The reproductive performance and health status of alpha-1, 3 galactosyltransferase gene(GGTA1) knockout pigs(Sus scrofa) is the key to success of xenotransplantation. Here we examined the genotype, litter size, blood routine, blood biochemical indexs of our group's GGTA1 knockout(GTKO) Bama minipigs(BMP) to assess their reproductive capacity and health status. GTKO pigs were mated with GTKO or wild type BMPs. The GTKO BMP have been bred to F3 generation. Firstly, the typing of GGTA1 gene knockout was identified by sequencing PCR products. α-1,3-Gal which is the product of alpha-1,3 galactosyl transferase gene was detected by flow cytometry after mixing Fluorescein isothiocyanate-Griffonia simplicifolia Isolectin B4(FITC-GSIB4) and peripheral blood mononuclear cells(PBMC). Then, reproductive performance was evaluated by litter size. Health status was assessed by blood routine and blood biochemical indexes. The results showed that the inheritance of GGTA1 gene was followed the Mendelian law; Low fluorescence expression were detected in GGTA1 double knocked (GGTA1-/-) genotype. The litter size of gilt was 7.33±1.70, and the litter size of sow was 9.43±1.68, which had no significant difference in the fecundity with wild BMP Meanwhile, compared with wild-type BMP, no obvious difference were found in blood routine and blood biochemical indexes. In short, our results demonstrated that GTKO BMPs' family was established, with stable hereditary, normal fertility and healthy physiological indicators. The GGTA1-/- Bama minipigs which effectively solved the hyperacute rejection, it can be used as ideal donor for xenotransplantation research.

β1,4 N-acetylgalactosaminyl transferase (β4GalNT2) and its products of porcine (Sus scrofa) are one of the important non-Gal antigens causing xenograft rejection. To further reduce the xenograft rejection, β4GalNT2 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) in α-1,3 galactosyltransferase (GGTA1) gene knockout pigs. Firstly, single guide RNA (sgRNA) targeting porcine β4GalNT2 gene was designed, sgRNA expression vector was constructed, and then it was electrically transferred to fibroblasts from GGTA1 knockout pigs, cells were individually cultured. The engineered mutation of colonies in the β4GalNT2 gene were identified by PCR, TA cloning and sequencing. The colonies of β4GalNT2 gene mutation were selected as the nuclear donor, and the β4GalNT2 knockout pig was generated by somatic cell nuclear transfer (SCNT). The method of identifying mutations in colonies was used to identify the β4GalNT2 gene mutations in piglet. Peripheral blood mononuclear cells (PBMCs) were isolated to co-incubation with fluorescein isothiocyanate conjugated Dolichos bi?orus agglutinin (FITC-DBA). Potential off-target sites were investigated by software named Optimized CRISPR Design, and PCR products of off-target sites were sequenced. The result showed that 14 of the 25 colonies occurred mutation. Reconstructed embryos were transplanted into 2 estrous recipient gilts. One recipient gilt was pregnant to the end and produced one piglet, the genotype of piglet in the β4GalNT2 gene was -6 bp/-13 bp/WT. No green fluorescence was detected in PBMC of β4GalNT2 knockout piglet after co-incubation with FITC-DBA, which indicated that β4GalNT2 gene of piglet was inactivated. No potential off-target sites was detected in the live pig via analysis. Thus, CRISPR/Cas9 was used to generate GGTA1/β4GalNT2 gene knockout pig domestically for the first time. It is expected to reduce the xenograft antibody mediated rejection and provide a good research material for the clinicalapplication of organ transplantation.

Potato (Solanum tuberosum) is one of the most important crops in the world and it can be infected by Potato virus S (PVS), which lead to reduction of quality and yield. The high detection rate of PVS, which can be improved by the sensitive and accurate detection technology, will control the PVS threaten for potato production because its introduction and spread is prevented, and the potato industry can be protected. For developing a sensitive and accurate technique, the droplet digital PCR (ddPCR) primers and probe were designed according to the conserved sequence of PVS coat protein (CP) gene and tested through specificity, sensitivity and reproducibility experiment. The results showed that the established method was specific to PVS, and it could effectively distinguish PVS from the control viruses Potato virus X (PVX), Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV), which was same with the result of qRT-PCR. The detection sensitivity of ddPCR was up to 20 copies/μL of RNA which was 10-fold higher than that of qRT-PCR. The standard error of three repeats droplets was less than 1.5% of the total number of droplets, indicating the established ddPCR method had good reproducibility. The ddPCR method established in this study can be used for the accurate detection of PVS and also provides a reference for the accurate detection of other plant viruses.

