农业生物技术学报

Construction of Recombinant Adenovirus Expressing the B,C Antigenic Sites of
Spike Gene from Transmissible gastroenteritis virus(TGEV) and Its Immunization

TANG Fang1 ; HE Kong-wang ; HOU Ji-bo ; JIANG Ping

2009, 17(1): 1-6 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The DNA fragment of 1.0 kb containing the B,C antigenic sites of the Transmissible gastroenteritis virus (TGEV) spike (S) gene was amplified by RT-PCR and cloned into the transfer vector pShuttle-CMV. The recombinant plasmid was co-transformed into Escherichia coli strain BJ5183 together with the Adenovirus (Ad) backbone vector pAdEasy-1 to generate the recombinant Ad plasmid by homologous recombination. A recombinant replication-defective human Adenovirus serotype 5 (Ad-5) containing 5' gene of TGEV S protein (rAd-TGEV-S) was generated by transfection and plaque purification in HEK293 cells. The transcription and expression of the target gene fragment was confirmed by RT-PCR and indirect immunofluorescent assay (IFA) subsequently. The recombinant virus rAd-TGEV-S was passaged by 9 times in HEK293 cells and the titer was stable with the average of 107.7TCID50. The immunization of mice with rAd-TGEV-S showed that TGEV-specific IgG and IgA were monitored.

Construction of a Recombinant Vector Expressing Avian influenza virus
Nucleoprotein with an Avian-derived Promoter and Detection of Its Immunogenicity

2009, 17(1): 7-11 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to investigate the immune responses to the nucleoprotein (NP) of Avian influenza virus, NP gene cut from recombinant plasmid pVAX-NP was subcloned into a eukaryotic expression vector pCAGGS with an avian-derived promoter. The recombinant plasmid pCAGGS-NP was obtained after PCR identification and sequencing. Then the positive pCAGGS-NP was subjected to transfect eukaryocyte and to immunize SPF chickens. Indirect florescent assay (IFA) was engaged to detect the transient expression of NP, and ELISA was used to detect the serum antibody of the immunized chickens which was against to NP protein. These results showed that NP was successfully expressed both in vitro and in vivo and had good immunoreactivity.

Development of the Monoclonal Antibodies and Immune Colloidal Gold-labeled
　　　　　Strip for Rapid Detection of Sulfamethoxazole

2009, 17(1): 18-23 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Balb/C mice（Mus muscalus） were immunized with bovine serum albumin and sulfamethoxazole （BSA-SMZ）, hybridoma lines that secrete monoclonal antibody against sulfamethoxazole（SMZ mAb） were filtered with cell fusion and the immunological traits of SMZmAb were characterized. A stript for detection of SMZ（SMZ-strip） was developed with SMZmAb and its traits were tested. The results showed that three hybridoma lines were filtered and the best one was 3G6 which secreted SMZ mAb with indirect ELISA titers of 1∶8.1 ×102 in supernatant and 1∶6.4×105 in ascites. SMZ mAb had a high affinity constant（Ka） with 3.07×109 L/mol, a good sensitivity with an IC50 of 12.4 μg/L to SMZ, 2.84% cross-reactivity to sulfadiazine and little or no cress-reactivity to other compounds. Its detection limit was 6 μg/L and its sensitivity was as same as that of competitive ELISA-kit and its coincidence rate was 100% compared with ELISA-kit. The SMZ-strip possesses rapidity, sensitivity, specificity and briefness and is proved to be used for the rapid test of SMZ residues.

2009, 17(1): 138-143 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To obtain the higher expression efficiency of producing recombinant protein in Escherichia coli, the prokaryotic expressing vector pET-S1, which contained whole antigenic sites of S gene from swine transmissible gastroenteritis coronavirus (TGEV), was constructed and transformed into E.coli strain BL21 (DE3). The target protein was expressed by the IPTG inducing expression system and auto-induction expression system, respectively. The highest amount of target protein accounted for 25.9% and 45.1% of total bacteria protein, respectively， indicating that the expression efficiency was significantly improved by auto-induction expression system.

