Plant heat shock transcription factors (Hsfs) plays a central role in thermotolerance, transgenerational thermomemory and many stress responsive processes. In this paper, a new gene TaHsfA2-12 (GenBank No. MK931301) was isolated from young leaves of wheat (Triticum aestivum) treated under heat shock (HS) at 37 ℃ for 1.5 h through homologous clone technique. The CDS length of gene TaHsfA2-12 was 1 134 bp encoding 377 amino acids residues. The amino acid sequence of TaHsfA2-12 contained a DNA-binding domain (DBD), a nuclear localization signal of RRKELAEALLSKKRGR peptide, a nuclear export signal of LLSLGLE peptide and an aromatic, large hydrophobic and acidic amino residues of ESFWKELLSL peptide. The TaHsfA2-12 protein shared 98% identities with the AtHsfA2 from Aegilops tauschii. TaHsfA2-12 protein was dyed with 4',6-diamidino-2-phenylindole (DAPI) and observed in tobacco (Nicotiana tabacum) epidermal cells under Confocal, the result revealed that the TaHsfA2-12 was localized in the nuclei under the normal conditions. qRT-PCR revealed that the gene TaHsfA2-12 constitutively expressed in different tissues of wheat under the normal conditions, such as young and mature root, shoot, leaf and pistil, stamen, sepal and embryos. The TaHsfA2-12 expression levels were lower in majority of tissues and higher in mature seed (P<0.05). TaHsfA2-12 was obviously up-regulated by heat shock (HS) and 20% PEG6000, but down-regulated by H₂O₂ and salicylic acid (SA), respectively. Phenotypes observation indicated that TaHsfA2-12 not only improved the basal thermotolerance and acquired thermotolerance of transgenic Arabidopsis seedlings, but also could rescue the thermotolerance phenotype defect of AtHsfA2 deletion mutant under HS. Chlorophyll content of different lines were consistent with phenotypes. TaHsfA2-12 could activate a suite of heat shock protein (Hsp) genes expression in transgenic Arabidopsis plants after different HS, suggesting molecular mechanism of regulating thermotolerances of TaHsfA2-12. These results will be benefited to deeply investigate the biological characteristics and functions of wheat Hsfs family members.

Glycerol-3-phosphate acyltransferases (GPAT) are rate limited enzymes that catalyze the initial step of de novo glycerolipid biosynthesis in plants. Thus, isolation and identification of GPAT encoding genes from Brassica napus will help to in-depth uncover the molecular mechanisms of glycerolipid biosynthesis and metabolism in oilseeds. In this study, 13 candidate genes, possessing conserved acyltransferase domains, were amplified by reverse transcription-PCR (RT-PCR) from B. napus, and constructed into yeast (Saccharomyces cerevisiae) heterologous expression vector yADH1-pYES2-Kan V2 (exogenous gene expression was induced by glucose, inhibited by galactose). Verification of digestion and sequencing of the recombinant yeast expression vector showed that the sizes and sequences of 13 gene fragments were as expected. According to alignment of amino acid sequences, 1~2 transmembrane domains and 4 conserved acyltransferase domains were in these BnGPATs, respectively. Then, yeast conditional lethal double knockout mutant strain ZAFU1 (BY4742, gat1Δgat2Δ+[pGAL1::AtGPAT1 LEU2]) was used to conduct genetic complementation verification. It was found that the growth of ZAFU1 could be rescued with heterologous expression of BnGPAT1-1, BnGPAT4-1, BnGPAT4-2 and BnGPAT9-1 induced by glucose, which indicated that the enzymes encoded by these genes might mimic the function of the yeast acyltransferase encoding gene YKR067w (named GAT1). The results of this study could provide reference data for the genetic improvement of oil content in B. napus.