Meat quality is affected by the type and percent of muscle fiber which is the basic unit of muscle tissue. In order to enrich the in vitro research mode of skeletal muscle growth and development, this study selected the hind-limb extensor digitorum longus of a newborn Landrace piglet (Sus scrofa) as sample, and the single muscle fiber was isolated through collagenase digestion. The type of myofiber was then accurately detected by RT-PCR and immunofluoresence. The single muscle fiber were isolated by collagenase digestion and these muscle fibers had smooth surface and the cells adhered to the myofibers originally, which indicated RNA was qualified for subsequent analysis. The result of RT-PCR indicated that myosin heavy chain Ⅱb (MyHC Ⅱb) mainly expressed in 8 myofibers. Furthermore, the result was confirmed by immunofluorescence with the antibody of myosin heavy chain I (MyHCⅠ). In this study, one simple and cheap detection method for isolation and identification of single muscle fibers of porcine skeletal muscle was established, and the result provides a muscle fiber model, which will be useful for studying the various functions and cellular processes of mature muscles in vitro.

Antimicrobial peptide(AMP), a burgeoning feed additive, is rarely reported when it apllies to the ruminant. The aim of this study was to evaluate the effects of antibacterial peptide (AMP) on rumen ciliate community structure by MiSeq high throughput sequencing technology. Twelve male Chuanzhong black goats(4-month-old) were randomly divided into 4 groups according to the similar weight. Four groups were normal concentrate group (A 300g /d), normal concentrate+AMP group (C 300g /d+3 g/kg), double concentrate group (D 600 g/d) and double concentrate+AMP group (E 600 g/d+3 g/kg), The rumen fluid samples were collected after 20 d, and then the total DNA were extraced for amplification 18S rRNA(547F-V4R), sequencing by MiSeq Illumina250. The results showed that: 1)A total of 471 580 valid 18S rDNA sequences and 1 398 operational taxonomic unit (OTU) across 12 samples were obtained. 2) The 99.17% of all OTUs belonged to eukaryotes, At phylum level, Ciliophora was the most abundant phyla in A and C groups(A: 46.0%; C: 48.2%), followed by incertae sedis (A: 35.5%; C: 42.83%); On the contrary, incertae sedis was the most abundant phyla in D and E groups (D: 49.93%; E: 60.27%), followed by Ciliophora (D: 45.33%; E: 35.23%), and no significant difference between the groups. 3) At family level, only two families (Ophryoscolecidae, Isotrichidae) were found belong to ciliates. And the differences between the groups were not obvious. 4) At genus level, a total of nine different ciliates genera were detected in every last sample. And the content of Ophryoscolex in C group were significantly higher than A group (P＜0.05), but no significant difference between D and E (P＞0.05); the content of Entodinium in C group are significant lower than A group (P＜0.05), but there have not significant difference between D and E (P＞0.05); The content of Polyplastron in E group are significant higher than C group(P＜0.05), and the content of Ophryoscolex were significantly lower than C group (P＜0.05). 5) There were no significantly difference between groups on alpha diversity index (Chao, ACE, Shannon and Simpson). In conclusion, Polyplastron was the most abundant genus in the rumen ciliate communities of young Chuanzhong black goats; there were still a lot of valuable information about eukaryotic microorganisms need to study in rumen. The contents of Ophryoscolex were significantly higher by the supplementation of 3 g/kg complex antibacterial peptide (P＜0.05), and the content of Entodinium were significantly lower by the supplementation of 3 g/kg complex antibacterial peptide (P＜0.05), but there were no significantly difference with feeding 600 g/d concentrate. The ciliate community structure was not affected when the amount of concentrate changed from 300 to 600. Moreover, alpha diversity index also was not affected by supplementation of 3 g/kg complex antibacterial peptide or the amount of concentrate changed from 300 to 600. The result of this study provides a theoretical basis for the application of complex antimicrobial peptide in ruminant.

