Bacterial fruit blotch of melons is an important disease on cucurbit plants which is caused by Acidovorax citrulli. Since now it has been reported that this disease can cause serious damages of local melons in United States, Australia, Brazil, China and many other countries. So far, to prevent bacterial fruit blotch of melons, we should find out the mechanism of Acidovorax citrulli. In this experiment, we constructed several type Ⅳ pili related gene-inserted mutants, and obtained two mutants with dramatically reduced virulence by seed-transmission assays which was identified for pilO and pilQ genes, then we tested some related phenotypes about them. By transmission electron microscopy, we found that wild type and pilO mutant could form type Ⅳ pili, whereas pilQ mutant lost type Ⅳ pili. Results of optical microscope observation showed that pilO and pilQ mutants, compared with wild type, both lost twitching motility completely, meanwhile their swimming ability and biofilm formation also reduced significantly. For complementary strains, virulence, twitching motility, swimming ability and biofilm formation recovered partly. Compared with wild-type, the growth of mutants showed no significant difference in MMX medium, and they could also stimulate hypersensitive response in non-host. PilO mutant lost some abilities mediated by type Ⅳ pili but still could form type Ⅳ pili showed that, pilO gene was not directly involved in synthesis of type Ⅳ pili, but it was functional gene of type Ⅳ pili in A. citrulli. PilQ mutant not only lost the abilities mediated by type Ⅳ pili but also couldn’t form type Ⅳ pili showed that, pilQ gene was directly involved in synthesis of type Ⅳ pili and was the synthetic and functional gene of type Ⅳ pili in A. citrulli. From this we knew that type Ⅳ pili of A. citrulli was related to twitching motility, virulence, swimming ability and biofilm formation.

Erwinia amylovora is a necrogenic phytopathogenic bacterium, the causal agent of fire blight in pear(Pyrus sorotina), apple(Malus domestica) and other members of the Rosaceae family, causing serious economic losses. LuxR transcriptional regulator is a key player in quorum sensing and plays important physiological and biochemical roles. In this paper, one of luxR homologous gene was amplified by PCR from E. amylovora. The mutant EaΔluxR was successfully constucted by homologous recombination, and the function studied preliminarily. The experimental results showed that, compared to the wild-type strain, the polyene macrolide biosynthesis, anti-oxidative stress, growth, and pathogenicity of mutant EaΔluxR declined. However, EaΔluxR still caused tobacco(Nicotiana tabacum) hypersensitive response, and there was no significant difference in antibiotic tolerance level and biofilm formation comparing to wild-type strain. These findings demonstrated that luxR gene in E. amylovora is involved in polyene macrolide biosynthesis, growth, and anti-oxidative stress and plays an important role in virulence. In addition, our results provid molecular evidence for in-depth understanding of the quorum sensing system of E. amylovora.

Diversity of branching types is produced during the evolution of Asteraceae, which is originated by the diversity of branching genes. BRC1 (BRANCHED1) genes, as the member of TCP family, are proved to play important roles in inhibition of lateral branches. BRC1-like genes were replicated during evolution, and different members were through different selective pressures, both of which may lead to the large family and diverse functions. Twenty-one BRC1-like gene fragments were isolated from 16 Asteraceae species, which contained the conserved TCP domain, R domain and ECE motif, and were in the CYCLOIDEA1 (CYC1) subclade of TCP family. With the sequences we cloned and sequences from gerbera (Gerbera hybrid) and sunflower (Helianthus annuus)(downloaded from NCBI), phylogenetic tree were constructed. All the BRC1-like genes from Asteraceae were clustered into two groups, BRC1a and BRC1b, which indicated that CYC1 subclade replicated and expanded during evolution. Maximum likelihood (ML) branch test was performed to detect the selective pressure, the results indicated that BRC1b group evoloved under a strong purifying selection, while the BRC1a group had experienced a decrease in evolutionary constrains. Results in the ML branch-site test showed that, relaxation of selective constrains rather than positive selection was the process associated with the evolution of BRC1a group. The amino acid residue targets of selection in all BRC1-like genes were identified with ML site test model. The results indicated that, residues evolved under strong purifying selections were located in the TCP domain, R domain and ECE motif, inferred that important domains were fixed during evolution. The evolution of BRC1-like gene in Asteraceae indicates that two groups were clustered through replication, then these two groups underwent different selective pressures, which may lead to different functions of genes.

