The characteristics related grain quality in Triticum aestivum L. are quantitative traits controlled by polygenic. To investigate the genetic basis of Triticum aestivum L. quality traits, the QTLs for grain hardness(GH), grain protein content(GPC), flour protein content(FPC) and wet gluten content(WGC) were analyzed in a population which consisted of 99 F10 recombinant inbred lines (RILs) derived from the cross Triticum polonicum L. line XN555 and T. aestivum L. line Zhong 13. The results showed that the differences between line XN555 and Zhong 13 about GH, GPC and FPC were highly significant difference(P＜0.01) in 2012 and 2013. WGC had highly significant difference(P＜0.01) between line XN555 and Zhong 13 in 2012 and significant difference(P＜0.05) in 2013. The analysis of variance (ANOVA) indicated that there were highly significant differences among RILs about GPC, FPC and WGC and significant differences about GH both in 2012 and 2013. Based on the linkage map constructed with single sequence repeat (SSR) makers, the software Icimapping v3.2 and the inclusive composite interval mapping were used to identify QTL for quality related traits. Two hundred and forty one markers were located to the 14 linkage groups including genome A and B. This map spanned 1 338.92 cM with the average distance of 5.56 cM between any 2 markers. The QTLs for quality related traits, i.e. GH, GPC, FPC and WGC , were detected based on the phenotypic in 2012 and 2013. A total of 24 additive QTLs with the genetic distance from 0.00 to 5.70 cM away from the nearest markers were detected on 9 chromosomes, including 1A, 3A, 4A, 5A, 6A, 1B, 2B, 3B and 5B. Five QTLs for GH located on chromosomes 1A, 1B and 5A, respectively. Single QTL for GH could account for phenotypic variations from 7.93% to 30.49%. Among these, the QTLs on 1A and 1B were identified both in 2012 and 2013. Seven QTLs for GPC were located on chromosomes 6A, 1B, 2B, 3B and 5B, respectively. Single QTL for GPC could account for phenotypic variations from 8.30% to 29.69%. Two of the seven QTLs for GPC were detected in the interval of Xbarc104~Xcfa2114 on chromosome 6A based on the phenotypic of the two years. Seven QTLs for FPC were found on chromosomes 3A, 4A, 6A, 1B, 2B and 5B, respectively. Single QTL for FPC could account for phenotypic variations from 6.90% to 29.50%. Two QTLs for FPC on chromosome 5B were found by using the phenotypic of the two years. Five QTLs for WGC were found on chromosomes 6A, 1B, 2B and 5B, respectively. Single QTL for WGC could account for phenotypic variations from 10.10% to 18.43%. In the interval of Xbarc104~Xcfa2114 on chromosome 6A, with genetic distances of 1.2 cM from the nearest marker Xbarc104, two QTLs for WGC were detected both in 2012 and 2013. Meanwhile, one QTL for FPC in 2012 and one QTL for GPC in 2013 were detected in the same interval as well. The identified molecular markers related to the quality traits in this study will benefit the marker-assisted selection in breeding programs.

Cytochrome P450 reductase (CPR) is a kind of electron donor in the oxidation system, which is a rate-limiting enzyme in the cytochrome P450-mediated redox reactions. CPR is the key enzyme in oxidation reaction in vivo. In this study, two degenerate primers were designed according to the sequence of CPR gene from other species, and partial sequence of CPR gene from Matricaria recutita was obtained. By RACE technologies, the 2 272 bp full-length cDNA sequence of CPR gene from Matricaria recutita was isolated, and GenBank accession was KJ004519. The full length CDs of CPR contained 2 106 bp nucleotides, which encoded a protein of 701 amino acids. The CPR protein was closely clustered with Artemisia annua in a phylogenetic tree, and they had 94% similarity of the amino acid sequence. The transcriptional expression of CPR was analysed with quantitative Real-time PCR. The results showed that the chamomile CPR mRNA expression was the highest in disc flower. Bioinformatics analysis showed that they had the most intimate relationship between Matricaria recutita and Artemisia annua, for 94% similarity of CPR amino acid sequence. The structure of CPR included P450, flavin mononucleotide(FMN), flavin adenine dinucleotide(FAD) and nicotinamide adenine dinucleotide phosphate(NADP) binding domains; The expressions of CPR were detected from radial flower, disk flower, stem and leave by qRT-PCR in Matricaria recutita, and the results showed that the expression of CPR in disk flower was the highest, which was 20 times higher than leaves, while radial corolla and stems had little expression of CPR. In this study, we had cloned the CPR gene from chamomile the first time, as we know, CPR was a key enzyme in the oxidative modification process of plant terpenes. So our results of the study may provide a molecular basis for the further research of the functions of cytochrome P450 oxidoreductase system in terpenes biosynthetic pathway in chamomile.