2009, 17(1): 114-120 | doi: | Full text (HTML) (1 KB) | PDF

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The regulative sequence(1 738 bp) of ferric chelate reductase (MxFRO2) gene from Malus xiaojinensis was cloned by genomic walking. In silico analysis of sequence suggested that the sequence contained several typical cis-acting elements, including multiple light responsive elements (LREs), ABA responsive element (ABRE), auxin inducible element, Cu responsive element (CuRE), TATA-box and CAAT-box. The promoter sequences of 8 members of Arabidopsis FRO gene family gained from TAIR website were predicted as well. A 1 644 bp and a 259 bp promoter sequence upstream 5' of translation start site of MxFRO2 were cloned and designated as MxFul and MxD1, respectively. MxFul-GFP and MxFul-GFP were constructed and transformed into Arabidopsis mesophyll protoplasts mediated by PEG. These results showed that MxFul and MxD1 could both drive the transient expression of GFP protein in Arabidopsis mesophyll protoplasts.

Construction of a BF2/Peptide Tetramer and Its Application on the Detection of Specific T Cell

2009, 17(1): 12-17 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

For the construction of BF2/peptide tetramer, large amount of recombinant chicken major histocompatibility complex (MHC) Ⅰ heavy chain (BF2-BSP) and light chain Chβ2m were expressed and purified. And a peptide originated from avian Infectious bronchitis virus (IBV) nucleoprotein (N71~78) was synthesized. Afterwards, BF2-BSP and Chβ2m were refolded with IBV N71~78 in refolding buffer to generate the monomer of BF2/peptide complex. Then the folded product was treated with enzymatic biotinylation by BirA enzyme and followed by a gel filtration column to purify the BF2/peptide fraction. Finally, the BF2/peptide tetramer was formed by mixing purified, biotinylated monomer with phycoerythrin(PE)-labeled streptavidin at a molar ratio of 5∶1. In order to test if the prepared BF2/peptide tetramer could be employed to detect the efficiency of specific T cell, the peripheral blood mononuclear cells (PBMCs) were isolated from the SPF chickens infected with IBV H52 at 10 days post infection and subjected to flow cytometry staining. The cytotoxitic T lymphocytes (CTLs) response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% of CTLs frequency specific to IBV N was detected. All these results indicated that the prepared BF2/peptide tetramer can be used to evaluate the specific T cell immune responses of IBV.

Construction of Recombinant Chicken AvBD5 - AvBD10 Double Molecule
Protein and Analysis of Its Physical and Chemistry Characterization

2009, 17(1): 41-46 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Chicken antimicrobial peptides, belong to β-defensins，are essential components of innate immunity and play significant roles in innate immunity in chickens. The cDNA of chicken avian β-defensins 10 (AvBD10) gene was sub-cloned into Sal Ⅰ and NotⅠ sites of AvBD5-pGEX-6p-1 vector to construct recombinant plasmid AvBD5-pGEX-AvBD10. The recombinant plasmid was transferred into Escherichia coli BL21. Then the bacteria was induced with IPTG at 37 ℃ and different time, and exogenous expression was detected by SDS-PAGE. Results showed that a 36 kD protein which was equal to chicken AvBD5-AvBD10 protein in molecular weight was expressed in E.coli BL21. The production of AvBD5-AvBD10 accounted for approximately 35% of the total protein. The recombinant fusion protein was purified and expressed as insoluble body. Two bacterials, E.coli and pathogenic Streptococcus of mid-log (108 cfu/mL) were used to evaluate antibacterial activity of the recombinant fusion protein by inhibition zone assaying. The results showed that the recombinant fusion protein exhibited anti-E.coli. and anti-pathogenic Streptococcus activitiy, and showed high stability at various temperatures -70℃～100 ℃ and pH 3～12.

2009, 17(1): 29-35 | doi: | Full text (HTML) (1 KB) | PDF

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Aeromonas hydrophila is a gram-negative opportunistic pathogen that can result in motile aeromonad septicemia in numerous freshwater fish species. In order to develop a safer vaccine strain for oral immunization, aopB-, aopD- and aroA- triple-deletion mutant was constructed. Firstly, the recombination suicide vector pREaopB/aopD with sacB gene (sucrose-sensitive) and 369 bp-deleted aopB-/aopD- gene (encoding A. hydrophila out membrane protein B, D) was constructed and conjugated with Ah2056. The aopB / aopD deleted strain was selected by two-step method and identified by PCR. Then, the other recombination suicide vector pSUParoA with gentamycin resistance gene and 1290 bp-deleted aroA gene (encoding 5-enolpyruvyl shikimate 3-phosphate synthase) was constructed and conjugated with the aopB-/aopD- deleted strain. The aopB-/aopD-/aroA- deleted strain was selected by gentamycin and identified by PCR. The growth properties and virulence in fish of the aopB-/aopD-/aroA- deleted strain were further characterized. Results showed that the aopB-/aopD-/aroA- deleted mutant was successful constructed. The mutant was lower virulence and aromatic amino acids dependent. The mutant can be potentially used as an oral vaccine strain.

