Marssonina leaf blotch of apple caused by Diplocarpon mali, is one of the main apple (Malus domestica) leaf defoliation diseases in apple production areas, which can result in severe etiolation and abscission of leaves during the growing season. In this study, a new generation of high-throughput sequencing technology Illumina Hiseq2500 transcriptome sequencing was used for transcriptome sequencing in M. baccata infected with Diplocarpon mali, and then bioinformatic methods were used for gene expression profile and gene function prediction. The results showed that 46.86 Gb sequence information was abtained. Through filtering, splicing, assembling and going redundancy, 48 868 unigenes were abtained. After assessing the length distribution, GC content, expression level and other aspects of the unigenes, the sequencing data showed good quality and high reliability. Homology of the 48 868 unigenes were predicted by common databases NR (Non-Redundant), Swiss-port Protein Sequence Database, Eukaryotic Orthologous Groups of Proteins (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and 25 797 unigenes were directly aligned onto the genes of apple (M. domestica). Compared with KOG database, the unigenes could be divided into 25 categories according to their function, including general function prediction for 19.45%, signal transduction mechanisms for 11.84%, and the cell motility was the least. Taking KEGG database as a reference, the unigenes were located to 145 branched metabolic pathways, including antibiotic biosynthesis for 842, purine metabolism for 740, starch and sucrose metabolism for 437, T cell receptors signaling pathways for 292. Then RSEM software was used to calculate the differential expression, and edgeR software was used for differential expression analysis. By setting the threshold false discovery rate (FDR) ＜0.05, log2(fold change (FC))＞ 2 or log2(FC)＜-2 to screen differentially expressed genes, all 1 230 upregulated genes and 1 869 downregulated genes were identified. The enrichment results of KEGG Pathway showed that 94 differential genes participated in tyrosine metabolism, plant-pathogen interaction, plant hormone signal transduction and biosynthesis of amino acids, etc.. Expression of some regulated genes validated by qRT-PCR showed consistence with transcriptome analysis. In this study, the second generation of high-throughput transcriptome sequencing technology was used to research the related genes of the Malus baccata in the resistence to Diplocarpon mali, which provides theoretical foundation for clearing the resistence mechanism.

In vitro culture system influences the development potential of mammalian early embryos. In the current culture systems, which simulating the hypoxic condition in vivo of uterus and fallopian tubes, the reactive oxygen species (ROS) is considered to be a major factor affecting embryo development. In this study, the effect of ROS on the mitochondrial function and the mitochondrial DNA (mtDNA) copy number in mice (Mus musculus) 2-cell embryos were studied. The fertilized eggs from the Kunming mouse were treated by 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH), then the mitochondrial activity, the mitochondrial membrane potential, the mitochondrial ROS (mtROS) and the distribution of mitochondrial were observed by fluorescent detection, meanwhile, the expression of ROS-related genes, the mitochondrial transcription factor A (TFAM) and the mitochondrial DNA (mtDNA) copy number were detected by real-time PCR. The results showed that mouse embryos had a higher blocking rate (66.16%) and a lower embryo damage rate (24 h malformation rate of 9.09%, 48 h mortality 10.39%) in 1.0 mmol/L treated group. ROS staining results showed that embryonic cytoplasmic ROS levels were increased significantly after AAPH treatment, and the mRNA expression of ROS-related genes were also increased. After treated by 1.0 mmol/L AAPH, the membrane potential, the mtDNA copy number and the level of mitochondrial ROS (mtROS) in 2-cell embryos were significantly higher than that of control group, and the expression of TFAM gene was also increased. The above results showed that the oxidative stress caused by AAPH resulted in embryo development block, the function of mitochondrial was promoted after treatment, with increased the mtDNA copy number and the expression of TFAM gene, which may participate in regulation of intracellular ROS dynamic balance in embryos. This study preliminarily explored the relationship between the ROS and the dynamic change of mitochondrial function, and might provide references for illustrating the mechanism of oocyte maturation and also optimization the culture system of mammalian embryos in vitro.