Vacuole processing enzyme(VPE) gene family plays an important role in mediating and executing programmed cell death in plants. β type VPE is specifically expressed in seed, which is indispensable in seed development. The ovules were harvested from V. vinifera cv. Pinot Noir in 8 different development stages. RNA was isolated from these ovules. VvβVPE gene was obtained by RT-PCR, and the mixture of cDNA was used as template. The results showed VvβVPE gene contained a complete ORF(open reading frame) sequence (1 485 bp) encoding 494 amino acid(GenBank accession number: KC136352). The full-length sequence was cloned into the prokaryotic expression vector of pGEX-6P-1. An about 81.3 kD fusion protein of inclusion body (GST-VvβVPE) was expressed in the Escherichia coli BL21 through IPTG inducing. After optimizing the inducing condition, two different methods were adopted to purify the protein, containing electro dialysis and dialysis renaturation by GdmCl treating, and then immunized the rabbit to obtain the polyclonal antibody. Western blot displayed a specific immunity-antigen signal, suggesting the obtained antiserum can be used for function analysis and detecting transgenic plants.

The objective of this study is to develop more molecular markers for construction of high density genetic map of the leaf rust resistance gene Lr45 in wheat (Triticum aestivum L.). Twenty-six pairs of expressed sequence tag(EST) primers located on the short arm of wheat chromosome 2A in combination with 4 kinds of restriction enzyme, resulting in a total of 104 pairs of EST-CAPS(expressed sequence tags-cleaved amplified polymorphic sequences) markers were used to analyze the polymorphism between TcLr45 and Thatcher. The EST primers BE426158 and BE442876 expressed polymorphsim of the PCR products between TcLr45(resistant leaf rust parent) and Thatcher(susceptible leaf rust parent) with the polymorphism rate of 7.7%. Four of 104 primers/restriction enzyme combinations (3.8%), CD454036 /MspⅠ, CD454036 /HaeⅢ, BE406923 /MspⅠ and BE425962 / RsaⅠ, were polymorphic between Thatcher and TcLr45. In addition, a pair of primers designed based on the sequence of band amplified with BE426158 in TcLr45 and converted successfully to a stable STS(sequence tagged site) marker, LR45-1. The linkage distance of the LR45-1 with Lr45 was 8.2 cM based on the data of a F2 population derived from TcLr45×Thatcher. The specificity and stability were validated by testing the wheat leaf rust resistance near isogenic lines and rye(Secale cereale) varieties. The results described above indicate that the EST-CAPS markers can be used in analysising of polymorphisms and development of molecular markers in wheat leaf rust resistance genes.

The aim of this study is to detect the expression of microRNA(miR)-27b in skeletal muscle development in goats(Capra hircus), and explore the roles of miR-27b in muscle development. Real-time quantitative PCR(qRT-PCR) was performed to detect the miR-27b expression in the longissimus dorsi muscle from the skeletal muscle tissues of different stages (neonate, 6 and 12 months) in Anhui White and Boer goats(C.hircus). Meanwhile, the expression of miR-27b in various tissues of 6 months goats was detected. And the conservation of miR-27b in different varieties was analysed by MEGA3.1. The miR-27b differential expression in skeletal muscle development exhibited different expression patterns between Anhui White and Boer goats. The expression level in longissimus dorsi muscle from 12 months goats was the highest, while, the expression level in neonate was the lowest. In 6 months goats, miR-27b was abundantly expressed in longissimus dorsi muscle and heart, moderately in liver and kidney, and weakly in other collected tissues. The results indicated that miR-27b may be involved in myofibril proliferation and differentiation and further contribute to skeletal muscle development and muscle phenotype with different genotypes, and the miR-27b was conservative in 10 different species. The expression of miR-27b has tissue and time specific, and the present result provides the theory base for study of miR-27b function.