The goal of this research is to assess the availability of SSR markers for variety testing in watermelon hybrids and try to establish a standard for variety uniformity testing by SSR markers. A set of 11 primer pairs were applied for 78 watermelon (Citrullus lanatus) varieties and 97 watermelon hybrids (48 individuals per variety) of Beijing regional trial from 2009 to 2013, and one SSR marker per chromosome was selected. In accordance with the statistics of 175 watermelons testing results by 11 core primer pairs and different varieties specifics characteristic on different SSR locus, it was revealed that there were different individuals uniformity ratio on different SSR locus in one variety, the one individual uniformity ratio on one SSR locus did not represent the variety uniformity, and variety uniformity could be assessed not only based on individuals uniformity ratio on one SSR locus, but also individuals average uniformity ratio on 11 SSR loci. Comprehensively analyzing the statistics result of the 175 varieties uniformity ratio on 11 SSR loci illustrated that varieties uniformity ratio on SSR loci could be the standard-assisted for watermelon uniformity assessment. It was necessary to assess watermelon uniformity in combination with one individual uniformity ration on one SSR locus and average uniformity ratio on 11 loci, and there were 5 levels for evaluation, and explained the criterion evaluating watermelon variety uniformity by SSR markers and testing methods in details, preliminary draw up uniformity criterion of watermelon (Citrullus lanatus) varieties.

In order to discover the effect of CCAAT/enhancer binding protein α (C/EBPα) and lipoprotein lipase (LPL) genes expression in tail fat adipose tissues among different types of sheep tails, primers were designed based on the full length of bovine (Bos taurus) C/EBPα gene (GenBank A: NM_176784.2). RT-PCR was used to amplify the C/EBPα gene, and bioinformatics methods were used to analyze the characteristics of genetic sequence and protein. Quantitative Real-time PCR and Western blot were used to evaluate the expression level of C/EBPα and LPL in tail adipose tissues among 5 sheep(Ovis aries) breeds (Shannbei Merino (long thin-tail), Tong sheep (long/short fat-tail), Tibetan sheep (short thin-tail), Tan sheep (long fat-tail) and Kazak sheep (hip fat-tail)) aged at 9-month-old, and 18 individuals were involoved. The results showed that the coding region of C/EBPα(GenBank No. : KF830871) was 1 062 bp in full length, encoding 353 amino acids, and the isoelectric point of the protein was 7.25, and the molecular weight was 37.15 kD, sharing 99% similar nucleotides with bovine sequences and its protein contained a typical leucine zipper domain. The expression trends of the protein and mRNA level of C/EBPα and LPL were roughly the same. Both C/EBPα and LPL highly expressed in fat-tailed (Tong sheep, Tan sheep) and hip fat-tailed (Kazak sheep) sheep, while lower expressed in thin-tailed (Shannbei Merino, Tibetan sheep) breeds. In the same breed, the mRNA and the protein expression levels of C/EBPα and LPL in adipose tissues of long fat-tail in Tong sheep were significantly higher than those of short fat-tail (P＜0.05). In this study, we successfully cloned sheep C/EBPα gene and the expression levels of C/EBPα and LPL genes had significant correlations with fat deposition in tail tissues of sheep. Our results provide the basic information for further exploring the mechanism of blocking excessive deposition of tail fat, and realizing the purpose that not only saving the cost of feeding, but also making full use of the excellent productivity of the sheep population.