Glucose uptake in the mammary gland mainly depends on the glucose transporter (GLUT), and the different GLUT has different effects on glucose uptake. The aim of this study was to investigate the molecular mechanism of glucose transporter that participate in lactation regulation of yak (Bos grunniens). Mammary epithelial cells were cultured in vitro using pancreatic enzyme digestion method, and the immunocytochemistry method was used to appraise mammary epithelial cells by detecting the expression of cytokeratin 18, β-casein and vimentin; GLUT1, GLUT3 and GLUT8 gene were cloned by PCR from purified cells and its biological characteristics were also analyzed. The mRNA and protein expression of GLUT1, GLUT3 and GLUT8 was detected by qRT-PCR and Western blot, and their location in yak mammary epithelial cells were detected by indirect immunofluorescence. The results showed that cytokeratin 18 and β-casein could be detected in the isolated cells, and vimentin was negative, which indicated the cells were mammary epithelial cells. The GLUT1, GLUT3 and GLUT8 (GenBank accession number: KU902419, KX094556 and KX268646) gene were successfully cloned from mammary epithelial cells of yak, which contained a complete coding sequence, the length of nucleotide were 1 479, 1 485 and 1 437 bp, respectively, and encoded 492, 494 and 478 amino acids, respectively. Nucleotide sequence analysis revealed that GLUT1, GLUT3 and GLUT8 genes were conserved. The physical and chemical properties of protein encoded by 3 genes were similar, all of them were hydrophobic membrane proteins including 12 transmembrane helical region. The expression of GLUT1, GLUT3 and GLUT8 in mammary epithelial cells of yak were extremely significant difference (P＜0.01), GLUT1 was the highest, followed by GLUT8, while GLUT3 was the lowest. GLUT1, GLUT3 and GLUT8 protein were mainly located in the nucleus of in mammary epithelial cells of yak. The results provide important information for understanding the physiological function of yak glucose transporters and a new theoretical basis for further studying the biological function of yak lactation.

The fertility of cytoplasmic male sterility (CMS) can be restored by nuclear-encoded fertility restorer genes. However, the number and modifications of these restorer nucleor genes remain poorly understood. In this study, using QTL IciMapping software an intraspecific genetic linkage map of Capsicum annuum was constructed, and 2 major and 7 minor quantitative trait loci (QTLs) were identified, which acting as restorer-of-fertility (Rf) genes counteracting CMS. The heritability of the 2 major loci reached 92.58%. The first most important allele, qIF-3-1, contributed to fertility with a phenotypic variance explained (PVE) of 46.68%, with dominant and additive effects of 1.28 and 0.84, respectively. The other major allele contributing to fertility, qIF-5-1, was associated with a PVE of 47.10% and improved fertility with higher dominant effects of 1.76. Among 7 minor QTLs, 3 QTLs mainly acted as additive effect, while another 4 as dominant effect. These results are beneficial for our understanding of the mechanisms regulating the function of Rf genes. Moreover, our results provide guidance for selecting molecular markers in the development of marker-assisted selection (MAS) fertility restoration breeding schemes in peppers.

Eukaryotic translation initiation factor (eIF2α) plays an important role in the regulation of protein synthesis in eukaryotes. To investigate the function of eIF2α and explore its regulatory mechanism of protein synthesis in tobacco (Nicotiana tabacum), the recombinant vector pET15b-NteIF2α for prokaryotic expression of NteIF2α was constructed. The results showed that NteIF2α of tobacco K326 was successfully produced by Escherichia coli BL21-CodonPlus-(DE3)-RIPL strain under induction of 0.2 and 0.5 mmol/L isopropyl-β-d-thiogalactoside (IPTG) for 4 h at 28 ℃, and the obtained protein was identified by Western blot using anti-His antibody. Further, the expression condition was optimized and the soluble protein was obtained under induction of 0.2 mmol/L IPTG for 13 h at 16 ℃. Also, this protein was purified by Ni2+-NTA agarose colunm and gelfiltration column, and a single protein band was obtained. Finally, the polyclonal antibody of NteIF2α was prepared and its antibody titer was 1∶400 000 by enzyme-linked immuno sorbent assay (ELISA). The antibody was used for detection of NteIF2α in different organs of tobacco K326, and the results showed that the NteIF2α level was the highest in leaf and the lowest in root, stem and flower. Prokaryotic expression and purification of NteIF2α protein was successful, and highly specific NteIF2α antibody was prepared in this study. The results lay a foundation for further study the function of eIF2a and its roles in regulation of protein synthesis in plant.