Anaerobic fungi (Neocallimastigomycota) are important fiber-degrading functional microorganisms in the herbivores' digestive tract. The purpose of the present study was to isolate anaerobic rumen fungi with high activities of plant cell wall degrading enzymes for practical applications to improve the utilization of roughage. Twelve anaerobic fungi strains were isolated from the ruminal contents of Xinong Saanen dairy goat (Capra hircus) using the Hungate roll tube technique. The fungi strains were identified using the morphological characteristics, nucleotide sequence analysis of internal transcribed spacer region and 28S rDNA D1/D2 region. Meanwhile, activities of xylanase, carboxymethyl cellulase (CMCase), avicelase, acetyl esterase (AE) and β-glucanase of 12 fungi cultures were assayed, and the enzymatic characteristics of xylanase and AE produced by fungus with the highest enzyme activities were also analysed. The results showed that 12 anaerobic fungi strains belonged to Piromyces, named Piromyces sp. CN1~ Piromyces sp. CN12, respectively. The ITS gene Genbank number is KY368100~KY368111. The xylanase, CMCase and AE activities of Piromyces sp. CN6 were 1655.3, 93.4 and 152.8 mU which were significantly higher than those of other fungi strains (P＜0.05). The avicelase activity of Piromyces sp. CN3 was the highest among these fungi, but its activity showed no difference with that of Piromyces sp. CN6 (P＞0.05). There was no significant difference on β-glucanase activity produced by fungi strains (P＞0.05). The xylanase activities had extremely significant positive correlation with CMCase and AE activities (P＜0.01), and the xylanase activities had significant positive with avicelase activity (P＜0.05). The optimum temperature of CMCase activity was 50 ℃, and the optimum pH was 5.0. The CMCase was stable at 40 ℃ and pH 5.0~8.0. K＋, Ca2+and Co2+ had active effects on its enzyme activity, whereas Zn2+, Cu2+, Mg2+, Fe2+ and Mn2+ had inhibitory be promoted on it. The AE activity preformed optimally at 50 ℃ and pH 9.0, and it was stable at 40 ℃ and pH 5.0~10.0. The AE activity could be promoted by Mg2+, K+ and Ca2+ and inhibited by Zn2+, Fe2+, Co2+ and Mn2+. Piromyces sp. CN6 isolated from rumen of Xinong Saanen dairy goats showed the highest plant cell wall degrading enzymes activity. Through enzyme activity assays and enzymological characterization, the enzyme system information of rumen fungi can be enriched and improved, which can provide basis for further application.

Phosphorylase kinase γ1 (PHKG1) and phosphorylase kinase γ2 (PHKG2) genes are important in the glycogen metabolism pathway, and have the function of decomposing glycogen to provide energy for muscle contraction and maintain blood glucose balance. In order to further explore the research on PHKG1 and PHKG2 genes genetic mechanism, the Guizhou Congjiang pig (Sus scrofa) and Large White pig PHKG1 and PHKG2 genes CDS sequences were cloned by the T-A cloning method. PHKG1 and PHKG2 genes structures, physicochemical properties of protein and homology comparision to other species were described by bioinformatic anyalysis software. Real-time quantitative PCR (qRT-PCR) technology was also be used to comparative analysis of these 2 genes in Congjiang pig and the Large White pigs in different tissues (heart, liver, spleen the lung, kidney, stomach, small intestine, large intestine, longissimus muscle and fat) in differential expression level. The results showed that the CDS of PHKG1 gene was 1 167 bp in length, encoding 388 amino acids, and the full sequence of PHKG2 gene was 1 221 bp in length, encoding 406 amino acids, both of which constituted a transmembrane with S_TKc domain Hydrophilic and non-secreted proteins. The phylogenetic analysis of PHKG1 and PHKG2 protein and phylogenetic tree analysis showed that the two proteins had some homology. The results showed that there were 4 mutations in PHKG1 gene, which were A371G, A525G, T945C and C1050T respectively, but there was a mutation in T945C compared with Large white pig, which did not cause amino acid change. PHKG2 gene sequence analysis showed that G236A, C431T, A726G, A807G, A816G and A867G were Large white pig, Congjiang pig and Bama miniature pig (KJ 186785) common mutations. Congjiang pig had a special A614G mutation, and caused 205th glutamic acid into alanine. Sequence analysis revealed that the amino acid phylogenetic relationship of PHKG1 and PHKG2 of Congjiang pig was close to pig(sus scrofa), cattle(Bos taurus), sheep(Ovis aries), ordinary people(Homo sapiens), Mus musculus, Rattus norvegicus (Rattus norvegicus)6 species, the homology was 92%~100%, and zebrafish (Danio rerio) was distantly related. qRT-PCR results showed that PHKG1 gene was present in longissimus muscle with highest ratio while heart, liver, spleen, kidney, stomach, large intenstine, small intenstine showed less expression in Congjiang pig and Large white pig. And in the lung, the expression was significant different (P<0.05). Relative expression of PHKG2 gene was high indication in spleen, lung, large intenstine, small intenstine, kidney and with lower ratio in heart and longissimus muscle. This study provides a theoretical basis for the follow-up eukaryotic expression of PHKG1 and PHKG2 genes.