There are abundant cold-adapted cellulose-degrading bacteria resources from soil in the Da Hinggan forest of Inner Mongolia. This study was aimed at analyzing the community structure of cold-adapted cellulose degrading bacteria. Cold-adapted cellulose-degrading bacteria were isolated by enrichment culture using sodium salt of caboxy methyl cellulose(CMC-Na) medium at 10 ℃. Cellulase activity was determined by 3, 5-dinitrosalicylic acid (DNS) method. The 16S rDNA genes (V3~V5 region) were amplified by using the universal primers (34lF and 907R). The PCR products were electrophoresed by denaturing gradient gel electrophoresis (DGGE). The 16S rDNA fingerprint was obtained and the bacterial community structures were analyzed. The results indicated that the relative enzyme activity increased constantly with the extension of incubation time during the process of enrichment, the 4th generation had the highest relative enzyme activity with 30.625 IU. Some bacteria died out or increased at each generation. Four DGGE profiles of microbial community structure of four generation enrichment cultures were obtained. There were five subgroups in enrichment culture, including α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Firmicutes, and Bacteroidetes, respectively. The subgroups of Firmicutes (65.5%) and β-Proteobacteria (13.8%) were the dominant bacterial floras. This study confirmed that there were abundant resources of cold-adapted cellulose-degrading bacteria in the Da Hinggan forest of Inner Mongolia. These bacteria will have a potential value of application.

Grape hyacinth (Muscari armeniacum) is an important ornamental bulbous plant with increasing economic, ornamental and scientific importance because of their outstanding blue color. Anthocyanins are principal flower pigments in M. armeniacum, and indispensible in plant biology and human/animal diets. Whereas anthocyanin synthesis is well characterized in numerous plants, the mechanism underlying the blue appearance of M. armeniacum is still far from understanding for the little knowledge of gene information of this plant. Here, two dihydroflavonol 4-reductase (DFR) genes, which encode a later enzyme for anthocyanin formation, were isolated from Muscari armeniacum petals using RACE techniques and designated as MaDFR2a and MaDFR2b(GenBank accession No. KJ619963 and KJ619964), respectively. Sequence analysis of cDNAs revealed that the full length sequence was 1 395 bp, containing a length of 76 bp 5' untranslated region and 215 bp 3' untranslated region，both of them contained a 1 101 bp open reading frame (ORF), which encoded a protein of 366 amino acids. The similarity of the two DFR was 98%. Homology analysis showed that the deduced DFR proteins were highly homologous to other DFR proteins from different plant species. The cluster analysis results showed that the two DFR genes clustered together with monocotyledons firstly. Bioinformatics showed that two DFR proteins were both hydrophilic proteins, containing two transmembrane domains and neither of them had signal peptide. Subcellular localization analysis showed that two DFR genes located in the whole cells. α-Helix and random coil were primary secondary structural components of the two DFR genes. The tertiary structures of two DFR proteins were similar. Both of them had a fissure which could do some catalytic reactions. High performance liquid chromatography (HPLC) analysis showed that high contents of color anthocyanins were detected in pigmented flower organs, while no anthocyanins were detected in root, stem, leaf and unpigmented flower buds. The result of Real-time fluorescence quantitative PCR analysis demonstrated that the DFRs expression was presented in flowers but little or very weak expression was also detected in the other tissues (leaf, stem and root). When flower pigmentation started, a steep rise of two DFR genes expression occurred and peaked at fully coloured flowers. Subsequently a slight decrease in the MaDFR2a and MaDFR2b expression was presented at later stage. In contrast, a relatively high-level of MaDFR2b expression was found in both unpigmented or fully-pigmented buds. This result suggested that DFR can play a role in the color formation of blue grape hyacinth.

Cydia pomonella granulovirus(CpGV) has been developed as one of the most efficient, environmental and safe insecticides for control of the codling moth. To enrich the research contents of CpGV GP37 protein and explore mechanism of synergism of Nucleopolyheroviruses(NPVs) and Bacillus thuringiensis (Bt) by the GP37 protein, a series of recombinant Autographa californica multiple nucleocapsid NPV (AcMNPV) bacmids containing egfp or CpGV gp37 fused with egfp were constructed. The resulting bacmids were named vAcGP37, vAcGP37EGFP, vAcEGFPGP37, and vAcEGFP, respectively. In vAcGP37EGFP and vAcEGFPGP37, EGFP was fused in frame with C terminal and N terminal, respectively. The bacmid vAcGP37 expressed GP37 protein and vAcEGFP expressed EGFP only. All recombinant bacmids were confirmed by PCR. Sf9 cells were transfected with the recombinant bacmids, and supernatants containing budded virus were collected at 96 h after transfection. Examinations were carried out for protein expression by Western blotting analysis using anti-GFP and anti-GP37 as primary antibody following SDS-PAGE of the cell lysates. An fusion protein about 50 kD, which was in consistence with the size of GP37 and EGFP, was detected in the cell lysates transfected with vAcGP37EGFP or vAcEGFPGP37. By contrast, an approximate 25 and 30 kD protein were detected in those cells lysates transfected with vAcGP37 or vAcEGFP, respectively. Sf9 cells (1×105) in 35-mm glass bottom cell culture dishes were infected with vAcGP37, vAcGP37EGFP, vAcEGFPGP37, and vAcEGFP, respectively. At 24 h after transfection, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed with PBS and then dealled with 0.25% Triton X-100 for 3 min at room temperature. Following a wash with PBS, the cells were stained with Hoechst 33258 working reagent for 5 min at room temperature. Cells were washed three times with PBS and visualized with confocal laser scanning microscope for fluorescence, respectively. In cells infected with vAcEGFP, EGFP was expressed without fusion of GP37 and fluorescence was dispersed uniformly throughout the cytoplasm and nucleus. In contrast, in cells infected with vAcGP37EGFP and vAcEGFPGP37, EGFP was expressed with fusion of GP37 and fluorescence distributed mainly in the cytoplasm. These results suggested that CpGV GP37 protein was localized mainly in the cytoplasm. The eukaryotic expression of CpGV GP37 protein may provide conditions for further study of this protein. The subcellular localization of CpGV GP37 protein in infected cells can lay a foundation for the study of synergic mechanism of this protein and enrich the theory of baculovirus GP37 protein.

