Ecotype targeting induced local lesions in genomes (EcoTILLING) is a high-throughput method of detection and analysis of single nucleotide polymorphism (SNP) variations in natural populations. However, it requires a substantial investment in sophisticated equipment and costly fluorescence labeled primers to detect CELⅠ- cut products that limit its use in laboratories with common financial resources. The technology of traditional EcoTILLING had been appropriately improved in this study. Here, we simplified extraction procedure of the CELⅠ enzyme, prepared heteroduplex DNA products for CELⅠdigestion by use of natural cooling method, and detected the CEL I-cleaved products through general silver staining. The results of activities of crude CELⅠenzyme extracts showed that the activity of CELⅠwas high enough for detection of mismatch bases. Using the simplified EcoTILLING technique, the G/T polymorphism at the first intron 5' splice site of the Wx gene among 18 rice(Oryza sativa L.) accessions were successfully genotyped. In addition, CELⅠdigested bands of a 417 bp fragment within the Wx gene of the 18 rice accessions were distinguished into several types. On comparisons with sequencing results, CELⅠcut bands could accurately reflect the SNP variations between the materials. The results suggested that the simplified EcoTILLING is an effective method for SNP discovery and genotyping in rice that will provide powerful tool for rice functional genomics research.

DNA methylation is an important component of the epigenetic network, and it plays a very important role in regulating gene expression. This paper aims to analyze the variation of methylation patterns during chilling induced endo-dormancy release process in tree peony(Paeonia suffruticosa), which will help to isolate and identify methylation regulating genes during dormancy release. The morphological changes of Luhehong enduring 0, 6, 12, 18 and 24 d of 4℃ chilling were observed to ascertain the stages of dormancy after transferred to greenhouse(18~22℃/10~18℃, 8 h light/ 16 h-dark cycle). The accumulation of chilling had a significant effect on the percentage of buds break and also florescence. As the amount of chilling accumulation increased, the percentage of bud break and florescence increased with faster germination. When exposed to less than 18 d of chilling, the percentage of bud break and flower was low, but after 18 d nearly 100% of the buds broke and finally flowered, and no difference between 18 and 24 d of chilling (P＞0.05). Therefore, physiological status of flower bud receiving less than 18 d chilling fulfilling was defined as endo-dormancy, and that receiving more than 18 d chilling treatment was designated as eco-dormancy. DNA methylation patterns were subsequently analyzed by methylation sensitive amplified polymorphism (MSAP) technology with 32 pairs of primer combination with EcoR Ⅰ and HpaⅡ/MspⅠ. Totally, 3 181 bands were amplified, averaging 99.4 bands per primers pair. The results showed that the level of methylation in dormancy buds was extraordinary high, and all exceeded 60%, among which hemi-methylation was dominant. The levels of hemi-methylation and fully methylation fluctuated during chilling duration. However, the level of total DNA methylation was down-regulated during bud dormancy release except a peak at 18 d chilling, and the methylation levels of 0, 6, 12, 18 and 24 d chilling were 78.6%, 76.3%, 74%, 79%, and 61.8% respectively. About 50% methylation sites varied during chilling duration when compared with no chilling treatments. After chilling treatments of 6, 12, 18 and 24 d, DNA demethylation sites were 97, 104, 148 and 218 with percentages of 17.1%, 18.6%, 24.6% and 36.1%. DNA hypermethylation were 197, 137, 95 and 79 bands with percentages of 34.7%, 24.6%, 15.8% and 13.1% respectively. In conclusion, DNA methylation plays an important role in the regulation of dormancy release in tree peony.