CRISPR/cas9 technology is widely used in gene knockout. It is helpful for early screening of experimental animals to knock in fragments containing fluorescent protein and target gene. Elongation of very long chain fatty acids 5 (Elovl5) is one of key enzymes to regulate the biosynthesis of fish highly-unsaturated fatty acids (HUFAs). Constructing a transgenic animal model expressing Elovl5 will provide a new strategy to study the in vivo function of this enzyme. Common carp (Cyprinus carpio) is one of the most widely cultured freshwater fishes in our country. Although common carp Elovl5 was identified and its tissue expressional pattern and correlation to growth were studied, its elongase activity has not been investigated. To study the in vivo function of common carp Elovl5 in zebrafish (Danio rerio), a vector including Tol2 sequence, zebrafish myosin light chain 2 (Mylz2) promoter, red fluorescence gene (DsRed2), zebrafish Elovl5 promoter, common carp Elovl5 gene, 2A cleavage peptide, and green fluorescence gene, was constructed. A fragment consisting of these promoters and genes with a length of 7.5 kb was amplified from this vector and then knocked into zebrafish genome using the CRISPR/Cas9 method. From 72 h after fertilization, the red fluorescence was observed. For the transgenic zebrafish screened using the red fluorescence, PCR amplification of DsRed2 confirmed the insertion of this fragment into the genome. The result indicated that using fluorescence to screen transgenic animals was reliable, simple and practicable. Among all the surviving zebrafish, the knock-in rate was 28.7%. The study also knocked this fragment into the genome by using Tol2 system with a rate of 32.8%, which suggested that the knock-in efficiency of CRISPR/Cas9 method was lower than the Tol2 system. Compared to the control group, common carp Elovl5 was expressed only in the CRISPR/Cas9 experimental group and the Tol2 experimental group. Furthermore, its expression level was comparable between these two groups, suggesting that the inserted gene expression efficiency of the CRISPR/Cas9 method was equivalent to that of the Tol2 method. The contents of Elovl5 substrates in the CRISPR/Cas9 experimental group decreased by 18.6%. The contents of Elovl5 products had an increase of 31.8% and the C20:4n-3 fatty acid content was 2.4 times as high as that in the control group. These results indicated that common carp Elovl5 could in vivo convert the substrates into HUFAs. In this study, CRISPR/Cas9 technology was used to construct transgenic zebrafish expressing common carp Elovl5. It demonstrated that the CRISPR/Cas9 method could effectively insert the long fragments into genomes. It will be applied into the simultaneous knock-in of multiple genes from one pathway or regulating multiple traits. The HUFA contents increased in the transgenic zebrafish, demonstrating the elongase activity of common carp Elovl5. The transgenic zebrafish will be useful to study the biosynthesis mechanism of common carp HUFAs and the regulation mechanism of Elovl5.

Reference materials are the key to quality control in the detection and supervision of genetically modified organisms (GMOs). However, at present, the positive controls for GMOs detection in China are mainly matrix reference materials and it's very limited, which is difficult to meet the needs of GMOs safety supervision and detection. As a new development direction of transgenic detection reference materials, the plasmid reference materials have wide application value. In this study, genetic information of 19 major transgenic rice (Oryza sativa) at home and abroad were obtained through collecting and collating relevant databases, By analyzing the composition and use frequency of the genetic elements in their foreign gene expression frames, and combining current technology standards of transgenic detection, a total of 9 regulatory elements/genes, including 2 rice endogenous reference gene of sucrose phosphate synthase gene (SPS) and sucrose phosphate synthase gene (SPS) and 7 targets, the CaMV35S promoter (35S promoter from Cauliflower mosaicvirus, P_CaMV35S), Ubiquitin promoter (ubiquitin promoter from maize, P_Ubiquitin), CaMV35S terminator, NOS terminator (terminator of nopaline synthase, T_NOS), bialaphos resistance gene (Bar), hygromycin phosphotransferase gene (HPT) and Bacillus thuringiensis gene (Bt) were used to construct the standard plasmid pUC18-RICE-screen. This plasmid was obtained by synthetic technology, verified by enzyme digestion, sequencing, PCR amplification , and the plasmid concentration suitable for detecting different regulatory elements/genes was comprehensively evaluated. The experiment results showed that the constructed plasmid could be used for the screening detection of 19 transgenic rice involved in this research, and the suitable concentration range was 1×10³~1×10⁵ copies/μL when it was used as a positive control for each screening element/gene PCR and qRT-PCR screening detection. This study provides a positive control for the safety regulation of genetically modified rice, which does not depend on the positive-control raw materials of genetically modified rice, and also provides technical support for solving the problem of lack of positive standards for GM rice.

