Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of hnRNP complex, can interact with nucleic acids and various proteins. The structure of hnRNP K protein mainly contains three K homology (KH) domains that can combine with DNA or RNA, and one K protein interactive region (KI) domain involved in protein interaction. It also contains a nuclear localization signal (NLS) in the N terminal that mediates its transport from the cytoplasm to the nucleus and a nuclear shuttling domain (KNS) in the C terminal that confers the ability to translocate bi-directionally through the nuclear pore complex. In eukaryotes, hnRNP K expresses in many type of cells and tissues, but the expression levels vary during different developmental phases, and it is located predominantly in the nucleus and cytoplasm, also in mitochondria and plasma membrane. In addition, hnRNP K is subject to several post-translational modifications, such as phosphorylation, methylation, sumoylation, ubiquitylatio, acetylation, and so on, which can regulate its interactions with different molecules and influence its functions. The complexity of structure, expression, location and the diversity of post-translational modifications in hnRNP K decide its abundant biological functions, which can participate in transcription, translation, RNA splicing, DNA repair, chromatin remodeling, and also have important roles in spermatogenesis, nervous system and ovary development, organogenesis and carcinogenesis process. Among them, spermatogenesis is tightly regulated and well-studied process consisting of a series of highly specific cellular processes and multiple genes involved in the final formation of the sperm. Many research have shown that hnRNP K may play a key role in spermatogenesis regulation by its special structure, selective splicing and expression pattern. This review focuses on the structure, expression regulation, post-translational modification and biological functions of hnRNP K, and its roles in spermatogenesis. It provides initial value?for?studying?the?biological?functions?of?hnRNP K especially in spermatogenesis.

The genes of pear (Pyrus) cultivars from different areas are different due to the natural self-incompatibility character. In order to identify the diversity of different cultivars and explore the important genetic traits, 134 core simple sequence repeat (SSR) markers with high polymorphism which covered all 17 lingkage groups of pear were used to study the genetic variability and the group structure of 45 European pears (Pyrus communis L.). 673 alleles were detected through all SSR primers, with mean of 5.02 per SSR primer. The mean of observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and Shannon's information index (I) were 5.02, 3.84, 0.73, 0.72 and 1.42, respectively. The results of clustering analysis and the genetic similarity coefficient showed that the 45 cultivars had high genetic diversity and the evolution trend of the varieties was even. The cultivars distributed in Europe and America and intertwined with each other, and had no much difference due to different location, which might reveal the close relationship with genetic background of Europion pears. The species with unknown sources of Kujieli, Feilaiyin and Dilibairui might originate from Western Europe. According to the analysis of population structure, when the K-value was 2, the population of European pear was divided into 2 groups: Pop1 and Pop2, and cultivars of Clapp's Liebling, Bo12, Radana, Dilibairui, Red Anjou and Le Conte showed high heterozygosity. Fingerprint identification of European pears showed that at least two markers should be used to separate different cultivars. The results provide scientific evidence and perfecct markers for comprehensively evaluating the genetic background and characteristic as well as identifying different germplasm correctly, and also provide basic data for protection of germplasm resources utilization and genetic breeding.

Acidobacteria is an important phosphorus removal bacteria in the sequencing batch reactors (SBR) system, and its effects on phosphorus removal process should not be ignored. Sludge retention time (SRT) is an essential parameter of the design and operation of the SBR process. If the SRT changed, the phosphorus removal effect of SBR would also change. In order to inspect the phosphorus removal effect, the SRT of SBR system was tested for 10, 15, 20 and 30 d. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial community structure of acidobacteria in the activated sludge. The results indicated that the removal rate of total phosphorus reached 90%, 74%, 52% and 31% at the sludge of 10, 15, 20 and 30 d, respectively. The removal effect was the best at the sludge of 10 d, and short sludge age was more advantageous to the total phosphorus removal. Acidobacteria was sensitive to the changes of the sludge age conditions, and the dominant strains of the Acidobacteria changed frequently. The diversity index reached 0.87 at the sludge of 10 d, and dropped to 0.81 at the sludge of 30 d. The results indicate that the Acidobacteria is more adapting to the environment of long sludge age.