Appropriate degradation of starch and cellulose in flue-cured tobacco leaves has become one of the key technologies to improve the quality of tobacco leaves. Bacterial strains Bacillus amyloliquefaciens A1 and B. pumilus C1 isolated from the surface of the tobacco leaves could effectively produce amylase and cellulase respectively. Two strains were cultivated at 37℃ for 60 h and the enzymes were isolated and concentrated from the fermentation metabolites and applied to tobacco(Nicotiana tabacum K326) leaves. Four variables were controlled to detect effects of two enzymes (quantity of enzyme, reaction time, relative humidity and temperature of the investigation). Results illustrated that the strain A1 mainly produced α-amylase which activity was 7×105 U/mL, while strain C1 produced exoglucanase which activity was 6×103 U/mL. In addition, at the temperature of 40℃ and relative humidity of 70% for 96 h, the total sugar and reducing sugar of tobacco leaves treated by 4×107 U/kg amylase from strain A1 increased 12.78% and 12.03%, respectively; as well as treated by 4×105 U/kg cellulase from strain C1 increased 13.87% and 18.07%, respectively. The results suggest that enzyme agents produced by strains isolated from the surface of the tobacco leaves can degrade cellulose and starch in flue-cured tobacco leaves, increase the content of reducing sugar in tobacco processing, and also can apply in the tobacco processing process.

Pyruvate dehydrogenase E1-α subunit (PDHA) plays an important role in the catalytic activity of pyruvate dehydrogenase of pathogens. In order to characterize the immunologic activity of the PDHA of Mycoplasma ovipneumoniae, we amplified and sequenced the pdha gene of M. ovipneumoniae. After optimized with TGG instead of TGA for coding the amino acid of tryptophane, the pdha gene was synthesized and inserted into pET32a (+) vector. The recombinant plasmid pET32-a(+)-pdha was transformed into Escherichia coli BL21 and was then induced to express. The recombinant PDHA was purified and subjected to evaluation of its immunologic activity with immunoblot analysis and immune test in mice(Mus musculus). The results showed that the open reading frame (ORF) of pdha gene of M. ovipneumoniae consisted of 1125 nucleotides, with a G+C content of 34.76%, encoding 375 amino acids. There were 8 TGA codons for tryptophane in the pdha gene (located in 304~306, 379~381, 586~588, 592~594, 625~627, 811~813, 889~891 and 964~966 sites). Comparative analysis of the nucleotide and amino acid sequences of PDHA of M. ovipneumoniae with those of 10 other Mycoplasma species revealed nucleotide sequence homology from 32.6% to 85.3%, with amino acid sequence homology from 39.3% to 90.6%. M. ovipneumoniae and M. hyopneumoniae showed the highest nucleotide and amino acid sequence identity (85.3% and 90.6%, respectively). The recombinant PDHA was expressed in the highest level when the recombinant BL21 was induced by 0.25mmol/L of IPTG at 33℃ for 6 h. The recombinant PDHA protein strongly reacted with the antiserum against M. ovipneumoniae based on the immunoblot assay, and the antibody titers in the immunized mice were increased significantly (P<0.05) compared with the control. We here cloned and expressed the pdha gene of M.ovipeumoniae for the first time. The results indicate the PDHA possesses strong immunologic activity and may be a candidate antigen for vaccine development.

Myoblast cell lines are regarded as progenitors of muscle cells containing filament and myofibrils, are characteristic of proliferation, self-renewal and multipotency. And they are recognized to contribute to reveal mechanisms of animal muscle development in vitro. In this study, both of the enzyme digestion and different speed adherence protocols were used to isolate and purify Ujumqin sheep(Ovis aries) myoblast cells derived from longissimu dorsi. The growth state of myoblasts was obtained with CCK-8 method, and myogenic differentiation was detected using H&E staining and Real-time PCR. The results showed that the highly refractile and slightly round cells adhered to the wall of the flask, but a few cells presented small projections after the cells were seeded into the culture flask for 24 h. Many elongated and spindle-shaped cells appeared 48 h after incubation. The growth curve showed that myoblast cells were in normal growth state. The standard curve was almost S-shaped, and the logarithmic phase occursed after 3 days. Myoblasts proliferated rapidly from the 3 rd to 8 th day, and the proliferation slowed down after 8 days, the stationary phase started on the 9th day. After the induction of myoblasts in 5 days, many mononucleate myoblasts entered into the plateau phase, and long bamboo-shaped primary myoblasts (myotubule), with centrally located nuclei, were formed. Gradually, the myotubule positioned themselves in a regular parallel arrangement and formed multinucleate myotubules. H&E staining of the differentiated cells showed blue-stained nuclei, pink-stained cytoplasm, and multinucleate long bamboo-shaped primary myotubule. These data suggested that the cells differentiated and fused to form myotubules. The expressions of MSTN(myostatin), FST(follistatin), BTG2 (B-cell translocation gene 2) and BTG3 (B-cell translocation gene 3) during differentiation were detected using Real-time PCR. The expressions of MSTN and FST gradually decreased with the progression of differentiation, but the expression of FST was higher than that of MSTN(P<0.05). The expression of BTG3 in the myoblasts was significantly higher than that in the differentiated cells at the early stage of differentiation(P<0.01); however, the expression of BTG2 in the myoblasts on the 3 rd and 7 th days was markedly lower than that in the undifferentiated cells(P<0.05). The expression levels of BTG2 and BTG3 were inversely proportional, and this indicated that these genes played different roles in muscle development. These findings confirmed that the separated myoblasts were myogenic and could form myotubules.The results suggested that Ujumqin sheep muscle-derived myoblast cells have the ablities to develop into myobubes in differentiation process. BTG2 and BTG3 expressed at different levels indicate that both gene maybe play different role in skeletal muscle growth.