Genetic Characterization of the 11 Isolates of Virulent Newcastle disease virus from Chicken Flocks

2009, 17(1): 36-40 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Eleven isolates of virulent Newcastle disease virus (NDV) prevailing during 2002～2007 from chicken flocks in Shanghai were collected. The F gene of these viruses were cloned and sequenced. Their sequences were submitted to GenBank，and accession numbers were as follows：NDV 022433（EU258661），022435（EU258662），0210149（EU258653），0210154（EU258654），0210227（EU258656），0210252（EU258658），03024（EU258637），04011（EU258639），05013（EU258640），07011（EU258642） and 07023（EU258643）,respectively. The results of sequence analysis showed that the F gene fragments from 11 NDV isolates consisted of l 654 bp，coding for 551 amino acids．The homologies of nucleotide and deduced amino acid sequence among the 11 NDV isolates were 94.6%～100% and 93.0%～100%, respectively. The sequences of cleavage site of F protein were 112R-R-Q-K-R-F117， all of the 11 isolates belonged to the genotype Ⅶ virulent NDV. There had the nearest distances among the 11 isolates as compared with that of the NDV vaccine strains（B1，La Sota，V4 and F48E9） by the phylogenetic tree. In addition，they had two RsaⅠ sites at position 973 and 1 249 nt which are characteristic to most strains of goose origin.

Preparation of Antiserum against Chicken C/EBPα and Its Expression Characteristics in Different Tissues of Chicken

2009, 17(1): 47-51 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The coding sequence (CDS) of CAAT/enhancer-binding protein α (C/EBPα) gene was obtained from T-C/EBPα plasmid by enzyme digestion. The recombinant C/EBPα was expressed in Escherichia coli with IPTG induction and purified by Glutathione Sepharose 4B affinity chromatography. The purified C/EBPα was used as immunogen to immunize the rabbit. The titer of the generated antiserum was detected to be 1∶12800 by ELISA. Western blotting result showed that the antiserum had high specificity. Expression characteristics showed that C/EBPα expressed abundantly in muscle stomach, glandulose stomach, kidney, intestine, liver, heart, fat, spleen and lung respectively while little in muscle. The antiserum was meanwhile used to analyse the C/EBPα expression characteristics between fat and lean line roosters，which showed that C/EBPα expressed higher in lean line rooster livers.

Establishment of Molecular Detection Methods for High Prolificacy Major Gene FecB in Sheep and Its Application

2009, 17(1): 52-58 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The Booroola gene FecB（Fec, fecundity; B, Booroola） is the first major gene for prolificacy identified in sheep (Ovis aries). The FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22-23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was fully associated with the hyperprolific phenotype of Booroola ewes. PCR-RFLP and PCR-SSCP methods for high prolificacy major gene FecB in sheep have been established in the present study. The detection results of 6 647 sheep including Small Tail Han, Hu, Chinese Merino, East Friesian, Dorper, Suffolk, Texel, Dorset, Corriedale, German Mutton Merino, South African Mutton Merino and crossbred sheep indicated that these two methods displayed accurately genotyping, good stability and reliability. Moreover, the detection results of 6 647 sheep using these two methods were identical. The detection results showed that both Small Tail Han and Hu sheep carried the same FecB mutation (A746G) as found in Booroola Merino sheep. Chinese Merino, East Friesian, Dorper, Suffolk, Texel, Dorset, Corriedale, German Mutton Merino and South African Mutton Merino sheep did not have FecB mutation.