Fusarium head blight (FHB) is one of the main diseases which caused a serious threat to the normal growth and yield formation of wheat (Triticum aestivum; 2n=6x=42, AABBDD). Rye (Secale cereal; 2n=2x=14, RR) with developed root system, strong stress resistance and disease resistance, is an important genetic resource for improving wheat traits. For the purpose of improving the FHB resistance in wheat, Mexican rye and common wheat W770B were hybridized to obtain several derived lines with genetic stability through multi-generation breeding. At the blooming stage of wheat, single flower inoculation (SFI) was used to identify the parents and progenies resistance to FHB. The results showed that the parents rye had moderate resistance to FHB whereas W770B showed high sensitivity, and the 12-5-1 derived line showed moderate resistance. Morphological and cytogenetic identification and analysis were conducted in 12-5-1 derived line by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) sequence amplification, sequence characterized amplified regions (SCAR), simple sequence repeats (SSR) molecular markers. The results showed that the root tip cell of 12-5-1 contained 42 chromosomes (2n=42). In the first meiotic metaphase of pollen mother cell, there is a proper chromosome pairing forming 21 bivalents(2n=42=21Ⅱ), and normal separation. GISH identification was performed on 12-5-1 using rye genome as a probe. Yellow and green signals were observed at the ends of two chromosomes of wheat, indicating that there is a translocation between wheat chromosome and rye chromosome. FISH results showed that the translocation of 1BS of wheat and 1RS of rye occurred in 12-5-1. Therefore, it is inferred that the hybridization of wheat and rye resulted in translocation of small chromosome segments. Moreover, Rye specific marker AF1/AF4 and SCAR marker of 1RS were used for identification and the corresponding bands were amplified in rye and 12-5-1 derived line. SSR analysis showed that three pairs of primers (Xgwm11、Xgwm31 and Xgwm550) on the 1BS chromosome of wheat could not be used to amplify the target bands in 12-5-1, while the remaining primers could be used to amplify the corresponding target bands, which indicated that 12-5-1 was a 1RS-1BS/1BL translocation line in Rye-Wheat. The creation of the derived line with FHB resistance provides a new germplasm resource for wheat breeding against FHB resistance.

The PCR detection of transgenic lines and the in-depth analysis of T-DNA flanking genome sequence can help to understand the specific molecular characteristics of transgenic lines. Three GmWRKY70 transgenic soybean (Glycine max) lines were developed using Agrobacterium-mediated soybean cotyledonary nod transformation method. Transformation events were confirmed by PCR detections of selective marker bialaphos resistance (Bar) gene, target gene GmWRKY70, as well as vector-construction-specific fragments and the integration site of T-DNA flanking sequence was analyzed by high-efficient thermal asymmetric interlaced PCR (Hi-TAIL PCR). The results showed that all 3 GmWRKY70 transgenic soybean lines were positive. T-DNA was reversely integrated into soybean chromosome Chr07 between 43 119 699 and 43 119 712 bp. The T-DNA integration led to a 12 bp deletion at the insertion site of soybean chromosome Chr07. For the introduced T-DNA, it was found that the 80 bp sequence at the end of the left border (LB) and the 22 bp sequence at the end of the right border (RB) were truncated. Micro-homologies of 2 base pairs were found between the introduced LB end and soybean genomic DNA at the integration site, while no homology was found for RB. New integration-site-specific primers were designed to validate this transformation events and could further facilitate molecular assisted selections in breeding program.