The sleeping beauty (SB) transposon is a Tc1/mariner family transposon. In order to examine the transposition efficiency of SB when it is used with different transposases and the same transposase at different ratios, our study constructed recombinant vector which was transfected into porcine(Sus scrofa) fetal fibroblasts cells (PEFs) and its transfection efficiency was detected by fluorescence microscope observation and qRT-PRC in an attempt to improve the transfection condition. The results of PT2-SV40-EGFP with different transposase at the same ratio of 10∶1showed that the transfection efficiency of EGFP(enhanced green fluorescent protein) was close, but the difference between expression of EGFP was significant, and SB100X has the highest expression. The results of PT2-SV40-EGFP with SB100X at different ratios showed that expression difference was significant at similar transfection efficiency. The expression level of PT2-SV40-EGFP and SB100X mixed at the ratio of 1∶1 was 50 times as high as the the group at the ratio of 5∶1 and the group at the ratio of 10∶1. This will provide technological platform for producing transgenic animals to further study SB transposon.

Heat shock protein(HSP)70 is synthesized in the preimplantation embryos of cow(Bos tarurs), ewe(Ovis aries) and mice(Mus musculus) under the conditions of heat shock in vitro, which enhances their thermotolerance to protect them from further heat damage. And Calmodulin(CaM)is the one of most important multifunctional receptor of Ca2+ and plays a key role in regulating the important gene expression in cell activities. To clarity whether the CaM participating in the expression of HSP70 in the mice embryos with heat shock and verify its mechanism, mice embryos were cultured in vitro to the blastocyst stage and then were randomly divided into control group(37℃), group treated with W7 without heat shock(37℃+W7), heat shock group(39℃)and heat shock group treated with W7. The method of RT-PCR was used to detect the gene expression of CaM, HSP70 and heat shock factor1(HSF1), and the method of Western blot was used to detect the expression of CaM, HSP70 and HSF1. The combinations of both HSP70-CaM and HSP70-HSF1 were detected by using co-immunoprecipitation(Co-IP). The results showed that the CaM and HSP70 mRNA expression significantly increased in the mice embryos treated with 39℃ for 1 h (P<0.05). However, the CaM and HSP70 mRNA expression significantly decreased in both groups of embryo cultured at 37℃ (P<0.05) and 39℃ (P<0.01) treated with W7; W7 had no effect on the HSF1 mRNA expression in any groups of embryo. The synthesis of CaM, HSP70 and HSF1 significantly increased in the embryos treated at 39℃ for 1 h (P<0.05), and the CaM synthesis significantly decreased in the embryos cultured both at 37℃ and 39℃ and treated with W7 (P<0.05). W7 had no effect on neither HSP70 nor HSF1 synthesis in the embryos cultured at 37℃ (P>0.05) while it inhibited both HSP70 and HSF1 synthesis in the embryos cultured at 39℃(P<0.05). The study also found that CaM combined with HSP70 and formed CaM-HSP70 complex in the mice embryos. The identification of interactions between CaM and HSP70,HSF1 and HSP70 in the embryos cultured both at 37℃ and 39℃ by using Co-IP showed that the quantity of HSP70-CaM complex had the tendency of increase while the HSP70-HSF1 complex decreased in the heat shocked embryos. The above results indicate that CaM regulates HSP70 expression by the way of competitive binding HSP70 and releasing HSF1, which finally activates HSP70 expression in heat shocked mice embryos. The research results reveal one of the ways that CaM participates in HSP70 expression in heat shock response of mice embryos which will provide a theoretical basis for the study of thermotolerance and heat shock signal transduction of the mice embryos.