Genetic Diversity of Domestic Strains of Common Carp
Based on Partial Mitochondrial DNA Sequences

2009, 17(1): 59-66 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Nucleotide sequences of 16S rRNA, Cyt b, and D-loop of mitochondrial DNA (mtDNA) for nine domestic populations and two wild populations of common carp (Cyprinus carpio) were amplified using PCR techniques and their genetic variability and phylogenetic relationships were analyzed. Thirty-two nucleotide variable positions, 21 parsimony-infomative positions and 16 haplotype were detected in 1 457 bp length sequence among the strains. Phylogenetic trees were constructed from the three different genes sequences as well as the combination sequences by NJ and MP method. There were no contradictions between these phylogenetic trees. The phylogenetic trees were all divided into two branches which Beier Lake wild carp (BE) was singleness and the others were clustered into one group. And a more particular phylogenetic relationship was obtained using D-loop sequences. Moreover, there were no contradiction between the phylogenetic trees constructed by combination sequences and different gene sequences, respectively. The estimated divergence time among breeding different populations was about 3.03 ×104～1.21×105 years ago. As a result, one variable position of 16S rRNA, 4 variable positions of Cyt b gene and 14 variable positions of D-loop region could be used as (SNP) to identify the different strains or wild populations. It was proved that the nucleotide sequences of mtDNA could be used in the identification of strains.

Spatio-temporal Expressed Analysis of Two Kinds of Chemical Communication
Related Proteins in Working Bee of Apis cerana cerana

2009, 17(1): 73-77 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The spatio-temporal expressed profiles of two kinds of chemical communication related to proteins genes of Apis cerana cerana: odorant-binding protein of Ac-ASP2 and chemosensory protein of Ac-ASP3, were identified by Real-time PCR. The results with the method of 2-ΔΔCt showed that Ac-ASP2 was a gene coding antenna specific protein, which did not express in larva and pupa, but had such discontinuous high abundance periods: 1, 9, 15, 27and 30 d, the expressing abundance at such periods was tenfold higher than that at other periods. From the distribution of Ac-ASP3 mRNA in different issues, the transcript amounts seemed to be higher in wings, abdomen and thorax (about 106 order of magnitude), and lower in legs, antennae and head (about 105 order of magnitude), the special relation of the original copy number was: wings >abdomen >thorax >legs >antennae >head. The results above showed that Ac-ASP3 had intimate relation with chemosensory behavior of wings and abdomen in Apis cerana cerana.

Discrimination of Milk Powder with Bovine or Ovine Meat and Bone Meal in Ruminant Feedstuffs

2009, 17(1): 67-72 | doi: | Full text (HTML) (1 KB) | PDF

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A method was established to distinguish milk powder and bovine or ovine meat and bone meal in feedstuffs, which were all ruminant-derived. Milk powder in feedstuffs was washed and removed by water and 0.5% sodium hypochlorite solution, subsequently, the ruminant-derived materials were identified using PCR according to 16S rDNA sequence. The detected limitation of bovine or ovine in feedstuffs with 25%, 50% and 75% milk powder concentrations were 2%, 0.5% and 0.1%, respectively. Taken altogether, this method is a simple and easily grasped way for identification and discrimination of the bovine or ovine-derived meat and bone meal and milk powder in feedstuffs.

Cloning and Expression Analysis of Tau Class Glutathione Transferase Gene from Polygonum sibiricum Laxm

2009, 17(1): 98-106 | doi: | Full text (HTML) (1 KB) | PDF

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Glutathione transferase (PsGSTU1) gene was isolated by RACE from Polygonum sibiricum Laxm, the PsGSTU1 gene had a complete open reading frame of 669 bp and encoded a peptide of 222 amino acid residues. Based on the comparison with other amino acids sequences among glutathione transferase family and the analysis of the phylogenetic evolution, this PsGSTU1 belonged to a glutathione transferase Tau kind of family. Expression analysis by RT-PCR showed that PsGSTU1 gene expressed in leaf, stem and root, especially higher expression in stems, next was leaf and root. After 3% NaHCO3 stress, this PsGSTU1 gene’s largest expression amount appeared at 48 h in leaf and root, but the largest expression within stem appeared at 72 h, When the stress was removed, the PsGSTU1 gene’s expression amount change within various organs were found as follows: root>leaf>stem. Subsequently, PsGSTU1 gene that had a complete cDNA was inserted into the pET28a plasmid to construct the prokaryotic vector and then transformed into Escherichia coli. The 26 kD protein was expressed after inducing by IPTG, and the resistance of E. coli expressed protein to NaHCO3 got enhanced remarkably.