Wap65-2 (warm temperature acclimation related 65 kD protein-2), a specific plasma glycoprotein in teleosts, plays a key role in fish immune response against pathogens. In the study, the large yellow croaker (Larimichthys crocea) Wap65-2 gene (LcWap65-2) was obtained with de-novo transcriptome sequencing of large yellow croaker liver. The cDNA sequence of LcWap65-2 (GenBank accession: KJ412463) was 1 585 nucleotides, which contained a large open reading frame that encoded a protein of 432 amino acids with a deduced molecular mass of 48.84 kD and a theoretical isoelectric point (pI) of 5.36. The N-terminus of LcWap65-2 contained a predicted signal peptide of 20 amino acids. Multiple sequence alignment of complete amino acid sequences showed that LcWap65-2 had the typical characters of fish Wap65-2. Sequence comparsion showed that LcWap65-2 shared the highest amino acid sequence identity (87.4%) with that of miiuy croaker (Miichthys miiuy). Phylogenetic tree analysis also confirmed that LcWap65-2 falled into the fish Wap65-2 cluster and was most closely related to that of miiuy croaker. Genome structure analysis showed that LcWap65-2 gene (GenBank accession: KJ412464) contained 10 exons and 9 introns. Real-time quantitative PCR (qRT-PCR) analysis showed that LcWap65-2 mRNA had the highest expression level in liver, and little amount in brain of the healthy large yellow croaker. Upon Vibrio alginolyticus infection, LcWap65-2 transcripts significantly upregulated in the liver, and reached its peak at 8 h. Then the mature peptide of LcWap65-2 (LcWap65-2m) overexpressed and the antiserum against LcWap65-2m was prepared. Western blot assay demonstrated that the serum LcWap65-2 showed no obvious difference at 4 h but significant increased at 8, 12, and 24 h in large yellow croaker infected with V. alginolyticus. In summary, our present study revealed a tight relationship between the LcWap65-2 expression and V. alginolyticus infection, which suggested that LcWap65-2 might play an important role in fish immune response against bacteria infection. The present study provides a theoretical basis for studying the functions of fish Wap65-2, and its molecular mechanism in regulating fish immune response to pathogens.

AM79 aroA (WO/2009/059485), an novel 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) gene, is isolated from glyphosate-polluted soil using metagenomics method and showed high glyphosate resistance. However, the function mechanism of the high glyphosate resistance of AM79 aroA remains unknown. Site-directed mutagenesis to uncover the function of some specific amino acids in AM79 EPSPS was performed. Phylogenetic tree analysis showed that AM79 EPSPS was a typeⅠEPSPS. Sequence alignment indicated that the 107th Alanine (Ala), the 114th Phenylalanine (Ala), the 355th Alanine and the 356th Histidine (His) were symbols to distinguish the AM79 EPSPS from other EPSPS. To explain the function of these specific amino acids in AM79 EPSPS, point mutations were carried out via overlap extension PCR. AM79 aroA and the mutant gene were transformed into aroA defective strain ER2799, then the glyphosate resistance of the transformed strains were identified. The results showed that mutants of Phe114, Ala355 and His356, respectively, led to the decrease of glyphosate resistance, indicating that these amino acids were essential for the enzyme activity and glyphosate resistance of AM79 EPSPS. Homology modeling analysis showed that the mutation of these amino acids changed the structures of the mutated AM79 EPSPS and this might be the reason for the decreased glyphosate resistance. We hypothesized that the highlighted amino acids in AM79 EPSPS might have similar function in other typeⅠEPSPS and reverse mutations on these amino acids of Pseudomonas fluorescens G2 EPSPS and Arabidopsis thaliana EPSPS (AtEPSPS) were performed. However, the mutations of these key amino acids didn't enhance the glyphosate resistance of G2 EPSPS and AtEPSPS. This study can be useful for enhancing the glyphosate resistance of AM79 EPSPS by directed evolution process.