Gonadotropin-releasing hormone (GnRH) and growth hormone (GH), which control the reproductive performance of animal, are secreted by hypothalamus pituitary gonadal axis and GH/IGF axis. The single nucleotide polymorphism of GnRH and GH genes was detected for the association with egg performance in white muscovy duck(Cairina moschata) RF line which came from Putian Guangdong Wenshi Poultry Co., Ltd.. PCR-SSCP method was used for identification of genotypes. The results showed that three genotypes AA, AB and BB were found in GnRH gene and a mutation G→A was found by DNA sequencing. Three genotypes CC, CD and DD were found in GH gene and a mutation A→G was found by DNA sequencing. Chi-square test indicated that the two polymorphism sites fitted Hardy-Weinberg equilibrium (P>0.05).The frequency of A/B alleles in the population was for GnRH 0.675 and 0.325, and for GH 0.667 and 0.333. GnRH genotype had been showed a significant difference in the egg number of 300 d (P<0.05)，but GH genotype had not. Both of genes genotype had significant differences in the highest clutch days but no significant difference in the age at the first egg (P>0.05). The general linear model(GLM) analysis results showed that both of GnRH and GH genes showed mainly in the pattern of additive effect and all the dominant effects were not significant. The additive effect of GnRH gene in the egg number of 300 d and the highest clutch days were -3.53 and -3.33, respectively. The additive effect of GH gene in the highest clutch days was -2.44. It implies that the GnRH and GH genes can be candidate genes locus or linked mark to the major gene, which affect the laying traits significantly in Muscovy duck.

The biological toxicity induced by the accumulation of ZnO nanoparticles in the body causes people's attention more and more. To study the genetoxic effect of ZnO nanoparticles with different concentrations on human embryonic kidney cells (HEK293), the human embryonic kidney cells were exposed to ZnO nanoparticles at the concentrations of 10, 25, 50, 75 and 100 mg/L for 12, 24 and 48 h, respectively. The modified comet assay and micronucleus test were performed to research the effect of DNA and chromosomal damage. The results showed that the DNA damage of treated groups was more serious than that of the control. The tail moment, olive tail moment and tail DNA percentage were significantly increased with the rising of ZnO nanoparticles concentration, but the head DNA percentage was decreased. Micronucleus assay showed that the micronuclear rates increased. The result showed that ZnO nanoparticles had the genotoxicity effect on HEK293 cells in vitro, leading to DNA and chromosomal damage. This study provides a scientific basis for evaluating the safty exposure to ZnO nanoparticles.