Anthocyanins belong to a kind of flavonoid and are widely found in different plants. The biosynthesis of anthocyanin is transcriptionally regulated by MYB transcription factor. In this study, the full length of MYB type transcription factor was cloned from flesh of blood orange (Citrus sinensis), then bioinformatic analysis and differential expression of the gene were performed. Furthermore, the prokaryotic expression vector PET28-CsMYB was developed and used to induce expression of MYB recombinant protein, which was used to immune rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody. Then Western blot was used to detect MYB expression between different samples. The results showed that the full-length of MYB nucleotide sequence was 789 bp, which encoded a protein with 262 amino acids. The protein had R2 and R3 MYB conserved domains at the position of 1~56 and 57~111 of amino acids residues, so that it was designated by CsMYB (GenBank No. KT757348). Multiple alignments were performed using different homologous MYB-type proteins. The phylogenetic tree showed that CsMYB protein was clustered with Litchi chinensis, an anthocyanin biosynthesis regulator (LcMYB1), and the amino acid identity between the two proteins was 56.1%. Comparison of differential gene expression in different tissues of blood orange showed that higher transcription level was found in the flesh only. In addition, chalcone synthase (CHS) and glutathione S-transferase (GST) also exhibited higher expression levels in the flesh. The relative expression of CsMYB significantly increased during fruit development. Compared with a mutant fruit with less anthocyanin contents, the relative expression of CsMYB in the red-fleshed fruit with normal anthocyanin contents was significantly higher, and the expression change of CHS was consistent with CsMYB. Western blot results showed that a weak hybridization band at 36 kD was occurred only in the red-fleshed fruit. Taken together, CsMYB might be involved in the anthocyanin regulation of blood orange. The present study could provide a reference for further studies on fruit coloring mechanism and genetic breeding of blood orange.

Leaf color mutants are ideal materials for studying a series of physiological metabolic processes such as chlorophyll metabolism, chloroplast development, photosynthesis, and hormone physiology in higher plants, as well as important materials in genetics and breeding researches. Porphobilinogen deaminase (PBGD) is the key enzyme in the process of plant chlorophyll biosynthesis. In this study, 4 PBGD genes were isolated using mottled leaf mutant of upland cotton (Gossypium hirsutum) 'Lumian 418', and named as GhPBGD1A (GenBank No. MN849924), GhPBGD1D (GenBank No. MN849925), GhPBGD2A (GenBank No. MN849926) and GhPBGD2D (GenBank No. MN849927), respectively. The sequence analysis showed that each of the 4 GhPBGD genes had no difference in sequences between the normal and mottled leaves. All GhPBGD genes had a similar gene structure with 5 exons and 4 introns, and were predicted to be located in chloroplasts. Phylogenetic analysis indicated that GhPBGDs had close relationship with the PBGD proteins from Arabidopsis thaliana and Brassica napus. In addition, the cis-elements analysis revealed that many elements involved in light, anaerobic and hormone responsiveness were identified in the promoter regions of GhPBGD genes. Furthermore, qRT-PCR analysis showed that GhPBGD genes expressed in leaves, flowers, bolls and sepals, and had the highest expression in the leaves. Compared to normal leaves, the expression of GhPBGD genes were significantly down-regulated in the mottled leaves. The above results suggested that GhPBGD genes might be involved in the regulation of leaf color mutation. This study could provide reference data for further exploration of the molecular mechanism of leaf color deletion mutants.

Stress associated proteins (SAPs) are zinc-finger proteins containing A20 and (or) AN1 domain which are involved in abiotic stress responses in plants. In this study, a stress associated protein gene, GhSAP8 (GenBank No. XM_016813997.1), was isolated from upland cotton (Gossypium hirsutum) via homology cloning. The full-length sequence of GhSAP8 gene was 519 bp, encoding a protein of 172 amino acids with a relative molecular weight of 18.22 kD and an isoelectric point of 7.51. GhSAP8 contained 1 conserved A20 domain in the N-terminus and 1 AN1 domain in the C-terminus which was typical SAP protein domain combinations. The expression of GhSAP8 gene was identified in different tissues, and qRT-PCR detection showed that the highest expression occurred in seedling leaf and it was induced by treatment of salt which suggested that GhSAP8 gene might be involved in responses to salt stress. The GhSAP8 gene was constructed into the plant expression vector pCAMBIA1300 and transformed into Arabidopsis thaliana. Under the treatment with 150 mmol/L NaCl, the survival rate (53%~70%) of Arabidopsis with overexpressed GhSAP8 gene was significantly higher than that of the wild type (27%), indicating the overexpression of GhSAP8 gene could enhance the tolerance to salt stress. In addition, the expression of salt-stress responsive genes, such as AtP5CS1 (Δ1-pyrroline-5-carboxylate synthetase 1), AtRD29B (responsive to desiccation 29 B), AtSOS1 (salt overly sensitive 1) and AtNHX1 (sodium/hydrogen exchanger 1), were significantly higher in transgenic Arabidopsis than that in wild type under salt stress. However, only a few of transgenic Arabidopsis lines showed increased expression of AtP5CS1, AtRD29B and AtSOS1 in wild type under non-salt stress. The above results indicated that GhSAP8 gene might improve tolerance to salt stress in transgenic Arabidopsis by regulating the expression of salt-stress responsive genes. The present study could provide superior gene resource for cultivation of transgenic salt-tolerant cotton.