Aconitum carmichaeli is a perennial herb of Aconitum in family Ranunculace, and it can be radix aconiti as medicine. Reports about Aconitum drug poisoning incidents have been very common, which are mainly caused by failure to identify species of Aconitum effectively. Through the research on development of Aconitum microsatellite primer and genetic diversity, the research realized fast and accurate identification of genetic resources and fake A. carmichaeli, which provided scientific guarantee for protection, development and successive research on superior genetic resources of A. carmichaeli. By extracting DNA of A. carmichaeli, establishing genomic library of A. carmichaeli, and sequencing A. carmichaeli with Illumina high-throughput sequencing technology, the research acquired plenty of 100 bp short-sequence microsatellite fragments. After screening the microsatellite fragments, microsatellite library of A. carmichaeli was established. According to the results, through second-generation high-throughput sequencing technology of Illumina, 38 942 660 strips of 125 bp pairing sequence were acquired. Through quality trim, 200 386 strips of splicing sequence were obtained after assembly. After filtration and screening, 12 095 strips of sequence fragments being larger than 299 bp were acquired finally. The fragments with repeat segment being larger than 100 bp were selected from microsatellite library of A. carmichaeli for primer design of microsatellite, and 55 pairs of microsatellite primers were obtained. By the use of these 55 pairs of microsatellite primers, amplification and polypropylene gel electrophoresis were carried out for 80 individuals from 4 wild Aconitum populations to detect the polymorphism of microsatellite primers and genetic diversity of population. After detection, 14 pairs of Aconitum microsatellite primers with clear stripe and rich polymorphism were acquired. For the 14 pairs of Aconitum microsatellite primers, average number of allele (A) was 4.85, average observation heterozygosity (Ho) was 0.613, average expected heterozygosity (He) was 0.583 and PIC was 0.528. Four sibling species of A. carmichaeli were used for universality detection of microsatellite primers. It was found that the stripe of 11 pairs of microsatellite primers were clear, and they occupied 78.57% of total microsatellite primers. According to the results of universality detection of Aconitum microsatellite primers, most of the microsatellite primers were proved amplifiable in sibling species of A. carmichaeli. Through analysis of genetic diversity of 4 A. carmichaeli populations, it was found that A. carmichaeli showed high genetic diversity at species level. Besides, for the Aconitum populations, A was 3.25, HO was 0.612, He was 0.493, Shannon's information index (I) was 0.851 and Nei's genetic diversity index (h) was 0.505. Genetic differentiation between populations showed that certain genetic differentiation (FST=0.149) existed in 4 populations of A. carmichaeli and gene flow between populations (NM)=1.432 was limited. According to research results, 14 pairs of microsatellite primers developed by the research showed high polymorphism and good universality, which could be used for identification of genetic resources, analysis of genetic diversity and construction of DNA fingerprint of A. carmichaeli. From the perspective of species level, A. carmichaeli had rich genetic diversity, which verifies abundant heritable variation ability of A. carmichaeli from DNA level and explained strong evolution potentiality of A. carmichaeli. The study will offer scientific guarantee for identification of genetic resources and fake A. carmichaeli, protection, development and successive research on superior genetic resources of A. carmichaeli.

The study was to investigate the lipid-lowering and anti-obesity function and mechanism of Malus xiaojinensis polyphenols (MXP). Hyperlipemia mice (Mus musculus) model was established by feeding high-fat diet and MXP gavage administration (50 and 100 mg/kg) for 7 weeks. The levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) in serum, the TG and TC in liver, the liver and spleen index were measured. At the same time, the transcription of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and the morphology of liver tissue were detected. Finally, the expression of related proteins (IRS, Akt) in the pathway of glucose and lipid metabolism was detected. The results showed that MXP could obviously resist mice body weight quick increase, MXP could significantly reduce the levels of TG, TC and LDL-C in the serum, TG and TC in liver, and increased the level of HDL-C in mice. MXP also could reduce the inflammatory cytokines including IL-6 and TNF-α and liver injury. Furthermore, MXP could improve the expression of IR and IRS significantly. The MXP seemed to improve the utilization rate of glucose and reduce the levels of inflammatory cytokines in liver, it has the potential to develop into a functional food and health care product. This study provides theoretical basis for scientific and rational development of M. xiaojinensis.

Bisecting N-acetylglucosamine (GlcNAc) act an essential role in a variety of biological processes, such as signal transduction, cell adhesion and cell migration. In this study, glycoproteins with bisecting GlcNAc structures were identified in normal mouse (Mus musculus) mammary gland epithelial (NMuMG) cells and mouse breast cancer cells (4T1) with lectin enrichment. Briefly, proteins from cell lines were reduced and alkylated, and digested with Lys-C/trypsin, and the peptides were subjected to PHA-E capture, PNGase F digestion and LC-MS analysis. 378 of bisecting GlcNAc N-glycosylation sites, 248 of glycoproteins were identified in two cell lines. The bisecting GlcNAc N-glycosylation consensus sequence was determined with WebLogo tool. Results showed that N-glycosylation consensus sequence containing NXS accounted for 38.9% of total N-glycosylation sites, the amino acid next to asparagine was most likely to be glycine and valine; consensus sequence containing NXT accounted for 61.1% of total N-glycosylation sites, the amino acid next to asparagine was most likely to be alanine and glycine. Gene Ontology classification of 248 glycoproteins with bisecting GlcNAc structures was performed as cellular component, molecular function and biological process with DAVID website using the whole Mus musculus proteome as background. The main cellular component categories were intrinsic to membrane, integral to membrane and plasma membrane; the major biological process categories were cell adhesion, biological adhesion and proteolysis; the top three most common molecular functions were ion binding, cation binding and nucleotide binding. For determination of the differentially expressed glycoproteins with bisecting GlcNAc structures in two cell lines, quantification of each glycosites was accomplished by comparative analysis between the intensity of MS signals derived from NMuMG and 4T1. Eighty glycoproteins were differentially expressed between NMuMG and 4T1. Fifty glycoproteins (including LAMP1 and LAMP2) with bisecting GlcNAc structures were up-regulated in 4T1 comparing to NMuMG, and 30 glycoproteins (including EGFR, ITA6 and ITA3) with bisecting GlcNAc structures were down-regulated. Functional enrichment analysis was performed to identify enrichment terms associated with the differentially expressed glycoproteins. The most highly ranked molecular function in the significant enriched GO terms of up- and down-regulated glycoproteins were respectively pigment qranule and integrin complex; the enrichment of GO terms of molecular function in up- and down-regulated glycoproteins were mainly hexosaminidase activity and calcium ion binding, respectively; the enrichment of GO terms of biological process in up- and down-regulated glycoproteins were mainly heterophilic cell adhesion and integrin-mediated signaling pathway, respectively. Functional network analysis of differentially expressed glycoproteins were performed by uploading protein lists to the String website. And we found that cell adhesion, lysosome and PI3K-AKT signaling pathway was involved. Glycoproteomics analysis based on lectin enrichment could profile differentially expressed glycoproteins during the disease process, and provide essential information for disease diagnosis, treatment and prognosis.