The α-tocopherol transfer protein (α-TTP) is a critical regulator of vitamin E status that stimulates the movement of vitamin E in hepatocytes and facilitates the secretion of tocopherol from hepatocytes to extrahepatic tissues. Heritable mutations in α-TTP gene cause ataxia with vitamin E deficiency (AVED), which characterized by low plasma vitamin E levels and progressive neurodegeneration. This review focused on the function and molecular aspects of α-TTP activity as well as the effect of vitamin E on the expression of α-TTP. In the end, further research focuses were also discussed. Overall, this review may provide theoretical references for α-TTP and vitamin E metabolism research.

As more and more genetically modified organisms (GMOs) and their products coming into all areas of human life, to detect GMOs and their products accurately, quickly and efficiently becomes basic requirements for the research and safety management of GMOs. The transgenic rice(Oryza sativa) EB7001S resistant to two kinds of non-selective herbicide was developed recently by our research team using Agrobacterium-mediated transformation method. In order to establish the event-specific detection method of transgenic rice EB7001S, the high efficient thermal asymmetric interlaced PCR (hiTAIL-PCR) and long distance PCR (LD-PCR) were adopted to reveal the flanking sequences of foreign genes in this research. Firstly, the right flanking sequence in length of 1 515 bp was revealed by hiTAIL-PCR, in which the upstream sequence from the 1st to 169th bp belonged to the vector sequence and the remaining was the rice genomic sequence of Chromosome 7. Secondly, the left flanking sequences in length of 1 460 bp was amplified by LD-PCR, which included a 777-bp (the 684th to 1460th bp) vector sequence and a 683-bp (the 1st to 683th bp) rice genomic sequence of chromosome 7. The BLAST analysis in NCBI database showed that the foreign genes were inserted into the position 1 470 725 of short arm of chromosome 7. The insertion of foreign genes resulted in 3 bases deletion (1 470 726~1 470 728). The event-specific qualitative PCR method were successfully developed by designing specific primer pairs based on the right and left flanking sequence, respectively. In the specific primer pair, one primer was designed based on the rice genomic sequence, and the other primer based on the foreign gene sequence. The event-specific detection methods could amplify a 458 bp and a 629 bp fragments from right border and left border respectively in transgenic rice EB7001S, but could not amplify in any other transgenic or non-transgenic rice, which showed strong specificity and high stability. When the DNA templates to be amplified by event-specific detection methods were the mixture of different percentage of genomic DNA of EB7001S, the sample only contained 0.1% EB7001S genomic DNA could be detected, of which the sensitivity could satisfy the detection demands of the Customs, Administration for Industry and Commerce, Ministry of Agriculture and so on. Then, the tri-primer PCR method based on the flanking sequence was also established to select homozygous genotype from transgenic offspring quickly and accurately. One primer GEB-F was designed according to the upstream of the insertion site on Chromosome 7, the 2nd primer and the 3rd primer were the event-specific primers (RB-F, RB-R) of right flanking sequence. When the tri-primer PCR run, the homozygous plants of foreign genes amplified a 458 bp fragment, the plants free of foreign genes showed a 808 bp fragment, and the heterozygous plants displayed both 458 bp and 808 bp fragments simultaneously, which made the homozygous plants identified from one segregative generation. The detection method established in this research will provide key technical supports for the utilization and detection of transgenic rice EB7001S.