Carboxypeptidase A (EC 3.4.16) includes a type of digestive enzymes that hydrolyzes the C-terminal aromatic or aliphatic amino acids of a protein. Single nucleotide polymorphism (SNP) analysis of genes functionally related to the growth traits of grass carp (Ctenopharyngodon idella) can provide useful information for grass carp molecular breeding. In this study, genomic fragments of carboxypeptidase A1 (CPA1) gene were amplified based on the sequences of two available contigs in the EST-SNP database of grass carp. Two SNP mutations were identified after sequencing the PCR prodcts, C+412A and A36C, which were located in exon 5 and intron 3 of the CPA1 gene, respectively. Regarding the two SNPs, the genotype and gene frequency of 296 grass carps were further assayed with the method of SnaPshot. The frequencies of AA, AC and CC were 26.7%, 52.0% and 21.3% at the A36C site, but 15.5%, 40.5% and 43.9% at the C+412A site, respectively. A general linear model was used to do the correlation analysis between the CPA1 gene SNPs and growth traits. The three different genotypes of the A36C site were significantly associated with body weight and eye distance (P<0.05). At this site, the genotype of AA significantly showed a higher value on body weight, body length, body width and eye distance compared to the genotype of CC (P<0.05). Although the C+412A site showed no significant correlation with body weight (P<0.05), the genotypes of CC and AC differed significantly for body weight and length before anus (P<0.05). Moreover, the genotype of CC always showed a greater values than that of AC or AA for six growth characters. Five diplotypes consisted of the two SNPs were different on body weight. For example, the body weight of diplotypes D3 and D5 differed significantly (P<0.05). Diplotype D3 showed a significant higher value of body weight, body length, body width and the ratio of body length/head length differences compared with genotype D8 (P<0.05).These results indicate that the CPA1 gene can be a candidate gene that can be useful for grass carp molecular breeding.

The intestinal microbiota plays an important role in the growth, nutrition and well being of the host, and it would be necessary to know the microflora structure in the gastrointestinal tract and the influence factors. The present study used 16S rDNA polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) technology to investigate the intestinal microbiota diversity of farmed fishes from three polyculture patterns with grass carp(Ctenopharyngodon idellus) as a major component. The results showed that there was significant differences among special growth rate of the same species from the different polyculture patterns after four months' cultivation and data analysis (P﹤0.05).Whereas PCR-DGGE fingerprinting of V3 region of 16SrDNA indicated that there was a higher similarity (>42.2%) of grass carp intestinal bacteria in different polyculture patterns. The intestinal bacteria between grass carp fed with compound feeds and the filter-feeding fish silver carp(Hypophthalmichthys molitrix), bighead carp(Aristichthys nobilis) and paddlefish(Polyodon spathula) feeding had the lowest similarity (<19%), while a higher similarity was shown between silver carp and bighead carp (>41.6%). As filter-feeding fishes, the intestinal bacterial community structure between paddlefish and silver carp, bighead carp had a lower similarity except the higher similarity (>50.3%) of intestinal flora structure between paddlefish and bighead carp in mode Ⅱ. Fourteen DGGE bands reflecting varying phylotypes were used for sequencing. Phylogenetic analysis of fourteen cloned bands showed that bacterial phylotypes were made up of four phyla bacteria, i.e. Proteobacteria, Bacteroidetes, Firmicutes and Fusobacteria. The study provides basic references to polyculture model optimization, diets development and disease prevention.

N-linked glycosylation site is critical for the infection and proliferation of most virus, elimination or inhibition of glycosylation is becoming a potent therapy of some virus such as Dengue virus. To elucidated roles of N-linked glycosylation in the infectivity and duplication of Japanese encephalitis virus (JEV), in this study, endo-glycosidase enzymes peptide N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) were used to digest JEV to remove the the N-glycosylation on the virus particles surface. Western blotting analysis showed that the glycan chains of JEV particles was successfully removed. To observe the changes in JEV infectivity and proliferation, plaque assay was performed using treated JEV on BHK-21 cells. In another exprement, BHK-21 cells were treated with tunicamycin and infected with JEV. The results showed that JEV digested by PNGase F or Endo H formed smaller plaques than that of control group, while JEV titer was reduced significantly. The plaques formed by JEV in cells treated with tunicamycin were not observed significant differences in size, but JEV titer was also reduced significantly. From these results we conformed that N-glycosylation plays an important role in the process of JEV infection and proliferation, without N-glycosylation, the infectivity and proliferative ability of JEV will be reduced remarkably.This study arises a new potential strategy on JEV therapy.