The objective of this study was to review the breast tissue structure and Insulin-like growth factor I receptor (IGF-IR) level of polythelia teats Hu sheep (Ovis aries)(test group), and then detected whether there were some differences between Hu sheep with 2 teats (control group) and polythelia teats and had a potential higher performance of lactation. In this study, the breast tissue samles were taken from 2 groups of Hu sheep during incubation, pregnancy, lactation and nonpregnant period. The changes of mammary gland tissue morphological between 2 groups during different development stages were observed by hematoxylin-eosin staining (HE), the qRT-PCR was used to detect the relative mRNA expression levels of insulin-like growth factor-I receptor gene (IGF-IR). The distribution of receptor protein was detected by immunohistochemistry (IHC). Western blot was used to measure the protein level in mammary gland during different stages. The main findings were showed as follows: During incubationperiod, the mammary stromal was rapidly growthed between the 2 groups, and also contained lots of connective tissue and adipose tissue, in connective tissue, numbers of catheter were denser; during pregnancy period, a large number of gland bubbles were rapidly growed and developed, lobular acini acinus cavity was filled with secretions, and that numbers of gland bubble in test groups were more than that in control groups. The volume of gland bubble was the largest between test and control groups during lactation, and gland bubbles were gradully collapsed and morphological structure of breast was changed after weaning. During lactation, the size and shape of gland bubbles were different, and acinar cavity were reched in milk, the volume of gland bubbles were bigger. The mRNA expression level and protein distribution quantity of IGF-IR intest groupgradually increased from incubation to lactation, however, it was gradually increaseed from incubation to pregnancy and slightly lower than that in pregnancy during lactation period; the expression level in nonpregnant period was lower than those in lactation. And during the entire process of physical development, the proteins of IGF-IR were mainly distributed in mammary epithelial cells and connective tissue between the test and control groups. The protein trend of IGF-IR in breast tissues of Hu sheep was consistent with its mRNA expression level. In summary, not only the breast tissue of Hu sheep with polythelia was more developed, but also the mRNA levels of associated with breast development hormone receptor gene and protein expression levels were higher than those in control groups. It is showed that providing theoretical support for cultivating high-yielding Hu sheep breeds.

Follicle stimulating hormone receptor (FSHR) is involved in follicle growth, development, differentiation and maturation, and is related to reproductive traits of animals, but the influence on genetic mechanism of reproductive traits in guizhou native goats is still unclear. To determine the correlation between polymorphism and mRNA expression levels of FSHR gene and reproductive traits in QianBei Ma goat (Ovis linnaeus), the high prolificacy breed QianBei Ma goat was selected as the candidate subject, and the low prolificacy breed Guizhou Black goat was selected as control, and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR direct sequencing were used to detect the polymorphism in exons 1 and 10 of FSHR gene. The results showed that the C126T and C1246A were identified in exon 1 and 10 of FSHR gene in two breeds. The litter size with genotype BB were significantly higher than genotype AA and AB of QianBei Ma goat (P＜0.05). However, there were no significant difference among different genotypes at two mutation sites in Guizhou Black goat (P＞0.05). Moreover, the mRNA expression levels of the FSHR gene in hypothalamus, pituitary, ovaries, fallopian tubes and uterus tissues in dioestrus and estrus of QianBei Ma goat with AA and BB genotypes were investigated by qRT-PCR, and the expression of FSHR gene in each tissues of BB genotype were higher than that of AA genotype QianBei Ma goat. The expression of the FSHR mRNA of BB genotype in hypothalamus of dioestrus and the ovaries of estrus were extremely significantly higher than that of AA genotype (P＜0.01). The significant differences of FSHR mRNA expression was also observed in uterus during estrus between two genotypes (P＜0.05). The results indicated that FSHR gene might play an important role. The results indicated that FSHR gene might play an important role in regulation of litter size in QianBei Ma goat. The results provide basic data for exploring candidate gene effecting reproductive traits of QianBei Ma goat and constructing prolific nucleus group in Guizhou native goats.