Peroxiredoxins(Prxs)are a superfamily of cysteine-dependent peroxidases that decompose hydrogen peroxide. Among the family, typical 2-cysteine Peroxiredoxins (2-Cys Prxs) are multifunctional proteins. In plant nematode, 2-Cys Prxs play an important role in nematode development in planta or in countering radicals produced by the plant as a defense response to parasitism. In order to understand the effect of low dose of nematicides on 2-Cys Prxs mRNA expression, a novel 2-Cys Prx gene was cloned from Ditylenchus destructor using RT-PCR and RACE techniques, and its mRNA expression of D. destructor treated by low dose(10、1、0.1、0.01、0.001 and 0.0001 μg/mL) nematicides of emanectin benzoate and aldicarb were monitored using Real-time PCR after 24 h. The results showed that the full-length cDNA was 784 bp with an open reading frame (ORF) of 591 bp encoding 196 amino acids residues, named Dd-prx-1(GenBank accession No. JQ609353). The protein had a predicted molecular weight of 21 850.09 D and a hypothetical pI of 6.83. The structure of Dd-Prx-1 genomic DNA include 6 extons and 5 introns. The N-terminal of predicted protein had no apparent signal peptide, but there was one transmembrane domain containing of 23 hydrophilic amino acids. Homology analysis showed that the deduced Dd-PRX-1 protein was highly homologous to the 2-cysteine peroxiredoxin in other nematodes. After treated by low dose emanectin benzoate and aldicarb, the expression levels of Dd-Prx-1 had no significant difference compared with control (P<0.05). The results provide the useful information for further research mechanisms of nematodes response to low dose of nematicides.

In order to investigate composition and diversity of intestinal bacteria of common carp(Cyprinus carpio L.) reared in saline-alkaline ponds, the 16S rRNA genes were amplified from the total DNA of intestinal bacteria in common carp by PCR with bacteria-specific primers and a clone library was constructed. Positive clones were analyzed by restriction fragment length polymorphism(RFLP), and the representative clones with unique patterns were sequenced, BLAST and then constructed phylogenetic tree. A total of 28 Operational Taxonomic Units (OTUs)(GenBank accession No. JX262557~JX262584) were sequenced from 176 clones of the clone library. The clones coverage value (C=88.6%) and rarefaction analysis both showed that the clone library covered the majority intestinal bacteria. Sequence alignment showed that the most bacteria exhibited high similarity (>98%) with known bacterial 16S rRNA genes retrieved from intestinal bacteria of fish and 4 potential novel taxonomic units exhibited low similarity (<94%) with known bacterial sequence were found. Phylogenetic analysis of the 16S rRNA sequences indicated that the intestinal bacteria in common carp reared in saline-alkaline ponds fell into the following major lineages, including Alpha and Gamma subclasses of the Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria, which accounting for 89.9%, 7.9%, 1.1% and 1.1% of the total number of clones, respectively. Seventeen sequenced OTUs(158 clones) affiliated within γ-Proteobacteria were the most diverse and dominant in our library, which had 99 to 100% similarity to known bacterial sequences belonging to Aeromonas, Shewanella, and Vibrio. The dominant genus of the intestinal bacteria was Aeromonas, which accounted for 50% of the 28 valid OTUs and 86.4% of the 176 clones. Three sequenced OTUs(3 clones) belonging to α-Proteobacteria had 99% identity to known bacterial sequences, including sequences of Devosia, Afipia, and Agrobacterium. Four sequenced OTUs(14 clones), with 99% similarity to known GenBank sequences, were affiliated with Firmicutes, which were related to the genera Fusibacter, Clostridium, and unculture bacteria. Two sequenced OTUs(2 clones) were affiliated with Bacteroidetes, which had 99% similarity to known bacterial sequences belonging to Bacteroides and Flavobacterium. The remaining 2 sequenced OTUs (2 clones) had 99% similarity to known GenBank sequences of Cetobacterium and uncultured bacterium belonging to Fusobacteria. The results showed that the fish intestinal microbiota harbored some bacteria related to pathogens, probiotics, synthesis and digestion of nutrient element. This study reveals intestinal microflora of health common carp reared in saline-alkaline ponds, and the composition of intestinal bacteria of common carp reared in saline-alkaline ponds is different from that of reared in other environments. These results can provide the basis for ecological control and prevention of the disease of common carp and the assessment of water environmental condition in ponds.