Ilicis Chinensis Folium is a traditional Chinese herbal medicine, which derived from one kind of ornamental plants named Ilex chinensis. In order to identify adulterants emerged in the market and closely related species in Ilex rapidly and reliably by DNA barcoding, ribosomal internal transcribed spacer 2 (ITS2) from 37 materials of I. chinensis, Ilex rotunda, Ilex asprella, Ilex cornuta and Ligustrum lucium was amplified and sequenced, respectively. Assembled by CodonCode Aligner 4.2.7, all of the 37 ITS2 sequences ampilified and 28 GenBank sequences of Ilex, L. lucium, Symplocos setchuensis were annotated based on hidden Markov model (HMM) to cut off 5.8S and 28S so that complete ITS2 region could be gained. Kimura 2-parameter (K2P) genetic distance analysis of intraspecific and interspecific accompanied by neighbor-joining tree (NJ tree) construction was performed by molecular evolutionary genetics analysis (MEGA6.0). Results indicated that unique bands were obtained from gel electrophoresis demonstrating the successful amplification of ITS2 sequences by the universal primer. Sequencing results showed that the aligned sequence length of I. chinensis was 239 bp and the interspecific one was 269 bp. Four variable sites of intraspecies were discovered, which divided the I. chinensis into 6 haplotypes. In contrast, there were 143 interspecific variable sites which were much more than those of I. chinensis. Sequence characteristics analysis of intraspecific and interspecific by MEGA6.0 showed the maximum K2P genetic distance of intraspecies was 0.013, which was much shorter than the minimum K2P genetic distance of interspecies of 0.094. L. lucium and S. setchuensis clustered into separate clade with bootstrap scores both of 100%. I. chinensis distributed to a single cluster that belonged to the cluster of the Ilex. Species in the same subgenus of Ilex could also cluster together. Therefore, ITS2, as one of the universal DNA barcodes, could differentiate I. chinensis from its adulterants and closely related species effectively. As a molecular biological method for discrimination, DNA barcoding possessed huge potential to authenticate the Ilex, meanwhile, it provides scientific foundation for clinical medication safety, exploration and application.

The high temperature is an important threat to aquaculture of rainbow trout (Oncorhynchus mykiss). In the present research, the effects of heat stress and recovery on non-specific immunity parameters were investigated in rainbow trout with average weight of 350 g under the laboratory conditions. Samples for detections of respiratory burst (RB), complement protein 3 (C3) content, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, cortisol (COR) content, catecholamines (CA) content, alkaline phosphatase (AKP) activity and lactate dehydrogenase (LDH) activity were taken before heat stress (18 ℃, 0 h), 2, 4, 8 and 12 h after stress (25 ℃) and heat stress recovery (18 ℃, heat stress recovery(HSR)), respectively. The results showed that compared with before stress, the stress group had the following significantly (P＜0.05) increased parameters: RB activity at 8 and 12 h; serum SOD activity at 2~12 h; MDA level at 4 and 12 h; C3 level at 2 and 12 h; AKP activity at 2, 8 and 12 h; the content of COR and CA at 8 h. The activity of LDH in the serum decreased significantly (P＜0.05) at 8 h after heat stress. Compared with the stress group, the HSR had the following variations: RB activity had no significant change (P＞0.05); MDA and LDH level were significantly lower than that of 4 and 12 h under heat stress (P＜0.05); C3 level was lower than that of 2 and 12 h of stress (P＜0.05); SOD activity was lower than that of 12 h under heat stress (P＜0.05); COR and CA level were lower than that of 8 h under heat stress (P＜0.05); AKP was at the same level as that of 2, 8 and 12 h under heat stress (P＞0.05). Compared with data before stress, the HSR had the following variations: RB, SOD, COR and AKP increased significantly (P＜0.05); C3, MDA, CA and LDH recovered to the level before stress (P＞0.05). These results indicated that 25 ℃ heat stress significantly affected the non-specific immunity of rainbow trout. The increase of RB and AKP indicated that heat stress could cause inflammation to rainbow trout. Most parameters decreased in the HSR group, especially, C3, MDA, CA and LDH reduced to the levels of pre-stress, which deduced that rainbow trout could restore normal function gradually under optimal temperature after 25 ℃ heat stress. However, the content of MDA significantly increased after heat stress and the high level in HSR group of AKP indicated that the fish cells were damaged. The death fish after only 2 h heat stress, showed that 25 ℃ approached to the upper limit of tolerance to temperature for rainbow trout. The above results provide basic data for resistance regulation to heat stress in rainbow trout and help producers taking good preventive measures based on better understanding about immune physiology and the adverse effects assess under heat stress.

Start codon targeted polymorphism (SCoT) is a novel genetic marker technique. In this study, a single factor experiment method was designed to optimize and establish a SCoT molecular marker system for pear (Pyrus spp.). Furthermore, the diversity and genetic relationship of 43 pear cultivars, which were collected from Dangshan, Anhui Province, were analyzed with the optimized SCoT system. The results showed that the optimized SCoT-PCR in pear was as follows: A total volume of 25 μL-reaction system including 2.5 μL 10×Easy Taq buffer (with 2 mmol/L Mg2+), 30 mg/L template DNA, 1.0 μmol/L primer, 0.2 mmol/L dNTPs and 1.0 U Taq polymerase. Sixteen primer combinations were selected from 67 SCoT primers for their amplification with clear bands and significant difference. A total of 145 bands were amplified, in which 126 bands were polymorphic (86.1%) with 9.1 bands per primer on average. The optimized SCoT-PCR system was applied to 43 pear samples. The result showed abundant polymorphism in the tested pear. The value of genetic similarity coefficient was ranged from 0.517 to 0.931, with the average of 0.685. Moreover, the 43 pear varieties could be classified into 2 groups including A and B based on the similarity of 0.610 using the cluster analysis. When the genetic similarity coefficient was set at 0.746, group A could be divided into 6 subgroups (Ⅰ~Ⅵ). These results indicated that the tested 43 pear varieties had relatively high genetic diversity and could be distinguished from each other by SCoT molecular marker technology, which provide basis for further research and application on genetic resources in pear from Dangshan, Anhui province.