Krüppel-like factors (KLFs) is a group of zinc finger protein which is important for gene transcriptional regulation in eucaryote. The aim of the study was to clone the CDS of KLF13 gene, and to determine the abundance of KLF13 mRNA expression in various tissues and differential stages of preadipocytes, which may provide the basic information about the fuction of KLF13 gene on regulating preadipocytes differentiation. Four 24-month-old healthy Jianzhou Big-Eared male goats (Capra hircus) were selected randomly in this study. After slaughtered, the tissue samples of heart, liver, spleen, lungs, kidney, subcutaneous fat, longissimus dosi muscle, biceps femoris muscle and triceps brachii muscle were collected for the total RNA extraction. The sequence of KLF13 gene was cloned by RT-PCR and analyzed using bioinformatics. The mRNA expression of KLF13 in various tissues, preadipocytes (0, 3, 5, 7 d) and myoblasts (0, 2, 3, 4, 5 d) were determined using qRT-PCR. A length of 991 bp cDNA of KLF13 gene was cloned (GenBank No.: KU041755), which contains 876 bp CDS, 12 bp 5' UTR and 103 bp 3' UTR, encoding 292 amino acids. The goat KLF13 protein shared 100%、97.9%、96.6% and 90.8% similarity with that of sheep (Ovis aries), bovine (Bos taurus), pig (Sus scrofa) and mouse (Mus musculus), respectively. Goat KLF13 mRNA sequence was closest to that of sheep, following by bovine, human (Homo sapiens), pig and mouse. The result of tissue expression showed that KLF13 gene was expressed high in spleen and lungs, and low in muscle. The expression of KLF13 was continually suppressed significantly with the differentiation of goat preadipocytes. However, excepted at 5 d, the expression of KLF13 was enhanced with the differentiation of myoblasts. These results showed that KLF13 gene may be involved in preadipocytes differentiation, and also will be beneficial for studying the role of KLF13 in regulating preadipocytes differentiation in goat.

Porcine oocytes in vitro maturation and embryos in vitro culture are of great importance for somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF) and isolation of porcine embryonic stem cells. Optimizing the in vitro culture medium will greatly facilitate the development of SCNT and IVF embryos. Glutamine (Gln) is the most abundant amino acid in porcine follicular fluid and is constantly used as an energy substrate throughout embryos development. But Gln is inherently unstable and spontaneously breaks down into ammonia, therefore interferes with proper development. This study compared the maturation efficiency and developmental competence of porcine oocytes and embryos, which were produced by parthenogenetic activation or SCNT and cultured in alanyl-glutamine (Ala-Gln) dipeptide or Gln containing medium. The results showed that Ala-Gln had no obvious effect on pig oocytes maturation((45.94±2.38)% vs (45.06±2.43)%), but improveed blastocyst formation rates((20.81±3.65)% vs (13.87±4.72)%) and blastocyst cell number (51.00±4.23 vs 37.71±3.94) of parthenogenetic embryos. At the same time, Ala-Gln could significantly improve the blastocyst formation rates((25.28±1.26)% vs (17.27±1.29)%) and cleavage rates((41.58±8.90)% vs (31.20±5.56)% for 24 h cleavage rate and (54.38±9.20)% vs (41.83±9.04)% for 48 h cleavage rate) of SCNT embryos. This study provides a new method to improve development competence of pig SCNT embryos.