Plant cells are highly compartmentalized into distinct subcellular structures, which requires proper assembly of relevant protein players in the right place at the right time. The nucleus is the control center of the cell, which entails protein expression and trafficking in a highly coordinated manner. Nuclear localization signal (NLS) promotes localization of proteins into nucleus that plays an important role in studying the cell biological functions of proteins. Based on the limitations of current NLS plant expression vectors, this study has obtained a new pRI101-NLS-GFP plant expression vector by fusing NLS with GFP based on the seamless cloning. To determine the subcellular localization of GFP, pRI101-NLS-GFP was transiently expressed in Nicotiana benthamiana. NLS-GFP fusion protein accumulated strongly in the nucleus, while the fluorescence of GFP spreaded the whole cell. In the meantime, the RXLR (arginine-x-leucine-arginine) effector gene PITG_04314 (PITG: Phytophthora infestans T30-4 gene, GenBank No. XM_002905913) was used to further investigate whether the vector could successfully introduce protein into the nucleus. Previous studies have shown that PITG_04314 localizes to the nucleus with additional cytoplasmic background and functions in thenucleus. To further confirm the localization, PITG_04314 was fused with pRI101-NLS-GFP and pRI101-NLS. Consistent with previous results, GFP-PITG_04314 was localized to the nucleus with cytoplasmic background, whereas NLS-GFP-PITG 04314 accumulated strongly in the nucleus. Transient overexpression GFP-PITG_04314 and NLS-GFP-PITG_04314 could both promote the Phytophthorainfestans colonization, indicated that PITG_04314 functions in the nucleus. Taken together, this study showed that NLS-GFP plant expression vector based on seamless cloning technology could promote the target protein into the nucleus, suggesting it could be widely used in the study of biological function of pathogen proteins.

Pond-drying can kill the pernicious microbe in the pond and decomposes organic matter in the sediment, which is a remediation method commonly used during the pond culture. Therefore, it is important to investigate the diversity, distribution and function of microorganisms in the pond before and after pond-drying. The present study investigated the effects of pond-drying on the aquaculture environment and microbial community structure in the water and sediment before and after pond-drying in the eel (Anguilla japonica) culture pond. The contents of ammonia nitrogen (NH₄⁺), nitrite (NO₂^-), nitrate (NO₃^-), total nitrogen (TN), total phosphorus (TP) were measured and the changes of microbial community structure were analyzed through high-throughput sequencing. The results showed that pond-drying had significantly decrease effect on the contents of NO₂^-, NO₃^-, TN and TP in water (P<0.05) as well as the contents of TP in sediment (P<0.05). The results of high-throughput sequencing analysis showed that Proteobacteria was the main kind of bacteria in the water and sediment after pond-drying, and the abundances of Proteobacteria, Nitrospirae and Bacteroidetes were increased, while the pernicious microbe such as Cyanobacteria was decreased in the pond at the level of the phylum. At the genus level, the beneficial bacteria such as Novosphingobium, Sediminibacterium, Limnohabitans and Rhodobacter were increased, while the pernicious microbe such as Microcystis and Ellin6067 were decreased. This current study showed that pond-drying could change the microbial community structure in the water and sediment, and have a significant effect on the pond environment remediating. It provides some theoretical references for improving the environment of aquaculture pond in the future.