Bowman-Birk trypsin inhibitor (BBTI) is a type of cysteine-rich serine protease inhibitors, which is widely distributed in Leguminousaes, Gramineaes and Asteraceaes plants, and it plays an important role in protein storage and plant defense. There are 13 members of BBTI in rice genome, among which BBTI-9 and BBTI-10 are both pseudogenes, while others all include different amounts of Bowmen-Birk domains. To research the expression of BBTI proteins, 11 BBTI specific antibodies were generated mainly by the method of peptide sequence design and synthesis, and the expression patterns at leaf growth and seed germination stages of rice(Oryza sativa L. ssp. indica) were separately revealed by Western blot. The results indicated that BBTI showed specific temporal and spatial expression at various development stages in leaves. Expression level of BBTI-1, -2, -11, -12, and a-13 increased steadily with the growth of leaves, while that of BBTI-5, -6, and -8 reached the highest level at seedling stage. BBTI-3, -4, and -7 expressed most in 1cm at early seeding stage, presented expression in forms of modification and polymer during the leaf growth. Expression of The BBTI-8 protein was high in seeds and declined to the lowest level at 2 h after germination, and a degrading band of smaller molecular weight was expressed at 24 h. However, others were not found obvious changes in expression during seed germination. In attempt to compare the corresponding relationship of RNA and protein level, we collected statistics about MPSS tags of leaves at both seeding and tillering stages and seeds with germinating period. In sum, this experiment revealed differential expression patterns of BBTI proteins during growth and development stages in rice, providing important clues for a better understanding their functions.

Manganese superoxide dismutase(Mn-SOD) is an important anti-oxidant enzyme, which bind manganese functions primarily to protect mitochondrial components from superoxide liberated as a normal byproduct of respiration. Fructose-1,6-bisphosphate aldolase(ALD/FBA) plays a critical role in the photosynthesis in plants. The physiological male sterility, induced by chemical hybridize agent, was different with genetic male sterility which caused by it's own genetic background of sterile cytoplasm. The wheat anthers and ovaries of genetic male sterility, normal fertility and physiological male sterility which induced by chemical hybridize agent SQ-1 were collected as materials for RNA extraction.Based on the differential expressed proteins identified by 2-DE technology, the Mn-SOD and FBA protein were significant and were analyzed by Real-time quantitive PCR. Compared to the normal fertility, the Mn-SOD was distinct down-regulated at young spike stage, uninucleate and trinucleate stages in physiological male sterility , while it's enzyme activity was especially higher than that of control at young spike stage and significantly decreased at binucleate stage. For the genetic male sterility, Mn-SOD was down-regulated at young spike and uninucleate stages, and the enzyme activity was increased at young spike stage but declined at binucleate stage. The FBA was up-regulated in both genetic and physiological male sterility lines at the young spike and uninucleate stages compared with the control, accordingly, it's enzyme activity was also fluctuated with the gene expression. Both the two genes were up-regulated in the ovary and anther of two male sterility lines at the trinucleate stage, and highest expressed in the genetic male sterility, but the expression of Mn-SOD in anther of physiological male sterility was lower than that of control. Mn-SOD and FBA genes were tissue-specific expressed in anther and ovary, and the protein abundance was hysteretic to gene expression. The Mn-SOD was over expressed during uninucleate and binucleate stages, which eliminated abundant active oxygen to keep the normal functions of cells. However, the abortion of anthers was also contributed to the decrease of photosynthesis capability, therefore, result in down-regulated expression of FBA.The study of the both enzyme activity and the gene expression level in different development stages among different male sterile lines contributed to the prefound learn about male sterility, and the consistence of this two index indicate a high relationship of male sterile and this two genes.