Myogenin gene (MyoG) is a member of myogenic regulatory factors (MRFs) family and plays an important role in regelation of muscle building. To explore the expression profile and expression pattern of MyoG gene and its possible mechanism of action, a pair of fluorescence quantitative primers according to the cloned coding sequence (CDS) of MyoG in Jinghai yellow chicken (Gallus gallus) was designed. Real-time fluorescent quantitative PCR was used to detect the expression level of MyoG in different tissues to draw the expression profile. Then, the expression levels of MyoG gene in high expression tissue of breast and leg muscle at four time points were detected to study the expression pattern. The results showed that MyoG mainly expressed in breast and leg muscle and the expression level was extremely significant higher (P＜0.01) than that in other tissues. Expression of MyoG was also detected in heart and ovary, however, the expression level was very low. Expression pattern of MyoG showed a change trend. The variation of MyoG expression in breast and leg muscle of female chicken was stable. The expression in breast and leg muscle of male chicken showed significant changes. Expression of MyoG gene achieved the highest level at 4 weeks of age and lowest level at 0 week of age. For male and female chicken, expression level of MyoG gene in breast muscle was higher than that in leg muscle. This study revealed the expression profile and expression pattern with the growth and development of muscle of Jinghai yellow chicken, and might lay a foundation for further study on mechanism of action and expression regulation of MyoG gene.

Addition with exogenous microbial inoculum has been proven to be an efficient method to promote lignocellulose biodegradation during cattle manure composting process. In this study, the effects of anctinobacterial community were investigated in compost with microbial inoculum compared with blank control compost under the same condition. The results showed that the inoculated compost was characterized by reaching the thermophilic phase on the third day and keeping high temperature phase for 20 d (blank control compost for 14 d). Moreover, the CMCase activities in the inoculated compost were higher than the blank control compost. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out and sequenced in order to assess the antinobacterial diversity in both inoculated compost and blank control compost. The phylogenetic tree was based on sequences of the actinomycic 16S rRNA gene sequencing fragments, which showed different actinobateria were dominant in different phases during composting process. A total of 10 dominant sequences were obtained during the different composting phases. The dominated band which identified by BLAST during the thermophiclic phase was arranged in 3 classes as follows: Nocardiopsis sp., Streptomyces sp. and Arthrobacter sp.. Most of the actinobacterial bands identified in inoculated sample were related to the reported cellulose-decomposing strains during thermophiclic phase. The results revealed that compost with exogenous microbial inoculum could raise the high temperature and prolong the thermophiclic phase. Moreover, it could promote the actinobacterial activities effectively, resulting in an abundant and diverse actinobacterial community. The present study provides the scientific basis and technical guidance for the research of actinobacterial functional diversity during the composting process.

The germplasm 24-3-1 is a wheat (Triticum aestivum)-Psathyrostachys huashanica disomic alien addition line which is selected by means of chromosome engineering, and has high resistance to wheat stripe rust. In order to improve its genetic stability, and promote the effective utilization of the agronomic genes of Psathyrostachys huashanica, 24-3-1 was treated by ethylmethylsulfone (EMS) for inducing translocation lines. M2 plants with translocation chromosomes were identified by cytology analysis and genomic in situ hybridization (GISH), and the inductive effect of different concentrations of EMS was analyzed. This study identified 61 plants with wheat-Psathyrostachys huashanica translocation chromosomes from 930 M2 plants, and the translocation frequency was 6.56%. Of the 61 plants, 7 plants were detected with 1 translocation chromosome and 5 plants with 1 translocation chromosome and 1 Psathyrostachys huashanica chromosome; in addition, 20 plants, 3 plants, and 26 plants were detected with 2, 3 and 4 translocation chromosomes, respectively. The cytological observation of root-tip cell and GISH analysis indicated that the plants with 2 translocation chromosomes were wheat-Psathyrostachys huashanica translocation lines; and the plants with 4 translocation chromosomes were wheat-Psathyrostachys huashanica translocation-translocation addition lines. This study also showed that 1.0% EMS was an optimal concentration to induce wheat-Psathyrostachys huashanica chromosome translocation. Furthermore, the research not only provides important genetic resources for wheat special genetic materials establishment, but also provides new germplasms in wheat breeding.

