roots. To determine the function of the gene, the full length of the gene was cloned from the genome of V592 strain. The target gene knockout plasmid was constructed based on homologous recombination, and its complementation plasmid containing the full length of the target gene encoding region was also constructed, then to generate the target knockout mutant and its complementary strain by Agrobacterium-mediated transformation. The growth phenotype characteristics and pathogenicity to cotton of the gene knockout mutant were assayed. The transcriptional expression of 4 genes involved in virulence in the target gene knockout mutants were measured by reverse transcription qPCR (RT-qPCR). The results showed that the target gene full length was determined to be 1 439 bp and the deduced protein contained 6 tandem Kelch motif and a galactose oxidase domains, and Kelch repeats formed a 6-bladed propeller construction. Thus, the gene was designated as VdKeR (GenBank No. MZ855770) due to the encoding protein containing Kelch repeats. On potato dextrose agar (PDA) media, VdKeR knockout mutant produced more aerial hyphae on the edge of the colony than the wild-type strain V592 and complementary strain, but the colony growth rate of VdKeR knockout mutant exhibited obviously lower than those of V592 strain and complementary strain, indicating that VdKeR gene knockout resulted in increased formation of aerial hyphae and slower growth rate in V. dahliae. VdKeR knockout mutant produced significantly less conidia than those of V592 strain and the complementary strain, suggesting that VdKeR gene knockout affected the conidiation in V. dahliae. In cotton seedling, VdKeR knockout mutant displayed significantly reduced disease severity compared with V592 strain and complementary strain, showing that VdKeR gene knockout led to reduced virulence to cotton. In VdKeR deletion mutants, the other 4 genes involved in virulence including the C subunit gene of cAMP-dependent protein kinase A (VdPKAC1), the G protein β subunit gene (VGB), the necrosis-and ethylene-inducing-proteins (Nep1)-like protein (NLP) family genes VdNLP1 and VdNLP2, the transcriptional expression of which were strongly down-regulated. These results indicated that VdKeR gene played an important role in the growth and pathogenicity and affected the transcriptional expression of other genes involved in virulence in V. dahliae. The results provide basic data for further study on the pathogenic mechanism of Verticillium dahliae.

Long non-coding RNAs (lncRNAs) are now considered as central regulators in normal biological activites and critically involved in the tumorigenesis. The maternally expressed gene 3 (MEG3) is located in DLK1-DIO3 imprinted region on human chromosome 14q32.2. MEG3 gene is composed of 34.9 kb size and encodes an approximately 1.6 kb lncRNA. The MEG3 is either lost or down-regulated in a many primary human tumors and tumors derived cell lines, and exhibits the function of tumor suppressor. MEG3 controls the expression of some tumor suppressor genes and oncogenes, such as p53, RB, MYC and TGF-β. MEG3 also affects epithelial-mesenchymal transition through regulating the Wnt-β-catenin signaling pathway. In addition, the methylation modification and polymorphic sites within the MEG3 locus are also associated with cancer risk of and drug therapeutic response of patients. In this review, several aspects of MEG3 participating in tumorigenesis, and the potential value in cancer diagnosis and prognosis are discussed. This article provides a basis for the follow-up study of the role of MEG3 in human tumor therapy.

Ustilago maydis is a well-established model system for biotrophic fungal plant pathogens. Domesticated maize and its wild progenitor teosinte (both Zea mays) are its obligated host plant. U. maydis establishes a comprehensive relationship with their host to support the infection process. Central to this strategy is to secrete an arsenal of protein effectors, which can suppress plant defense responses, modulate host metabolism, or induce plant tumors to facilitate the growth and development of the pathogen. Since the functional redundancy and the lack of recognizable domain, it was very difficult for effector identification and functional study. With the development of next-generation sequencing, comparative transcriptome and comparative genome provide an excellent chance for effector research. In this review, we discuss new insights into the biological function, transcriptional regulation, and post-translation modification of effectors. Highlight its functions in a preventing cell wall reinforcement, suppressing reactive oxygen species (ROS) accumulation and programmed cell death, regulating hormone metabolism, and inducing plant tumors. This paper is helpful to understand the infection strategy of biotrophic fungi and identify relevant effectors.

Cotton (Gossypium hirsutum) is an important cash crop, by the end of 2019, 76% of the total cotton acreage was cultivated on genetically modified (GM) cotton globally. GM cotton event COT102 is a transgenic cotton event with insect resistance developed by Syngenta, Switzerland. This event has been approved by the Ministry of Agriculture of China, but has no detection method in relevant standards in China. In order to ensure the development of cotton industry in China and the orderly conduct of import and export trade, it is very important to establish an effective detection method for GM cotton event COT102. In this study, multiple sets of primers and probes were designed according to the exogenous inserts and adjacent sequences of genomic DNA of GM cotton event COT102 based on duplex real-time PCR technology. After screening and optimizing the reaction conditions, the proportion of primers and probes and other indicators, a duplex real-time quantitative PCR method was developed for the detection of GM cotton event COT102. The specificity, sensitivity and reproducibility of the method were measured. The results showed that the method was specific for the detection of GM cotton event COT102, without any nonspecific amplification. The sensitivity of the method was high, and the lower limit of detection and quantification was as low as 5 copies of cotton genomic DNA. The deviation between repeats of multiple PCR amplification was small and reproducibility was good. The results showed that the bias between the measured value and the expected value was between -0.42% and 4.63%. The standard deviation (SD) was less than 0.5, and the relative standard deviation (RSD) was less than 25%, both were within the acceptable range. Therefore, the duplex real-time quantitative PCR method established in this study was suitable for qualitative and quantitative detection of the components of GM cotton event COT102 in cotton and its products. The high-throughput detection method could effectively improve the detection efficiency, reduce the detection cost, and improve the customs clearance efficiency. This method provides effective technical support for the effective supervision of imported GM cotton and its products, and promotes the implementation of GM product labeling policies in China, meanwhile provides necessary technical support for the smooth and orderly conduct of import and export trade.

Wheat leaf rust is one of the most serious diseases of wheat (Triticum aestivum). It is of great significance to create single-base mutants of Puccinia triticina (Pt) for the study on virulence genes and pathogenic mechanisms. Therefore, ethyl methyl sulfonate (EMS) was used to mutate the Pt. It was effective to inhibit the germination of Pt urediniospore by treating with 0.04 mol/L EMS for 8 min, and the inhibition rate reached 83.69%. Nineteen mutant isolates of 2008-5-9-2 (KHHT), 2013-5-72-1 (THSN), 2013-5-28-1 (JHKT) and 2017-8-1465 (PHST) induced by EMS were obtained, and the mutant rate was 95%. The infection types of the mutant strains were detected on 18 wheat leaf rust resistance near-isogenic lines (cultivars). It was found that the infection types of the mutants changed on 15 near-isogenic lines. Strain Mu2008-5-9-2② was virulent to leaf rust resistance gene 24 (Lr24) and strains Mu2013-5-28-1④, Mu2008-5-9-2②, Mu2008-5-9-2④, Mu2008-5-9-2⑤, Mu2013-5-28-1③, Mu2013-5-28-1⑥, Mu2013-5-28-1⑦, Mu2013-5-28-1① and Mu2013-5-28-1② were avirulent to Lr2a, Lr14a, Lr16, Lr17 and Lr33. The results provide basic materials for the analysis of mutant loci in leaf rust and the cloning of avriulence gene of Lr24, Lr2a, Lr14a, Lr16, Lr17 and Lr33 in future.