To investigate the genetic basis of excellent wheat (Triticum aestivum L.) cultivar, three high-yield breeding lines (Bainongjinguang 588, BainongAK 58-18 and Bainong T5) and their sib lines (Bainong 0487 and Bainonggaoguang 3709) were evaluated using SSR markers in this study. Among the 5 breeding lines, Bainongjinguang 588, BainongAK 58-18 and Bainong T5 had high yield potential and disease resistance. These accessions were evaluated using 701 SSR markers distributing across the whole genome of wheat for their effective utilization in future. The result indicated that the genetic contribution from parents to different sib lines in either of the two crossing combinations were various. The total rates of genetic contribution from Bainong AK58 to Bainongjinguang 588, BainongAK 58-18 and Bainong 0487 were 51.2%, 57.0% and 54.0% separately, which were higher than that from Zhoumai 18 (48.8%, 43.0% and 46.0%, respectively). The total rates of genetic contribution from Bainong 160 to Bainonggaoguang 3709 and Bainong T5 were 41.9% and 36.4% separately, which were higher than that from Zhoumai 16 (27.5% and 29.5%, respectively) and Wenmai 8 (30.6% and 34.1%, respectively). Similarly，the rates of genetic information inherited from the parents to their derivatives were also unbalanced on either genomic level or chromosomal level. On genomic level, BainongAK 58-18 and Bainong 0487 from the crossing combination "Zhoumai 18/Bainong AK58" inherited nearly equal genetic component (53.1% and 53.8%, respectively) from their parent Zhoumai18 on A genome. The rate of genetic information inherited from Zhoumai 18 was 52.6% for Bainongjinguang 588, which was higher than that for Bainong AK 58-18 and Bainong 0487 (26.6% and 36.4%, respectively) obviously on B genome, whereas Bainongjingguang 588 and BainongAK 58-18 inherited nearly equal genetic component (49.1% and 48.1%, respectively) from Zhoumai 18 on D genome. Among 21 chromosomes of wheat, the rates of genetic information inherited from Zhoumai 18 for Bainongjinguang 588, BainongAK 58-18 and Bainong 0487 varied from 9.1% to 100%, from 0 to 100% and from 0 to 75.0%, respectively. Similar result could be observed in Bainonggaoguang 3709 and Bainong T5 from the crossing combination "Zhoumai 16/Wenmai 8//Bainong 160". This indicated that the same combination can produce very different varieties during the breeding of wheat. Furthermore, Bainongjinguang 588 had 109 specific SSR loci compared to other 2 sib lines (BainongAK 58-18 and Bainong 0487), which formed 11 specific chromosomal regions. Whereas BainongAK 58-18 had 36 specific SSR loci compared to its sib lines (Bainongjinguang 588 and Bainong 0487), which formed 3 specific chromosomal regions. Bainong T5 had 55 specific SSR loci compared to its sib line (Bainonggaoguang 3709), which also formed 3 specific chromosomal regions. Most of these observed chromosomal regions above are located with genes and QTL associated with agronomic traits such as yield and resistance to disease, which play an important role in yield components of wheat.

Plant farnesyl pyrophosphate synthase(FPPS) is a key branch-point enzyme in mevalonate pathway, and it is involved in the synthesis of isoprenoids viz. chlorophylls, carotenoids, cytokinins, abscisic acid, gibberellins, dolichols, terpenoids, ubiquinones, sterols and phytotoxins required in the essential biological processes such as growth, development, reproduction and adaptation to the environmental challenges. To provide the theory basis of FPPS in plant evolution study and genetic improvement, this paper summarizes the advances in the cloning and expression of FPPS gene, and its enzyme kinetics.

Agrobacterium tumefaciens VirD2 protein performs functions in binding a single-stranded T-DNA (ssT-DNA) covalently, transferring it into plant nuclear, and integrating it efficiently into the nuclear genome. If it can be modified to target plastid, the VirD2 can pilot foreign genes there, and a new method of plastid transformation can be established. In this study, the localization signals of VirD2 were modified, which included the coding sequence of the nuclear localization signal (NLS) at its N terminal was point-mutated and its C terminal containing a bipartite-type NLS was truncated using an overlapping PCR method. This mutated VirD2 (mVirD2) was then fused with an eGFP (enhanced green fluorescent protein) reporter gene at the 3' end, and with or without the coding sequence of plastid-targeting peptide from Arabidopsis thaliana cold-regulated gene (AtCOR15A) at the 5' end, to construct chimeric genes pt-mVirD2-eGFP and mVirD2-eGFP, respectively. The chimeric genes were controlled by CaMV 35S promoter and were integrated into tobacco (Nicotina tabacum) nuclear genome via Agrobacterium-mediated transformation. The fluorescence was only concentrated in chloroplasts for the transformants expressing pt-mVirD2-eGFP while dispersed among the cytoplasm for those with mVirD2-eGFP, and was not observed in the nuclear for both of them. The results showed that the expressing fused protein pt-mVirD2-eGFP targeted plastid specifically without nuclear localization, which bring forth an idea to explore efficient plastid transformation mediated by protein pt-mVirD2 as a guider of foreign DNA.