In order to establish the flow cytometry (FCM) method for estimation of Salvia Genomic C-value, fresh leaves of Salvia japonica, S. barrelieri, S. chinensis, S. patens, S. repens were taken as samples. Based on the improved isolation method and DNA reference standards from Oryza sativa subsp. japonica ‘Nipponbare’, Galbraith's、WPS、GPB、Tris-MgCl2、LB01 was used as buffer separately, and ultimately we selected the best results of LB01. In the linear scale measured, the 1C content of S. japonica, S. barrelieri, S. chinensis, S. patens, S. repens was: 0.604, 1.158, 0.634, 0.579 and 0.954 pg, respectively. The results showed that different Salvias with significant different genomic C value, and C value range of similar plants reported in the literature wassimilar. The results provides basic data for further studying on genomics, cytobiology and germplasm resource for Salvia plants.

Flo8 is a key transcription factor at the downstream of cAMP signal pathway and plays an important role in fungal development. In this experiment, in order to clarify the function of transcription factor Flo8 and cAMP singal pathway in regulating the pathogenicity of Setosphaeria turcica, the genes encoding transcription factor Flo8 was cloned and the expression pattern of the gene was analyzed during the development of infective structures and the early stage of infecting the host. The homologous search was carried out in the database of S. turcica using the Candida albicans Flo8 amino acid sequence as a probe. The gene encoding the transcription factor Flo8 was identified by PCR. The structural characteristics of the gene and protein were analyzed through GSDS, ProtParam, SOMPA and SMART software, respectively. Subcellular localization of protein was analyzed by WoLF PSORT online software. Quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression patterns. The results showed that there was a protein sequence containing the conserved domain of LisH which belonged to the typical FLO8 protein, and its coding gene was named StFlo8. The total length of the gene was 2 384 bp, and that of the cDNA was 2 037 bp, this gene contained 6 introns and 7 exons, encoding 678 amino acids. The results of expression pattern analysis showed that, compared with the mycelial period, the expression level of StFlo8 was significantly decreased in the conidia and germ tube period, and was significantly higher in appressorium and invade mycelial period, reaching more than 4 times of that in the mycelia. During the interaction of S. turcica with the susceptible host, the expression of StFlo8 was down-up-down regulated, and reached the highest level at 18 h after inoculation, which was as about twice as that of the beginning of inoculation. The results indicated that StFlo8 was involved in the regulation of appressorium development and infected mycelial formation. The results provide a theoretical basis for the further study of the function of StFlo8 gene and the infection mechanism of S. turcica.

Amylopectin synthesis-related genes are important genes to determine quality of rice. In order to study the effect of starch synthesis-related minor genes on rice (Oryza sativa) quality as well as to provide foundation for improving rice quality, rice physicochemical properties and stach paste viscosity profile were investigated under genetic background with same major genes of starch synthesis. A backcross recombinant inbred line (BIL) of B1C1F11 generation was used as materials, which was constructed by indica photo-thermo-sensitive gene male sterile (PTGMS) line Guangzhan 63S (Oryza sativa ssp. indica) and rice potential restorer line CG173R, both with the same alleles of granule bound starch synthase gene (Wx) and starch synthase IIa gene (SSII-3, SSIIa, ALK). Effect of soluble starch synthaseⅠgene (SSI), soluble starch synthaseⅢ-1 gene (SSIII-1) and pullulanase gene (PUL) on eating and cooking qualities (ECQs) were analyzed. Meanwhile, split plot design was used to analyze interaction between two genes and among 3 genes on rice ECQs. SSI had significant influences at 0.05 or 0.01 level on physical/chemical qualities and rapid viscosity analyzer (RVA) profile characteristics except for apparent amylose content (AAC) and PeT. Interaction among 3 genes had significant influences on AAC at 0.05 level and significant influences on other physical/chemical indicators and RVA profile characteristics except for PaT at 0.01 level. Effects of minor gene SSI and interaction among 3 genes of SSI, SSIII-1 and PUL had significant influences on GC, PKV, HPV, BDV, CPV, CSV and PaT at 0.01 level under the genetic background with same major genes of Wx and SSII-3. These findings provide an important theoretical basis for molecular marker assisted breeding.