Chinese cherry (Prunus pseudocerasus) is one of the most important deciduous fruit tree in China. In order to enrich stable and reliable reference genes and analyze the gene expression more accurately in Chinese cherry by quantitative Real-time PCR (qRT-PCR), the present research selected 7 housekeeping genes of tubulin (TUB), β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1β (EF1B), ubiquitin-conjugating enzyme (UBCE), 18S rRNA and translation initiation factor 5A (TIF5A) based on Chinese cherry flower bud transcriptome data. The expression stability of 7 candidate reference genes were analyzed by Genorm, Bestkeeper, NormFinder and Delta Ct, respectively, under 4 ℃, NaCl (300 mmol/L) and abscisic acid (ABA) (100 mmol/L) treamtent as well as during endodormancy release. This result indicated that the expression of the above 7 genes was not significantly different. GAPDH could expressed stablly under 4 ℃ and NaCl stress, and ACTB and UBCE should be selected as internal controls during ABA treatment and dormancy transition, respectively. Identifying stably expressed housekeeping genes for candidate reference genes from transcriptome database was found to be reliable and efficient. The reference genes selected in this study will be helpful to improve the quality of gene expression studies under various stresses in Chinese cherry.

Most quality traits of wheat (Triticum aestivum L.) are quantitative traits controlled by multiple genes, and discovery of main effective quantitative trait locis (QTLs) which control quality related traits in wheat can provide useful information for molecular breeding. The 585 QTLs of wheat quality related traits reported were collected, and 264 meta quantitative trait locis (MQTLs) were predicted by meta-analysis which included 34 MQTLs for water absorption (ABS), 26 MQTLs for flour protein content (FPC), and 84 MQTLs for grain protein content (GPC), 52 MQTLs for kernel hardness (KH), 39 MQTLs for sedimentation value (SV) and 29 MQTLs for wet gluten content (WGC). Then the technology based on BioMercator software and marker projection were used to integrate QTLs on the wheat quality related traits which were from different genetic mapping population into a reference map. The MQTLs on wheat quality related traits were inhomogeneous distribution on chromosomes, and some QTLs concentrated clusters. The finding of real QTLs section (or site) which controlled wheat quality related traits and the construction of integration map provide reference data for exploring genes on wheat quality related traits, candidate genes and marker-assisted breeding.

To quantitatively detect and analyze transgenes in genetically modified (GM) crops, development of endogenous reference genes with taxon-specificity, low copy number and no allelic variation in varieties is prerequisite. In this study, bioinformatic analyses of genes from database and literatures revealed that one pollen-specific protein gene, PSG719 (GenBank accession: FJ497025.1), from hexaploid wheat genome had high specificity for wheat and low copy number; based on its gene sequence specific primers were designed. These primers were used for qualitative PCR detection of 16 plant species including durum wheat and other closely related cereal species, and no PCR products were obtained from all the plant species except that from common wheat indicating a good taxon-specificity. And the homogeneity was verified on 16 widely different eco-systems wheat varieties by quantitative real-time PCRs. This gene was then used for subsequent qualitative and quantitative PCRs and relevant studies. In conventional qualitative PCRs, the limit of detection (LOD) was about 2 copies per wheat haploid genome per reaction. In the quantitative Real-time PCR assays, LOD and limit of quantitation (LOQ) were about 2 and 5 copies haploid genome, respectively, the latter of which was lower than other reported wheat endogenous reference genes. Furthermore, standard curves through five-fold serial dilutions corresponding to 10 000, 2 500, 625, 156 and 39 copies wheat haploid genome per reaction were performed by the above-developed PSG719 quantitative PCR system, and the results showed ideal slope (-3.395) and highly linear co-efficiency (R2=0.999) between Cross point (Cp) values and the amounts of DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. Taken together, these results indicated that the PSG719 gene was a new endogenous reference gene suitable for the specific detection and quantification of genetically modified materials in common wheat. The developed PSG719 system will facilitate the enforcement of genetically modified organism labeling for wheat in the near future.