Overexpression of thymosin beta 10 (Tβ10) gene is detected in many of neoplastic tissues and cell lines compared to the respective normal tissue types. So Tβ10 is considered to be an anticipated marker for tumorigenesis and tumor progression. Deer (Cervidae) antlers are not a cancerous organ with the astonishing growth rate up to 2 cm/d. Tβ10 gene was highly expressed in the growing tips of antlers by using in situ hybridization. Therefore, tests of the effects on biological properties (proliferation, migration and senescence) of endogenous Tβ10 gene expressed in the antler stem cells by using a lentiviral vector were performed in this study. In the experiment, antlerogenic periosteum (AP) tissue from a 8 months old male sika deer was collected by using surgical operation in a relative sterile environment and primary culture for AP cells was carried out by using typeⅡcollagenase. Total RNAs of AP cells were extracted by using PureLinkTM mini RNA kit and Tβ10 gene was cloned using RT-PCR. AP cell lines with the overexpression of Tβ10 gene were successfully established by using lentiviral vector guidance system and determined the effects on proliferation, migration and senescence, which may be influenced by endogenous Tβ10. AP cells could proliferate in vitro, morphology of these cells was bipolar spindle shaped under a phase contrast microscope. Tβ10 gene was cloned accurately based on sequencing and inserted into the lentiviral vector plvx-ires-mCherry-puro successfully. Recombinant lentivirus was packed and produced successfully by using co-transfection of three-plasmid lentiviral vectors and X-treme GENE HP transfection reagents in ERK 293T cells. Transfection efficiency was evaluated through the expression of red fluorescent protein (RFP) by using an inverted fluorescence microscope. The lentivirus from culture medium were collected and concentrated by using Lenti-X concentrator regents, and the titer of lentivirus achieved 4×108 TU/mL detected with 293T cells. AP cells infected by recombinant lentivirus were selected and enriched by applying puromycin (2.5 μg/mL) in culture medium for 10 days. AP cell lines with overexpression of Tβ10 gene were established and identified by qRT-PCR and Western blotting. The results of methyl thiazolyl tetrazolium (MTT) assay showed that Tβ10 gene inhibited proliferation of AP cells significantly (P＜0.01), compared to the control; β-galactosidase staining showed that Tβ10 gene promoted senescence of AP cells significantly (P＜0.01) and the migration assay showed that Tβ10 gene inhibited human (Homo sapiens) umbilical vein endothelial cells (HUVEC) migration significantly (P＜0.05). This study would lay the foundation for revealing the underlying mechanism of rapid growth of deer antlers without becoming cancerous.

The aim of this study was to obtain the complete coding region sequence (CDS) and explore its expression in different tissues of the myocyte-specific enhancer binding factor 2A gene (MEF2A) in duck(Anas platyrhynchos). In this study, Xingyi ducks (A. platyrhynchos) were chosen to the samples for the coding region cloning of MEF2A gene by the Real-time fluorescent quantitative PCR (qRT-PCR) and A/T cloning technology, and we obtained the pGEM-T-MEF2A recombinant plasmid successfully, and the mRNA expression of MEF2A gene in different tissues by qRT-PCR was also researched. The results showed that the coding sequence of the MEF2A gene in Xingyi duck was successfully cloned, the length of the open reading frame sequence was 1 479 bp, and 5 SNPs were found. The qRT-PCR results showed that the MEF2A gene had the similar expression mode in pectoral, thigh muscle and heart. They all had the same trend that from rise to decline, but there was different expression in gender. The expression amount of the female duck in myocardium showed that, it decreased from 0 to 30 d age, then it rised from 30 to 90 d, it decreased again after 90 d, whereas the expression of the myocardium in drake was keeping descending. The expression of pectoral in different sex duck had the same pattern, it decreased from 0 to 30 d age, and it began rised from age of 30 d, it reached maximum value at 120 d, after that, it decreased again.The expression amount of thigh muscle and myocardium were similar, they were both keeping the downward trend between 0 and 150 d age. The expression amount of thigh muscle in female duck was from the decline to a little rise, and then keeping the downward state. Although the MEF2A gene existed expression difference in different sex, on the whole, the expression of the myocardium and skeletal muscle was higher than that in smooth muscle. The results of these experiment showed that the MEF2A mRNA could be expressed in different tissue of Xingyi duck, and it existed some difference. And this study also found that the expression trend between female and male ducks in different tissues of the same growth stage was not consistent, moreover, it also existed significant expression difference in different growth stage of the same tissues. Therefore, this study provides the basis for the construction of prokaryotic expression vector and tissue expression profile of MEF2A gene in the follow-up research.

Blue shell trait is not only a protective coloration that can avoid predation, but also is one of the important economic traits of poultry. Due to the differences of evolutionary and genetic backgrounds, the formation mechanism of blue shell egg are divergent in different poultries. By using the high-throughput sequencing, the oviduct shell gland of blue egg shell ducks and white egg shell ducks were studied to enrich and analyze microRNAs (miRNAs) and its target genes involved in formation of blue shell egg traits. The analysis result of sequencing data quality showed the GC content of each sample was not less than 42.60%, and the proportion of clean reads that was ≥ 30 bases (Q30) was not less than 89.02%. Compared with the chicken (Gallus gallus) genome sequence (Gallus_gallus-4.0, GCF_000002315.3) using the miRDeep2 software, all 269 miRNAs were screened in the shell gland of blue shelled ducks and white shelled ducks, including known 245 miRNAs and new 24 miRNAs. Based on the expression of miRNAs in each sample, the correlation analysis was performed between the all of each samples. The result showed that the correlation coefficient were higher between all of the shell gland samples in blue and white shelled ducks (r＞0.99), which suggested that miRNAs expression profiles might be similar in the shelled gland of blue and white shelled ducks. Compared with white shelled ducks, the shell gland of blue shelled ducks had 169 differently expressed miRNAs, including 85 up-regulated and 84 down-regulated, and total 4 872 target genes were obtained, but the miRNAs were not significant difference (P＞0.05). The Top Gene Ontology (TopGO) analysis of target genes showed that molecules function including heme binding (count=23, P=0.008) were existed, and most of them were accompanied by function of electron and Fe2+ transport. In addition, the analysis of differently expressed miRNAs found that biliverdin synthetic gene — heme oxygenase 1 (HMOX1) and a large number of organic matter transport and metabolism genes were predicted by gga-miR-133b, gga-miR-193b-3p and gga-miR-449a (log2 (fold change (FC))＞1, P＞0.05). These results suggested that some miRNAs might participate in the formation of blue shell egg traits, but miRNAs might not be an important role in the formation of blue shell egg traits in duck. The study provides a theoretical basis for revealing the molecular mechanism of the formation of the blue shelled eggs in poultry, and basic information for the poultry cultivation of blue shelled eggs which based on gene manipulation technology.