Eukaryotic translatoin initiation factor 4E (eIF4E) and its iso form (eIF(iso)4E) have been demonstrated to play central roles in plant-vorus interactions. Broad-spectrum turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa ssp. pekinensis) has been demonstrated to be related to BraA.eIF(iso)4E.a and BraA.eIF(iso)4E.c. By now only two double allelic mutant lines (RLR22 and BP8407), were reported, and they shared the same pattern of mutation in eIF(iso)4E.c. To explore diversiform allelic variation in BraA.eIF(iso)4E.a and BraA.eIF(iso)4E.c, germlines with confirmed TuMV resistance or susceptibility were analyzed in this study using candidate gene re-sequencing approach. One line (He102) with new double mutation of eIF(iso)4E.a and eIF(iso)4E.c resistant to TuMV was identified. Polymorphisms between wild and mutant sequences showed that in line He102 eIF(iso)4E.a was a pseudogene with exons 4 and 5 deleted, while eIF(iso)4E.c contained a frame-shift mutation which resulted in early termination. These mutations were responsible for the loss of TuMV resistance as wild-type. Chinese cabbage genes could recover virus susceptibility in Arabidosis lsp1 mutant while mutated genes could not. Codominant functional markers related to the mutation on both alleles were developed. The categorized double allelic mutated line was a more valuable resistant resource with a diverse background, and the developed markers could be used directly for germplasm screening and broad-spectrum TuMV-resistant variety breeding through marker-assisted selection. The study provides a representative exaple exemple for allelic-variation identification by candidate gene re-sequencing with mini-core germplasm collection as materials, which were carefully selected based on target traits.

Epsilon - lycopene cyclase (LCYE) is involved in synthesis of the key enzymes of the carotenoid and plays important roles in carotenoid synthesis. In order to explore Hibiscus esculentus LCYE in gene function, In this study, the full-length cDNA sequence of lycopene epsilon-cyclase (LCYE) gene that encoded carotenoid biosynthesis downstream enzymes lycopene epsilon-cyclase was isolated from H. esculentus. by RT-PCR and RACE (rapid-amplification of cDNA ends) technique. The cDNA sequence was 1 845 bp in length and consisted of a 1 605 bp ORF that encoded a polypeptide of 534 amino acids, with a predicted molecular weight (MW) of 59.73 kD and a hypothetical isoelectric point (pI) of 6.22. The total average hydrophobicity (GRAVY) of the LCYE protein was 0.036, and it was predicted as hydrophobic protein. It shared over 80% identity with the homologous proteins from Gossypium arboretum, Theobroma cacao and Jatropha curcas, proving that it was highly conservative. This gene was named HyLCYE, and the GenBank accession was KX257999. The fluorescent quantitative analysis indicated that the expression level of HyLCYE gene was the highest in mature leaves and the lowest in roots. During the development of leaves, the highest expression level was found in mature leaves and the lowest in old leaves. During the development of fruits, the highest expression level was found in 3 d after flowering and the lowest in 1 d after flowering. The ultra performance liquid chromatography (UPLC) analysis showed that lutein contents were the highest in mature leaves and 3 d after flowering. These results suggested that the expression level of LCYE was positively correlated with lutein contents. The total carotenoid contents among different genotypes okra varieties were highest in green varieties, followed by red varieties, and was lowest in white varieties. The results provide a scientific base for the further development and utilization of Hibiscus esculentus.

China is one of the richest countries of chicken (Gallus gallus domesticus) genetic resources, with the more and more cultivated varieties appearing constantly, so how to distinguish these breeds is very important. The aim of this study was to explore the utility of mtDNA D-loop as DNA barcoding for identifying the chicken breeds. Four chicken breeds about Anka chicken, Recessive white chicken, Henan Game chicken and Silky were used as materials. By PCR amplification and sequencing technique, completed mtDNA D-loop sequences of 115 chickens were studied, and the genetic polymorphism characteristics and genetic distances between species and within population were analyzed. The results showed that, the D-loop region of four chickens were 1231~1232 bp, and the 1231 bp samples existed a base C deficiency in 859 site. In the 115 samples assay, there were 26 mutation sites and 15 haplotype. Anka chicken had 3 especial haplotypes. Recessive white chicken had 4 especial haplotypes, Henan Game chicken had 3 especial haplotypes, and Silky had 2 especial haplotypes. Haplotype diversity(Hd) and nucleotide diversity(Pi) of 4 chickens were 0.467~0.799 and 0.00 217~0.00 561 respectively. The biggest values of average number of nucleotide differences(K)(6.910), Pi(0.00561) and Hd (0.799) were all from Recessive white chicken, and the smallest value of Hd was from Silky(0.467). Kiumura 2-parameter distance and Net distance of 4 chicken breeds were 0.435%~0.940% and 0.150%~0.712% respectively. Clustering analysis showed that the cluster results of four chickens were consistent to the morphological class. According to the public standard of haplotypes classification, Anka chicken and Recessive white chicken mainly converged into clade E, Henan Game chicken mainly converged into clade B and Silky mainly converged into clade A. The results indicated that the divergence between 4 chicken breeds is much farther, and it was feasible to identify chicken breeds using DNA barcoding of D-loop region. This study can provide theory basis for chicken variety identification, genetic resources protection and the development and utilization.