Cloning of Carnation GA20-oxidase Gene and Construction of Plant RNAi Vector

2009, 17(1): 107-113 | doi: | Full text (HTML) (1 KB) | PDF

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A pair of degenerate primers were designed based on the conserved domain of the GA 20-oxidase reported in other plants. A full-length cDNA (1 179 bp) of carnation (Dianthus caryophyllus L. cv. Marster) GA20-oxidase named Dc20ox was cloned by RT-PCR and RACE methods. The deduced amino acids of this sequence shared a high homology. Blast showed that the amino acids sequence presented a high homology (66%~75%) with the GA20-oxidase in other plants. A RNAi vector (pART400) was constructed by a high homology 400 bp fragment.

Cloning and Expression of Gene Encoding Gqα-protein in the England Grain Aphid (Sitobion avenae )

2009, 17(1): 78-83 | doi: | Full text (HTML) (1 KB) | PDF

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Based on conserved homologous amino acid sequences of Gq protein α subunit in arthropods, a pair of degenerate primers were designed to amplify the gene from the England grain aphid(Sitobion avenae) by RT-PCR and (3′/5′)-RACE techiques. A Gqα was obtained from the alate adult aphids. The open reading-frame was 1 062 bp, encoding 352 amino acid residues with calculated molecular weight of 40.8 kD. The cDNA sequence was deposited in GenBank with the accession number EF638906. The deduced amino acid sequence of Gqα shared high identity (≥82.17%) with reported Gqα from other insects and even vertebrates, and with the typical characteristics of Gqα protein. In order to explore the function of Gqα gene, a eukaryotic expressional system(Baculovirus expression vector system, BEVS) was constructed by TOPO and Gateway technique. After the recombinant reaction between pUC-Gqα and Gateway-adapted Baculovirus DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV), the constructed recombinant viruses containing V5-His6Gqα were transfected singly into an insect (Trichoplusia ni) cell line of Tn-5B1-4. After collecting the infected cell, an expected protein (about 42 kD) was detected by SDS-PAGE and Western blot. The results showed that the system of recombinant Baculovirus and Tn could express Gqα protein successfully.

Cloning of S-adenosylmethionine Synthetase Gene from Malus xiaojinensis （MxSAMS） and Its Expression under Iron-deficiency Stress

2009, 17(1): 121-125 | doi: | Full text (HTML) (1 KB) | PDF

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S-adenosylmethionine synthetase gene (SAMS) cDNA from a iron-deficiency subtracted library of Malus xiaojinensis was isolated ,which showed 90% identity to the other homolog gene of S-adenolyl-L-methionine synthetase enzyme in GenBank. The full length cDNA of SAMS gene from M. xiaojinensis was obtained using RACE and named as MxSAMS (GenBank accession No.EU639408). The full cDNA of MxSAMS was 1 479 bp with an open reading frame of including a complete ORF of 1 176 bp that encoded a predicted polypeptide of 392 amino acids with the predicted MW of 42.99 kD. MxSAMS showed 88.1%～92.6% amino acids sequence similarity with the other SAMS in plant. The deduced product of MxSAMS had the conserved domain among all the known SAMS, including a ATP binding domain. Southern blot showed that there was only one single copy of MxSAMS gene in the genome. The result of semiquantitative RT-PCR indicated that the MxSAMS in root and leaves of M. xiaojinensis was induced by iron-deficiency stress.

Relationships Between the Tolerance Difference to High Temperature Stress and Superoxide Dismutases (SODs) in Different Cultivars of Celosia cristata

2009, 17(1): 126-131 | doi: | Full text (HTML) (1 KB) | PDF

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To investigate the relationships between the tolerance difference to high temperature stress and superoxide dismutases (SODs) in different cultivars of Celosia cristata, ten-leaf stage seedlings of the heat-tolerant cultivar, Century Green and the heat-sensitive cultivar, Century copper were pulsed for 48 h with diethyldithiocarbamate (DDTC) pretreatment（specific inhibitor of SOD） and subjected to high temperature stress under controlled conditions of relative humidity of 75%~90%, 12 h/24 h photoperiod with 1 500 lx and 45 ℃, to research the changes of external morphology and interior physiology. The results showed that SOD activity was remarkably inhibited by 20 mmol/L DDTC pretreatment and the tolerance of Celosia cristata seedling to high temperature stress was obviously decreased, heat-sensitive cultivar was more obviously than that as heat-tolerant cultivar. During the high temperature gradient stress, the band intensity of all SOD isoforms was reduced differentially, in which the band of MnSOD was significant reduction, followed by Cu/ZnSOD， and one new band named as Cu/ZnSOD was appeared at 35 ℃ and 40 ℃ in heat-sensitive cultivar and heat-tolerant cultivar, respectively. When the temperature was 45 ℃, all bands disappeared expect for FeSOD2 , weak FeSOD3 and Cu/ZnSOD1 in heat-tolerant cultivar; while only FeSOD2 band was remained in heat-sensitive cultivar. It was suggested that SOD isoforms of heat-tolerant cultivar had more activity and stability than that of heat-sensitive cultivar. However, the new bands were inhibited by DDTC pretreatment. It is showed that the tolerance to high temperature was highly related to SOD activity and the difference of cultivars to high temperature stress was relative to activity of FeSOD3 and Cu/ZnSOD1.