As growing quickly, big body size and delicious, triangular bream (Megalobrama terminalis) is an important economic fish, which is suggested to breed in open water in China. It is also an important catch species in Qiantang River Basin. However, M. terminalis resources have reduced recently, due to overfishing, environmental damage, water pollution, construction of water conservancy and transportation and so on. Restocking enhancement is considered as an effective method to recover the wild resources. However, there was rarely report on the effect of restocking enhancement of M. terminalis. In order to analyze the effect of restocking enhancement of M. terminalis, 12 pairs of microsatellites primers with good amplification effect were selected from 31 pairs of microsatellite primers in this study. Based on the microsatellite marker paternity test technique, genetic typing and polymorphism analysis were carried out on 301 parents from 4 breeding farms and 371 recaptured offspring individuals. The results showed that the number of alleles of microsatellite marker ranged from 15 to 68, and the expected heterozygosity was 0.726~0.969 (mean 0.863). The range of polymorphism information content was 0.693~0.967 (mean 0.849) and all 12 loci were high polymorphic (PIC>0.5). Among them, the frequency of invalid alleles of 4 microsatellite loci was greater than 0.2, which would affect the paternity analysis by software, so the data of the 4 loci were not used in following analysis. The parentage assignment for the situation of unknown sexes showed the combined non-exclusion probability of 8 loci was 99.997 5%, according to Cervus 3.0. The results showed that there were 4 recaptured individuals from offspring of brood stocks. The study suggested that the contribution of the released populations to nature resources of M. terminalis was 1.08% in 2018. This study discussed the enhancement and releasing effect of M. terminalis, which has great significance in protecting the resources and guiding the scientific breeding and releasing of M. terminalis in the Qiantang river in the future.

The possible impacts on nontarget organisms of transgenic crops are important in environmental risk assessment for genetically modified organisms (GMOs), such as Bt maize (Zea mays). Because Bombyx mori belongs to the same order as the target pests of transgenic Bt maize, i.e., the Lepidoptera, it may be affected by consuming mulberry leaves covered with pollen from nearby transgenic Bt maize. In this study, laboratory experiments were conducted to assess the potential effects of transgenic maize 'DR12-5' pollen on growth and reproduction of B. mori. In the assay, B. mori larvae were fed mulberry (Morus alba) leaves covered with different densities of pollen from 'DR12-5' or its corresponding near isoline (control). When B. mori larvae were exposed to 40 g/L (10000 grains/cm²) transgenic maize 'DR12-5' pollen, the survival rate dropped significantly, and no larvae could survive after 8 d. When the 'DR12-5' pollen concentration on the leaves of mulberry was 4 g/L or 0.4 g/L,no lethal effect was observed but the development of the neonate larvae was inhibited. The weights of different instar larvae were significantly decreased, and the development time was significantly prolonged than control larvae. However, there were no significant differences in pupation rate, emergence rate, cocoon weight, pupa weight, cocoon shell weight and oviposition between GM maize pollen and its non-transgenic cron pollen. The activity of the anti-oxidation enzyme system in 5th instar silkworms was detected, and it was found that the activities of superoxide dismutase (SOD), peroxidase (POD) and hydrogen peroxide (CAT) in 'DR 12-5'-treated silkworms were significantly increased, and the higher the pollen concentration, the higher the enzyme activity. The above results showed that 'DR 12-5' pollen densities ≥ 100 grains/cm² leaf had negative effects on the growth of the silkworms and the adverse effect was improved with higher pollen density. In the natural conditions, the drift of maize pollen is affected by various environmental factors such as distance, weather conditions and so on. And the exposure levels are far lower than in the current study. Therefore, it is generally recognized that Bt maize pollen will pose a low risk to B. mori. This study provides a theoretical basis for evaluating the potential risks to silkworm posed by insect-resistant genetically modified maize