Despite the greatly agricultural importance of pepper (Capsicum annuum) and the ubiquitously involvement of MAPK (mitogen-activated protein kinases) cascades in a broad range of plant biological processes, the roles of MAPK cascades in pepper remain unclearly. In the present study, a MAPK of pepper, designated as CaMAPK9 (Accession no.: CA04g21490), was expressed and functionally characterized. The results showed that CaMAPK9 shared more than 95% deduced amino acid sequence identity with its homologues in other plant species including tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum) and potato(Solanum tuberosum). By subcellular localization assay through transient overexpression of CaMAPK9-GFP (Green Fluorescent Protein) in leaves of Nicotiana benthamiana plants, CaMAPK9 was found to target to the nuclei. Total RNA was extracted from abiotic stresses imposed pepper plants, as salt, mannitol and foliar application of salicylic acid (SA), methyl jasmonate (MeJA) or abscisic acid (ABA) and used for first-stand cDNA synthesis. cDNA RT-PCR showed that CaMAPK9 was induced under salt or mannitol and foliar application of SA or ABA stresses but impeded by SA, showing that CaMAPK9 may be involved in salt stress pepper response. In addition, two T3 transgenic Arabidopsis lines were developed and the ectopic overexpression of CaMAPK9 was confirmed. The 2 transgenic lines consistently showed a enhanced tolerance to salt stress manifested by the higher germination rate and longer root length under salt stress compared to that of the wild type seeds of seedlings. The transcriptional expression of ABI5, an ABA maker gene which was responsive to signaling in plant under salt-stress tolerance, was higher in two transgenic Arabidopsis types that in wild plants under salt-stress. All these results collectively suggested that CaMAPK9 acted as a positive regulator in the response of pepper to salt stress possibly regulated by signaling mediated by ABA and JA. This study provides a theoretical basis for further research on the molecular breeding mechanism of salt stress response in pepper

High salt is one of the major constraint that adversely affects wheat yield in China. The zinc transporter (ZTP) belongs to the ZRT, IRT-like protein (ZIP) family, and ZTP is involved in the response to salt stress through regulating induced the unfold protein response pathway, zinc levels to induced the unfold protein response pathway. In this study, the TaZTP29, which is the orthologous gene of AtZTP29, was isolated from wheat (Triticum aestivum) by RT-PCR and the expression pattern was analyzed through qRT-PCR. The subcellular localization of TaZTP29 was also investigated through green fluorescent protein (GFP) method. Meanwhile, the salt resistance of TaZTP29 was confirmed by over-expressing of the TaZTP29 in Arabidopsis thaliana. The sequencing analysis showed that TaZTP29 gene (GenBank accession: KY610283) consisted of 834 bp CDS, 81 bp 5'UTR and 117 bp 3'UTR, and encoded 277 amino acids. The conserved domain analysis demonstrated that TaZTP29 had the typical ZIP domain, which was consisted of 8 transmerabrane domains and the V transmerabrane domain had the completed conserved amino acid histine (His). TaZTP29 was localized to the endoplasmic reticulum with proyien predicted. The subcellular location were validated by means of fusing TaZTP29 with N-terminal GFP and then expressed in wheat protoplast using polyethylene glycol (PEG) transfection method. The green fluorescence was observed in endoplasmic reticulume. The information of conserved domain proved that TaZTP29 belonged to the ZIP protein family. The 16 homologues genes from 16 different species of TaZTP29 were searched and found in NCBI database, and the identity of amino acid between 16 homologous genes and TaZTP29 ranged from 64.03% to 94.5%. The highest identity of amino acid was between Aegilops tauschii (EMT27056.1) and TaZTP29, while the lowest identity of amino acid was between Arabidopsis thaliana (Q940Q3.1) and TaZTP29. Then phylogenetic tree of TaZTP29 was constructed using these 16 genes and TaZTP29. The expression pattern disclosed the expression level of TaZTP29 was increased immediately under ZnCl2 treatment and NaCl stress. However, TaZTP29 in response to heat stress was passive. When wheat suffered heat stress and drought stress, expression of TaZTP29 dropped rapidly. Tissue expression pattern analysis showed that TaZTP29 expressed in multiple tissues. Root accumulated higher TaZTP29 transcription when comparing to the other tissues of ground. The seed germination rate and total root length of wild type was similar with that of TaZTP29 transgenic Arabidopsis lines in normal growth condition. While, the seed germination rate and total root length of TaZTP29 transgenic lines were significantly higher and longer than those of wild- type plants under salt treatments (100 and 150 mmol/L NaCl), respectively. These results indicated that TaZTP29 transgenic lines had higher salt tolerance than wild type, and TaZTP29 could increase the salt tolerance of Arabidopsis thaliana, and could be used as a candidate gene for salt tolerance. This study provides a new foundation for further understanding the molecular mechanism of wheat stress resistance.