Identification of A Marker Closely Linked to Rice Stripe Disease Resistance Gene in a Japonica Rice Variety

2009, 17(1): 144-147 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Markers related to the gene of rice stripe resistance in Oryza sativa ssp.japonica Zhendao88 were screened. The inheritance of rice stripe resistance in F2 lines and F2:3 lines from the cross Zhendao 88/ Wuyujing No.3 were studied using tiller inoculation and inoculation at the seeding stage, respectively. A marker OPO11, which was coseperated or complete linked to rice stripe disease resistance gene in Zhendao 88, was obtained by RAPD analysis. The amplification results of OPO11 primer in several resistance japonica varieties, such as Xudao No.3, originated from Zhendao 88, were the same as that of Zhendao 88. The above marker linked to the rice stripe resistance gene in Zhendao 88 could be used in marker-assisted selected program for rice stripe resistance breeding.

SSR Markers Linked to Leaf Rust Resistance Gene Lr2c in Wheat

2009, 17(1): 148-152 | doi: | Full text (HTML) (1 KB) | PDF

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SSR analysis was performed based on 7 wheat (Triticum aestivum) near iso-genic lines (NILs) with Lr genes located on chromosome 2D including two multiple alleles of Lr2c, the susceptible control Thatcher, and a population of 215 F2 plants derived from TcLr2c × Thatcher, respectively to identify the SSR marker linked to wheat leaf rust resistance gene Lr2c. Twenty-nine SSR primer pairs on wheat 2D chromosome were screened and four of them could generate polymorphisms to the other near isogenic lines. Result showed that Xgwm261 and Xgwm296 were tightly linked to the gene Lr2c with a distance of 1.9 and 3.6 cM respectively using test and verify of the population of 215 F2 derived from TcLr2c × Thatcher, which could be used in molecular assisted breeding against wheat leaf rust.

Expression of β-agarase ⅠDagA in Prokaryotic Cell and Its Activity Identification

2009, 17(1): 132-137 | doi: | Full text (HTML) (1 KB) | PDF

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dagA gene and dagA(▽) which is a dagA gene encoding sequence without signal peptide were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by PCR. After ligation with pET21 vactor, dagA and dagA(▽) were expressed in E. coli ER2566, respectively, using molecular chaperone DsbC and FkpA. Strain of ER2566- pET21a-dagA(▽)-DsbC was screened as high effective expressing system, in form of the inclusion body in which had the target protein up to 60 % of total bacterial protein. DagA protein was renaturated and purified by dissolving in 8 mol/L of urea, Ni-NTA resin affinity chromatography and refolding by urea gradient method. DagA was about 30.8 kD identified by SDS-PAGE and had the ability to digest agarose. At the range of pH 4.8 to 6.8, DagA could hold an bio-activity of beyond 60 %, with 5.8 as optimum pH value and its activity was available under 37 to 60 ℃, with 55 ℃ as optimum temperature.

Detection of Apple fruit crinkle viroid in Xinjiang Apple and Its Specific Fragment Sequencing

2009, 17(1): 164-169 | doi: | Full text (HTML) (1 KB) | PDF

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Branches, leaves and phloem of apple (Malus domestica Brkn) collected from an old orchard in Yanji, Xinjiang were used for total RNA isolation specific fragments of Apple fruit crinkle viroid (AFCVd) were amplified by RT-PCR using the primers disigned according to the sequence AB104558 on GenBank. Ten sequences were obtained through recovering, cloning and sequencing, and also registered in GenBank (accession No. EU031507~EU031516). Then cDNA probe laballed by Digoxigenin using plasmids containing targent DNA of AFCVd was composed and used for AFCVd detection. At the same time, in situ RT-PCR of AFCVd for further detection proved the presence of AFCVd in apple leaf, mainly located in nucleus of mesophyll cell.