To accurately identify exogenous chromosome fragments or genes plays an important role in crop genetic improvement. Although the molecular markers have been widely used in detection of exogenous chromosome fragments or genes, the efficiency of identificating a few positive plants in large group is very low. In this study, DNA samples of 147 plants in BC2F1 population which was established by Gossypium hirsutum Xinluzao 48 and G. barbadense chromosome segment substitution lines CSSL-122 through hybridization and backcross were used as materials, which were arrayed by constructing 2 dimensional matrix. The test samples were mixed according to the row and column, respectively, to get m rows of mixed samples and n columns of mixed samples, taking m=n=8 matrices as example: The m×n=64 mixed samples were obtained to test. The positive samples containing exogenous chromosome fragments in the matrix could be identified accordingly to the detection results. Results showed that exogenous Gossypium barbadense chromosome fragments in G. hirsutum could be detected quickly by using the test method of establishing a matrix mixing bank based on PCR-SSR. In addition, the accuracy of the method was verified by means of sample detection one by one furtherly. Statistical analysis showed that frequency of positive sample detection was less than 7.81%, cost saving ratio of detecting the samples could be maintained at more than 50%. Detection method of constructing the matrix mixed pool based on PCR-SSR will provide a new method for the rapid identification of exogenous Gossypium barbadense chromosome fragment in Gossypium hirsutum.

Seasonal oestrus is one of main restricted factors in the development of sheep industry. The ovaries are the important reproductive organs of sheep, and the periodicly expressed gene in ovary is likely to regulate the ewe seasonal breeding directly or undirectly. In order to discuss the molecular genetic mechanism of the sheep seasonal oestrus, the subtracted cDNA library of the Chinese Merino sheep(Ovis aries) ovary(estrus and anestrus) was conducted by the suppression subtractive hybridization(SSH). Random sampling of 386 positive clones were identified by PCR, and obtained about 306 effective clones and 126 non-redundant sequences after sequencing. The sequences were studied through BLAST on line with the GenBank dbase and GenBank EST dbase and the results showed that 21 of them were not matched to any of the databases which might represent new genes related to Chinese Merino sheep ovary tissue seasonal reproduction trait. Retinoic acid receptor response element1(Rarres), thymosin beta 4(TMSB4X), eukaryotic translation elongation factor 1 alpha 1(EEF1A1), prothymosin alpha(PTMA), and No.12 and No.89 genes were selected from the library, and tissue-specific detection of the 6 kind of genes screened was performed in heart, liver, spleen, lung, kidney, ovary, hypothalamus, pituitary, uterus and fallopian tubes tissues. The results showed that RARRES1 and No.89 gene over-expressed in ovary tissues, however, TMSB4X, EEF1A1, PTMA and No.12 gene expressed in all tissue. In order to further validate the obtained results, RARRES1, TMSB4X, EEF1A1, PTMA, No.12 and No.89 genes were selected for expression analysis by quantitative Real-time PCR. The results showd that their expression were all up-regulated in ovary of estrous Chinese Merino sheep. In this study, we got potential genes related with the seasonal reproduction trait of the Chinese Merino sheep, wihch provides a foundation to research the molecular mechanism of interaction between ovary function and seasonal reproduction trait of sheep in future.

The sensitivity of chemiluminescence immunoassay method mainly depends on the quality of chemiluminescence analyzer. This paper reports a chemiluminescence immunoassay apparatus that was developed independently. In this research, we employed the optical fiber conduction as the detecting system and fiber-optic probe which was more close to the analyte as the movement system to improve the detection performance. And then a clenbuterol chemiluminescent immunoassay kit (CLEN-kit) was also developed to evaluate the analyzer quality. A set of quality evaluation tests were designed to evaluate the comprehensive quality of the kit. Results showed that the linear detection range of CLEN-kit was 0.313~40.000 ng/mL, the sensitivity was 0.397 7 ng/mL, and the detection limit was 0.023~0.041 ng/mL. Besides, its cross-reactivity with its structural analogues, terbutaline, tulobuterol and adrenaline was not appeared. It was validated that CLEN-kit has a good ability to detect trace clenbuterol and the self-developed chemiluminescence immunoassay analyzer has a good performance when applied in CLEN-kit's development.