microRNA-490-3p (miR-490-3p) is down-regulated and may act as a tumor inhibitor in many tumors, such as gastric cancer, ovarian carcinoma, endometrial carcinoma and so on. However, specific roles of miR-490-3p are not well understood in cancers. This study aims to investigate the effects of miR-490-3p on the proliferation and metastasis of human (Homo sapiens) cervical tumor cells HeLa. In order to investigate the effects of miR-490-3p on proliferation, migration, cell cycle and EMT (epithelial to mesenchymal transition) of HeLa cells, miR-NC mimic or miR-490-3p mimic was transfected into HeLa cells. The expression level of miR-490-3p was detected using qRT-PCR, then, cell counting was used to measure the cell proliferation, and wound healing assay was applied to detect the ability of migration. Moreover, cell cycle was assayed by flow cytometry, and EMT proteins were detected by Western blot, including E-cadherin, β-catenin, vimentin and glycogen synthase kinase 3β (GSK-3β). The results showed that, in comparison with negative controls, the expression level of miR-490-3p was significantly improved by miR-490-3p mimic transfection (P＜0.01), which showed that miR-490-3p mimic was effctive to express miR-490-3p. Then, the HeLa cells transfected with miR-490-3p mimic exhibited signicantly slower growth than negative controls (P<0.05), which indicated that the overexpression of miR-490-3p reduced cell proliferation. Wound healing assay showed that, after transfected with miR-490-3p mimic, HeLa cells migration was reduced (P＜0.01), which suggested miR-490-3p transfection inhibited migration ability of HeLa cells. Flow cytometry analysis confirmed that, comparing with control group, the proportion of HeLa cells transfected with miR-490-3p in G2/M phase displayed a significant increase (P<0.01). This induced cell cycle arresting at G2/M phase. miR-490-3p probably inhibited cells growth through arresting cell cycle of HeLa cells. Western blot showed that the EMT proteins were also regulated through transfection with miR-490-3p into HeLa cells, the introduction of exogenous miR-490-3p was followed by an increase in expression of E-cadherin (P＜0.05) which was a biomarker of epithelial cells and decreases in β-catenin (P＜0.01), vimentin (P＜0.05) and GSK-3β (P＜0.01) expression. The result showed that the EMT of HeLa cells was inhibited and indicated that miR-490-3p may inhibit cell invasiveness by repressing EMT, which was a key mechanism in cancer cell invasiveness and metastasis. Collectively, these results displayed the overexpression of miR-490-3p reduced cell proliferation and migration and promoted G2/M phase arrest, which suggested that miR-490-3p may act as a tumor suppressor gene in HeLa cells. These data indicates that miR-490-3p can be used as a potential therapeutic target and a molecular marker for cancer prediction in human cervical cancer.

Bovine leukemia is a kind of chronic tumor disease caused by Bovine leukemia virus (BLV) in cattle(Bos taurus), sheep (Ovis aries) and other animals. In order to verify whether there is an association between BLV and SNPs of CXCR1 gene coding sequence (CDS), bovine leukemia infection of 866 Chinese Holstein was detected by fluorescence resonance energy transfer - quantitative polymerase chain reaction (FRET-QPCR) in this study. The SNPs (CXCR1-c.642A>G, CXCR1-c.816C>A, CXCR1-c.980A>G and CXCR1-c.1068G>A) in the CDS of CXCR1 gene were detected by MALDI-TOF-MS (matrix-assisted laser desorption/ ionization time of flight mass spectrometry). The relationship between genotype and haplotype of CXCR1 SNPs and bovine leukemia infection was analyzed by Logistic regression. The results showed: 1) Of the 866 cows, 42 had bovine leukemia, with a positive rate of 4.85%. 2) The predominant genotypes of CXCR1-c.642 A>G, CXCR1-c.816C>A, CXCR1-c.980A>G and CXCR1-c.1068G>A were GG, CC, AA and GG, with frequencies of 0.580, 0.466, 0.60 and 0.658, respectively. The relative risk of leukemia was significantly affected by CXCR1-c.980A>G locus (P=0.016). The individual of GG genotype was 5.04 times suffering from leukemia than that of AA genotype. CXCR1-c.642A>G, CXCR1-c.816C>A, and CXCR1-c.1068G>A have no significant effects on the susceptibility to bovine leukemia. CXCR1-980 A>G can be used as a marker for disease resistance breeding of dairy cows, and selecting AA genotype individuals can help to reduce the susceptibility of bovine leukemia and improve productivity efficiency.