Przewalski's wild horse (Equus ferus ssp. przewalskii) is a subspecies of E. ferus and is considered as endangered animals. The healthy growth and continuous expansion in their population have special value for germplasm resource protection. The parasites in Przewalski's wild horse can produce different degrees of pathological damage to the organism. The present study aimed to identify the intestinal parasite species in Przewalski's wild horses which were taken from the State Forest Administration Gansu Protection Centre of Threatened Species. During sampling procedures, the worms were separated in the faeces of Przewalski's wild horses. Morphological characteristics were identified and ITS (internal transcribed spacer) sequences were compared using bioinformatic methods. The results showed that 2 kinds of worms named PE and CN were isolated from intestinal tracts of Przewalski's wild horses, and preliminarily identified as Parascaris equorum and Cylicocyclus sp. by morphology observation. The full length of PE ITS region was 865 bp (GenBank No. MT579850), and the length of ITS-1, 5.8S rDNA and ITS-2 was 421, 156 and 288 bp, respectively; and the homology with Parascaris equorum was 99.88%. The full length of CN ITS region was 843 bp (GenBank No. MT581488), and the length of ITS-1, 5.8S rDNA and ITS-2 was 370, 153 and 320 bp, respectively; and the homology with C. nassatus was 99.41%. The above results showed that PE sample collected from Przewalski's wild horses was Parascaris equorum and the CN sample was C. nassatus. The results of this study could provide a reference for healthy breeding of Przewalski's wild horses.

Wheat leaf rust, caused by Puccinia triticina (Pt), is one of the most destructive diseases of wheat. The haustoria of Pt is the main place where the effector proteins were secreted, and the effector protein is the key of the pathogenetic of Pt. To clarify the expression characteristics of these effector proteins in the stage of interaction with the host, 635 candidate effector proteins have been harvested with bioinformatics analysis based on the sequencing of transcriptome. qRT-PCR analysis was used to test the expression characters of 10 candidate effector proteins. The results showed that these genes were consistent with the basic structural properties of fungi effector proteins and were highly homologous to wheat leaf rust BBBD and wheat stem rust (P. graminis f.sp. tritici), three of these genes contained a known conserved domain [Y/F/W]xC, one gene contained [L/I]xAR domain, two genes were glycosyl hydrolase family, and one gene had dioxygenase activity. Six of them was induced to be up-regulated in the late stage and the others were up-regulated dring 6 hpi to 48 hpi respectively. The results lay a foundation for revealing the interaction mechanism between wheat and leaf rust, and also provides favorable conditions for the study of wheat leaf rust effector proteins.

The GATA transcription factor family contains a zinc finger protein conserved domain that recognizes and binds the 5'-(A/T) GATA (A/G)-3' motif to regulate the transcription and expression of multiple functional genes in eukaryotes. It is closely related to the growth and development of pathogenic fungi and the utilization of nitrogen sources. In order to explore the function of the GATA transcription factor family of Setosphaeria turcica, the Softberry database (http://linux1.softberry.com/berry.phtm) was employed to carry out genetic structure analysis, prediction of conserved domains and promoters; qRT-PCR was used to detect the effects of nitrogen source concentration levels and species on gene transcription levels of family members. The results showed that there were 7 members in the GATA transcription factor family of S. turcica. Compared with KNO₃ as the nitrogen source, in the medium with NH₄Cl as the only nitrogen source, the growth rate of hyphae was accelerated and the conidia production was reduced by about 15 times, and the transcription levels of all GATA transcription factors were increased by 2 to 4 times. Compared with the normal Richard's medium, when KNO₃ content was 0, the growth rate of hyphae was increased and the yield of conidia was reduced by about 10 times. At the same time, the expression of all GATA transcription factors was increased by 20 to 40 times, indicating that GATA transcription factor expressions was highly induced under the completely restricted nitrogen source. The above results preliminarily clarified the response of GATA transcription factors to nitrogen metabolism, revealed the availability of nitrogen and the regulatory role of GATA transcription factors in the development of S. turcica, and provide new ideas for the control of northern corn leaf blight.