Ganoderma leucocontextum is morphologically similar to certain species from its genus in nature. And the so-called distinguishing white flesh within its basidiocarp seemed non-unique according to Ganodermataceae sampling around Southern China. ITS region was reported a DNA barcode marker for macrofungi, anunique DNA markers are yet expected for species identification within genus or genus group. This article designed pair of specific PCR primers for G. leucocontextum identification. Mycelium samples of the 17 selected species phylogenetically closed to G. leucocontextum were harvested and their ITS were isolated and amplified, based on the universal primers. The alignment result showed multiplenon-conservative sites located at 120~140 bp and 450~470 bp. Two oligo sequences, BRLZ-F and BRLZ-R, were designed to match the 2 differentiate fragments as the forward and reverse primers for G. leucocontextum PCR identification. BRLZ-F and BRLZ-R bind efficiently to the DNA templates at 58 ℃ in annealing and only a 348 bp fragment was capable for amplification. Confirmatory experiment illustrates BRLZ-F and BRLZ-R is effective for G. leucocontextum identification within the Ganoderma genus.

Citrus tatter leaf virus (CTLV), a species of the family Capillovirus, is an important citrus (Citrus reticulate) virus and aquarantine object in obtaining virus-free citrus seedlings, which causes significant losses to citrus industry worldwide. It is very important to prepare sensitive and specific monoclonal antibodies (MAbs) against CTLV for the diagnosis and detection of CTLV in citrus groves. For this purpose, the 714 bp coat protein gene (CP) of CTLV was cloned from a CTLV-infected citrus sample collected from citrus grove in Chongqing municipality, which shared 100% nucleotide sequence identity with a Chinese isolate of CTLV deposited in GenBank. The CTLV CP then was cloned into the His-tagged prokaryotic expression vector, pET-28a. The resulting expression vector was transformed into Escherichia coli BL21 (DE3) strain. After induced by isopropylthio-β-D-galactoside (IPTG), E. coli BL21 (DE3) cells harboring the recombinant vector expressed an approximately 30 kD fusion protein. The recombinant fusion protein was purified with Ni2+-NTA agarose and used to immunize BALB/c mice (Mus musculus). Three hybridoma cell lines (6C5, 14E11 and 15F8) secreting MAbs against CTLV were prepared by fusing mouse myeloma cells (SP 2/0) with spleen cells from the immunized BALB/c mouse. The hybridomas were injected into pristine-primed BALB/c mice to prepare the ascetic fluids contained the MAbs. The titers of 3 MAbs in ascitic fluids ranged from 10-6 to 10-7 in indirect-ELISA. Isotypes and subclasses of all 3 MAbs belonged to IgG1, κ light chain. The IgG yields of 3 MAbs (6C5, 14E11 and 15F8) in ascetic fluids were 9.41, 7.83 and 9.93 mg/mL respectively. Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) analysis indicated that the 3 MAbs could specifically react with CTLV-infected citrus leaf and CTLV-infected Chenopodium quinoa leaf crude extracts, had negative reactions with Citrus tristeza virus, Apple stem grooving virus, healthy citrus leaf and Chenopodium quinoa leaf crude extracts. Western blot demonstrated that all 3 MAbs could specifically react with an approximately 30 kD CP of CTLV in CTLV-infected citrus leaf crude extracts, but no hybridization signal was observed on the lane of healthy citrus leaf crude extracts. A dot enzyme-linked immunosorbent assay (dot-ELISA) was developed using the prepared 3 MAbs, and the established dot-ELISA could successfully detect virus in plant sap diluted at 1∶640 (W/V, g/mL). Fourteen grove samples collected from Chongqing municipality and Zhejiang Province were tested for CTLV infection using the dot-ELISA and RT-PCR. The detection results indicated that the developed dot-ELISA could accurately, reliably and sensitively detect CTLV in citrus trees in groves. The prepared anti-CTLV MAbs and the developed dot-ELISA provide technical and material support for the diagnosis, production of virus-free citrus seedlings and scientific prevention and control of CTLV in citrus groves in China.