Alfalfa (Medicago sativa) is widely grown and is one of the most important forage crops in the world, but its growth and biomass production are markedly reduced under salt stress. The objective of this study is to identify the inner molecular mechanisms of southern type alfalfa in response to salt stress, and mine these genes closely related to salt responsiveness. Illumina HiSeqTM 2000, a high-through transcriptome sequencing technology, was used to obtain the anscriptome differential expression data of southern type alfalfa (Medicago sativa 'Millenium') leaves under 72 h treatment at 250 mmol/L NaCl. Function and pathway of those different expression genes were also investigated using Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway biological analysis in order to obtain some of the potential target genes to salt stress. Eight randomly selected DEGs (differentially expressed genes) were used to validate the reliability of sequencing results. The results showed that after filtration of reads, a total of 60 395 324 control (CK) and 60 303 692 salt stress (ST) reads were acquired, among these reads, 54.18% and 53.77% could be precisely compared to the reference sequence of M. truncatula. After 250 mmol/L NaCl stressed for 72 h, in total, 30 900 DEGs were authenticated among which 4 187 and 3 507 were regarded as raise-and lower-regulated genes. GO analysis showed that these DEGs were mainly referred to binding, catalytic activity, cell part and cell. KEGG pathway analysis showed that these DEGs were mainly referred to biosynthesis of secondary metabolites, metabolic pathways and phenylpropanoid biosynthesis. Besides, we discovered many candidate genes, like glutathione S-transferase, superoxide dismutase [Cu-Zn] protein, L-ascorbate peroxidase, receptor-like kinase, stress-induced receptor-like kinase, sucrose nonfermenting 1(SNF1)-related kinase, Calmodulin-like protein, choline monooxygenase, delta-1-pyrroline-5-carboxylate synthetase 3, 2Ccatalytic/protein phosphatase type 2C and Trehalose-phosphate phosphatase, and many transcription factors were identified, to be related to salt tolerance, like ANTAP2-like ethylene-responsive, Transcription factor bHLH36, Transcription factor NAI1, bZIP transcription factor, Zinc finger C-x8-C-x5-C-x3-H type family protein, Nucleic acid binding transcription factor activity, Myb transcription factor, NAC transcription factor-like protein, Sequence-specific DNA-binding and WRKY family transcription factor, This study affords the initial value for the molecular mechanisms of salt tolerance in alfalfa.

Porcine (Sus scrofa) Pasteurella multocidain (Pm) infection caused serious diseases including swine plague, hemorrhagic septicaemia and infectious atrophic rhinitis. The present inactivated vaccine had poor cross protection effect and short immune protection period, and the recombinant vaccine still did not provide high protection rate. Based on Pm methionine transporter Q (metQ) sequences in GenBank, the primers were designed after removing the corresponding nucleotide sequence of expressing hydrophobic amino acid regions to express metQ in Escherichia coli. The partial sequence of Pm metQ was amplified by PCR from Pm genome. Bioinformatics analysis showed that the Pm metQ gene was composed of 768 bp, encoding a protein of 255 amino acids. This protein was an extracellular toxic protein and high homology among different Pm serotypes. Pm metQ gene was subcloned into pET32a (+) to construct pET-metQ. After induced by isopropylβ-D-1- thiogalactopyranoside (IPTG) rMetQ expressed in E.coli BL21, and determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that rMetQ with 28 kD was detected. Animal experiment was conducted by Kunming mice (Mus musculus). The serum immunoglobulin G (IgG) level was determined by enzyme-linked immuno sorbent assay (ELISA). The results showed that serum IgG level significantly increased in immuned group (aluminium adjuvant+rMetQ) at 14 d after third immunization. The infected mice in control groups were 20% survival rate at 3 d and all died at 7 d. The mice in the immunized group (aluminium adjuvant+30 μg rMetQ) showed 40% survival rate at 3 d and 10% survival rate at 7 d. But the mice in the immunized group (aluminium adjuvant+50 μg rMetQ) showed 80% survival rate at 3 days and 40% survival rate at 7 d. Autopsy of dead mice showed that the liver was the main diseased organ with haemorrhagic spot and even necrosis.Mice subcutaneously vaccinated with rMetQ revealed a partial protection against the challenge infection of Pm. This study provides data for screening new molecular vaccines against Pm infection.