Bacterium QDC01 producing chitinase was isolated from a Rhopilema esculenta in Qingdao sea water. It was identified as Aeromonas hydrophila by morphological and cultural characteristics as well as 16S rDNA sequence analysis. The chitinase gene, named as ahchi (GenBank accession number: JX863407), was cloned from QDC01. Analysis of the gene sequence and its encoded protein sequence showed that the open reading frame (ORF) of ahchi was 2 100 bp in size and the chitinase gene encoded a protein composed of 699 amino acids. The molecular weight of Ahchi was 74.875 kD and the predicted theoretical isoelectric point was 5.81. Sequence alignment and homology analysis indicated that the gene ahchi had the highest similarity (98%) to the chitinase gene (GenBank accession number: CP000462) of A. hydrophila subsp. hydrophila ATCC7966T and the corresponding amino acid sequence homology was also 98%. The phylogenetic analysis of the conserved domains of Ahchi and related chitinases revealed that Ahchi belongs to the typeⅠchitinase and glycosyl hydrolase family 19. The prokaryotic expression vector pET-30a(+)-ahchi was constructed by the method of restriction enzymes (BamHⅠand HindⅢ) and induced with IPTG, and the ahchi gene was successfully expressed in Escherichia coli BL21. Grown in the basic fermentation medium, the chitinase activity of QDC01 was 0.21 U/mL, while in the optimized fermentation medium, the chininase activity was 0.58 U/mL which was much higher than that (0.39 U/mL) of A. hydrophila strain SWCH-6 and also higher than that(0.41 U/mL) of Aeromonas sp. strain CJ-5 reported as chininase producers in the previous literature, suggesting that A. hydrophila strain QDC01 is another good resource for producing chitinase.

Tripterygium wilfordii Hook. f. is a kind of perennial woody vine plant, and it's period of growth is very long and the content of secondary metabolites is low. The natural resources can’t supply enough needs in medicine and agriculture. The way that modern cell engineering technology which produces the secondary metabolites can solve the natural resource shortage and protect the ecological environment for wild T. wilfordii. The leaves of one-year-old T. wilfordii were used to induce calli and suspension embryoid was established using the loose calli. The results showed that green embryoid could be easily induced from calli in 1/2MS+60 mL/L coconut juice liquid medium. The separate embryoid would grow and propagate in 1/2MS+0.5 mg/L NAA. The suitable condition for embryoid culture was 1/2MS basal medium with 1.0 mg/L NAA and 30 g/L sucrose, the initial inoculum was 1.5 g/100mL, pH 5.8, and the medium volume per flask was 120 mL /250 mL after screening inoculum size, pH, the medium volume per flask. The two culture systems were obtained according to the biomass accumulation after adding endophyte fungal elicitors. The triptolide was 1.07 and 1.44 times in embryoid than that in the leaves and in the natural root bark in 1/2MS+0.5 mg/ L NAA+30 g/L sucrose+Y1(leaf endophytes)，pH 5.8; the total alkaloids were 17.02 and 1.46 times in embryoid than that in the leaves and in the natural root bark in 1/2MS+0.5 mg/L NAA+30 g/L sucrose+G4(root endophytes)，pH 5.8. The death rate of embryoid extracts was over 90% after 72 h on Plutella xylostella L. 3rd, the biological activity of embryoid extracts were the same as root bark. The content of triptolide and total alkaloid in embryoid which was induced from leaf calli matched the wild leaf and root bark, there was no significant difference of insecticidal activities between the embryoid extract and root bark. These results show that the culture of embryoid may be one of the pathways to solve the shortage of natural resource in T. wilfordii.