Deer antlers are the only mammalian organs that can fully grow back once lost from their pedicles, the permanent bony protuberances. Studies have demonstrated that it is the proliferation and differentiation of pedicle periosteal cells (PPCs) that give rise to the antler blastema, from which antler regeneration takes place. PPCs express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro, so are called antler stem cells. Further studies have found that PPCs can initiate antler regeneration only when they have interacted with the pedicle skin. Histologically, the process of early antler regeneration resembles that of healing of a mouse (Mus musculus) leg stump wound. However what sets these two apart is the difference in proliferation potential between the PPCs and the periosteal cells of the mouse long bone. We believe that if we can impart a greater proliferation potential to the long bone periosteal cells, we might be able to achieve the dream of regenerating limbs in mammals including humans. This article has reviewed the progress on histogenesis of antler regeneration including the inner and outer tissue components and antler stem cell research, made comparison between regenerations of deer antlers and newt limbs and identified their similarities and differences, pointed out the future direction toward the possible use in clinics, and provides new way of thinking for how to ultimately solve the problem of human limb regeneration.

VQ protein family is a plant-specific transcription regulation cofactor, which plays an important role in regulation of plant growth, development and responses to various external environment stresses and it is named after the invariant valine-glutamine (VQ) dipeptide. SIB1 (sigma factor binding protein 1) was the first VQ protein that found from Arabidopsis thaliana in 2002. Subsequently, VQ multi-gene families had been identified in Arabidopsis thaliana, rice (Oryza sativa), soybean (Glycine max), grape (Vitis vinifera), Chinese cabbage (Brassica rapa), maize (Zea mays) and so on. The analyses of protein characteristic show VQ proteins contain a highly conserved VQ domain ‘FxxxVQxLTG’ (where x is any residue), however, the amino acid sequences of other regions have diversity. In addition, basing on the difference of L and G residues, VQ domains of different plant VQ proteins could further have been divided into different types. For example, there are six (LTG, LTS, LTD, FTG, VTG and YTG) and four (LTG, VTG, FTG and ITG) types of VQ proteins in Arabidopsis thaliana and rice, respectively. Further results indicate more than 80% VQ proteins contain 300 amino acids or less and most proteins are localized in the nucleus. The analyses of gene structure display more than 80% VQ genes have no intron. Furthermore, VQ genes are dispersedly distributed on all chromosomes of plants except for several chromosomes of soybean and grape. The researches of VQ gene functions show that not only they participate in regulating the growth and development of seed, hypocotyl, flower and leaf, but also play an important role in response to the stresses of drought, salt, temperature and pathogen. For example, in Arabidopsis VQ genes, over-expression of VQ8, VQ10, VQ17, VQ18 and VQ22 inhibited plant growth; vq14 mutant cause the production of small seeds; over-expression of VQ29 influence the length of hypocotyls and flowering time; VQ9 and VQ15 impact plant response to high salt and osmotic stresses and VQ4, VQ12, VQ16, VQ21, VQ22, VQ23 and VQ29 affect plant resistant to pathogen infection. The researches of regulation mechanisms indicate that VQ proteins widely take part in regulating plant various physiological and biochemical process through interacting with other proteins, such as interacting with WRKY transcription factor to affect plant seed size and resistance to salt injury and pathogen infection, such as VQ14-WRKY10, VQ9-WRKY8, VQ22-WRKY28/51 and VQ23-WRKY33; forming ternary complex with MAPK and WRKY to provide specific, such as VQ21-MPK4-WRKY33, accurate and effective regulation mechanism for plant response to biotic stress, VQ29 interacting with phytochrome binding factor to impact plant photomorphogenesis, and also forming homologous or heterologous dimer among these VQ proteins. In addition, the researches of VQ domain discover that it playing an important role in biological functions of VQ gene family, and it also influences cellular localization and protein interaction, for example, VQ domain of VQ14 influence the production of small seeds and protein interaction, VQ domain of VQ9 affect subcellular localization and protein interaction. Here, this review focused on VQ protein, genetic characteristics, biological function and regulation mechanisms, which was aimed at providing us with some hints and inspiration for further study of plant VQ proteins.

DNA methylation is an essential epigenetic modification closely correlated with the mechanisms underlying cell growth, differentiation and transformation in eukaryotes. Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. With the development of technology and further study of DNA methylation, more and more new technologies have been used in the detection of DNA methylation. In this review, this study introduced some typical and novel technologies in different methodologies of DNA methylation analysis. A description of the development of these methods in principle, processes, strengths and weaknesses, optimization and other aspects were given for further study of the detection of of DNA methylation. A description of the development of these methods in principle, processes, strengths and weaknesses, optimization and other aspects were given to offer more options for DNA methylation detection in disease and cancer related study.