Enterotoxigenic Escherichia coli (ETEC) F4ac is a major pathogenic bacteria causing diarrhea in newborn piglets (Sus scrofa). MUC4 and SLC12A8 influence the susceptibility of piglets to ETEC F4ac, and these 2 genes are located on the loci controlling F4ac receptor towards QTL mapping. In this study, 5 MUC4 and 6 SLC12A8 gene knockout vectors were constructed through CRISPR/CAS9 system. First, 5 and 6 single guide RNAs (sgRNAs) targeting to porcine MUC4 and SLC12A8 genes were designed and inserted into the pX330 plasmid. There were 3 and 5 knockout vectors, separately, which were verified efficiently using the T7E1 enzyme digestion assay after transfected into IPEC-J2 cells. The results of T7E1 enzyme digestion assay showed that the mutant efficiency for 3 knockout vectors of MUC4 amounted to 8.4%, 6.7% and 6.2%. The mutant efficiency for 5 SLC12A8 knockout vectors amounted to 18.8%, 19.1%, 12.2%, 18.1% and 13.1%, respectively. Then Western blot analyses were performed to further detect the expression of targeted genes after transfection. The results showed that the expressions of MUC4 were downregulated with varying degrees after transfection with pX330-MUC4-3, pX330-MUC4-4 and pX330-MUC4-5, and pX330-MUC4-4 vector had the highest knockout efficiency. Among the 5 target vectors of SLC12A8, transfection with pX330-SLC12A8-2, pX330-SLC12A8-5 and pX330-SLC12A8-6 downregulated the expressions of SLC12A8 with varying degrees, and pX330-SLC12A8-5 vector had the highest knockout efficiency. This study could provide experimental materials and technical support for cultivation of MUC4 and SLC12A8 gene knockout cell lines, and contribute important materials for understanding of the gene function in the future.

Potato common scab, a seed tuber-born disease, is caused by Streptomyces in temperate regions, which seriously threatens the potato (Solanum tuberosum) production and tuber quality. In this study, the specific primer pairs Aci-3/4 were designed according to the toxin gene sequence of Streptomyces acidiscabies, which is one of the pathogens causing potato common scab. A target fragment of 192 bp was specifically amplified using S. acidscabies genomic DNA as a template. The sensitivity of real-time quantitative PCR (qPCR) method obtained in this work showed 1 000 times higher than that of conventional PCR. The results of genomic DNA amplification of the tested samples showed that the cycle threshold (Ct) values in qPCR detection of diseased potato and diseased soil were in the range of 25~32, while conventional PCR did not detect DNA bands in diseased soil. These results suggested that the PCR detection method established in this work had the advantage of strong specificity and high sensitivity, which could quickly detect S. acidiscabies from diseased potato tissues and soil samples. This detection method for potato scab pathogens could provide an effective technical means for early monitoring and effective control of potato scab.

Japanese encephalitis virus (JEV) is an important zoonotic pathogen theating human health, and still lacks sensitive antigen detection method currently. NS1 protein is a secreted protein expressed by JEV and is an ideal target for diagnosis of the virus. In this study, a double antibody sandwich ELISA (DAS-ELISA) method for detecting non-structural protein 1 (NS1) was established using the constructed NS1 protein monoclonal antibodies. Monoclonal antibody hybridoma cell lines (5H2 and 2F6) against different sites of the JEV NS1 protein were used to prepare ascites. After purification by Protein G, the ELISA plate was coated with 5H2 as the capture antibody, and 2F6 labeled with HRP as detection antibody. The optimized ELISA conditions showed that the optimal concentrations of capture antibody and enzyme-labeled antibody were 25 and 2.296 μg/mL, respectively. Using this method to detect serially diluted NS1 protein in concentration of 78.125~625.000 ng/mL, there was a good linear relationship between the OD₄₅₀ value and the protein concentration, indicating that this method can be used for quantitative detection of NS1 protein. Detection of porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, and porcine parvovirus by this method showed negative results, indicating good specificity. The reproducibility test results showed that the repeatability within the batch was 0.7%~2.6% and the repeatability between batches was 0.6%~2.1%. Using this method, brain tissue homogenates of JEV-infected mice were detected, and NS1 protein was clearly detected during the onset of mice (Mus musculus). This study established an ELISA method for detecting JEV NS1 protein, which provides a tool for the diagnosis of the virus.