Identification of genes differentially expressed during development, ripening and softening of strawberry fruit is needed to improve strawberry fruit quality and to extend shelf life. In this study a forward and a reverse suppression subtraction hybridization(SSH) cDNA libraries with 516 and 524 positive clones were constructed respectively using cDNA from strawberry(Fragaria×ananassa Duch. cv.Toyonoka) and large green stage fruit as the tester and cDNA from strawberry turning stage fruit as the driver. After removing repeat and redundancy sequences, 707 uniESTs including 369 uniESTs in forward library and 338 uniESTs in reverse library were obtained. The assembling provided a total of 123 contigs and 584 singletons by cluster analyses of the uniESTs. Nucleotide homology searched with Blastn in NCBI non-redundant nucleotide database and 537 uniESTs (75.95% of total uniESTs) were homologous with known genes. Protein homology searched with Blastx in NCBI non-redundant protein database and 509 uinESTs (71.99% of total uniESTs) were homologous with known proteins. The results of gene ontology(GO) annotation showed that 193 uinESTs were involved in biological process with 315 times, molecular function with 332 times and cell component with 409 times respectively. Among these ESTs, putative proteins were related to cell vegetative growth, division, early embryonic development and nutrition transport in large green stage library, and related to metabolites biosynthesis and cell wall-related proteins during fruit softening, pigment synthesis, seed maturation in turning stage library. These results were generally consistent with physiological processes during strawberry fruit development and ripening. In two SSH libraries six genes including ripening-related protein, polygalacturonase(PG)，MYB transcription factor(MYB)，isoflavone reductase-like gene(IRL)，later embryo abundant protein(LEA)and ethylene receptor Etr1(Etr1) genes were analyzed by qRT-PCR in different tissues and stages of fruit development and ripening. The information generated in this study provides new clues to aid the understanding of the development and ripening process in Fragaria×ananassa fruit.

Current detection technologies for diagnosis of animal diseases is mostly targeted at single pathogen, but the prevalence of animal diseases is characterized by mixed infection with more than one pathogen. In order to resolve the trouble, here we describe the new multiparameter assay, which is based on multiplex ligation-dependent probe amplification(MLPA) technology with the advantages of specificity, sensitivity and high-throughput. Five pairs of specific probe targeted at Swine influenza virus(SIV), Pseudorabies virus(PRV), Foot and mouth disease virus (FMDV), Transmissible gastroenteritis virus (TGEV) and Porcine reproductive and respiratory syndrome virus (PRRSV), were designed, respectiviely. The mixture of five standard RNA/DNA was used as template, together with the mixture of these probes as probe and the PCR universal primer, one MLPA method for simultaneous detection of the five porcine viruses was developed. The result of specificity test showed that the designed probes had good specificity without mismatch between each virus-specific probe pair and other six viruses, and that the mixture of the five pairs of probe only amplified the corresponding one specific fragment from the eight virus templates, respectively, no amplification signal was produced among Porcine parvovirus(PPV), Classical swine fever virus(CSFV) and Porcine epidemic diarrhea virus(PEDV) with the same probe mixture. The result of sensitivity test showed that the concentration of nucleic acid of single virus in one MLPA reaction was up to 3 000~6 000 copies. All the results showed that the developed MLPA method in this article accomplished the simultaneous detection of five viruses in one reaction, which indicates MLPA technology may be an alternative to simultaneous detection of many pathogens in the future in the field of veterinary medicine.

Nongda No.3 laying hen is the new layer breed with saving grain and small size features, which is bred by College of Science and Technology, China Agricultural University. This new cultivator got second prize of agricultural advancement in 1998, second prize of national technology advancement in 1999. And it was officially approved by national varieties committee in 2003. Nongda No.3 laying hen gets good comments from society for its excellent performances and benefits. In this paper, the cultivator selection process, features and extension situation of Nongda No.3 laying hen were described, at the same time the current situation and problems of Chinese breeding work were discussed.