Genetic Identification of Wheat-rye 1R Alien Disomic Additional Line with
Novel Resistance Gene to Powdery Mildew

2009, 17(1): 153-158 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Rye (Secale cereale) with multi-spikelet and resistant gene to powdery mildew, is beneficial for wheat genetic improvement. Wheat germplasm N9436-1 is bred from the progeny of Austria rye × common wheat （Triticum aestivum） Shaanmai 611. In the present study, cytology, Genomic in situ hybridization (GISH), Giemsa C-banding, sequence characterized amplified region (SCAR) and A-PAGE were used to detect rye chromatin of N9436-1. The result showed that the morphology and cytology of N9436-1 was stable with chromosome configuration of 2n=44=22Ⅱ. N9436-1, with multi-spikelet, was immune to powdery mildew. GISH and C-banding showed that N9436-1 was wheat-rye 1R disomic additional line. SCAR and APAGE analysis further displayed that the line N9436-1 had the genetic material of rye. Pm8 and Pm17 of 1R chromosome were not resistant to powdery mildew, while N9436-1 was resistant to it. So there is probably a new gene on 1R chromosome of the line N9436 resisting to powdery mildew and can be used as resistant resource of powdery mildew for wheat breeding.

Effect of Nano-TiO2 on the Bacterial Community of the Cucumber Phyllosphere

2009, 17(1): 159-163 | doi: | Full text (HTML) (1 KB) | PDF

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The impact of nanometer titanium dioxide(nano-TiO2) to the phyllosphere bacterial community were assessed using culturable-depend and PCR-DGGE methods. Results showed that quantities of the culturable phyllosphere bacteria were reduced with the increasing concentrations of the nano-TiO2. The quantity of the culturable phyllosphere bacteria decreased from 1.8×107 to 3.1×106 cfu/g along with the concentrations of the nano-TiO2 from 0.002 to 20 mg/mL. The diversity of the phyllosphere bacteria was also analyzed by PCR-DGGE. When the concentrations of the nano-TiO2 were higher than 0.02 mg/mL the DGGE bands was significantly lower than that of the control. Sequencing results of the bands from the DGGE gel showed that there were at least 7 bacterial genera on the cucumber phyllosphere. And only one phyllosphere uncultured bacterium was not influenced by the concentration of the nano-TiO2.

2009, 17(1): 84-89 | doi: | Full text (HTML) (1 KB) | PDF

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A cDNA library was constructed using fibers of Gossypium hirsutum 7235 line. The library was randomly sequenced and an expressed sequence tag (EST) with the function of lipase was obtained. The full-length cDNA encoding lipase in 7235 line was cloned by the rapid amplification of cDNA ends (RACE) technique and named GhLipase. Sequence analysis showed that the full-length cDNA was 1 286 bp which contained a major open reading frame of 368 amino acids, 5′ un-translated regions, 3′ un-translated regions, and a Poly (A) tail. The sequence was submitted to GenBank, and accession No.EU273298. It had high homology to reported lipase genes from other plants. RT-PCR analysis confirmed that the GhLipase gene was up-regulated in ovules and fibers compared with roots, stems, and leaves of 7235. Southern blot analysis showed that there were two copies of the GhLipase gene in the genome of G. hirsutum. GhLipase gene was mapped on chromosome A13 in [(TM-1 × Hai7124) TM-1] population.

Establishment of Real-time RT-PCR Assay to Differentiate O and AsiaⅠType Foot-and-mouth disease virus

2009, 17(1): 24-28 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Several genome sequences of Foot-and-mouth disease virus (FMDV) were obtained from GenBank. After multiplex alignment, three sets of primers and probes were designed according to 3D, VP3 and 3C region of FMDV. Three sets of primers and probes were used to establish FMDV species-specificity, O type-specificity and AsiaⅠtype-specificity Real-time RT-PCR, respectively. Standard S shape curves occurred after Real-time RT-PCR amplification on Lightcycler machine. All three newly established methods had good specification for they could differentiate FMDV from Infectious bovine rhinotracheitis virus, Swine transmissible gastroenteritis virus, Akabane virus and Porcine respiratory corona virus. FMDV species-specificity Real-time RT-PCR could successfully amplify O type(O1 strain and O2 strain) and AsiaⅠtype (AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV. O type-specificity Real-time RT-PCR could successfully amplify O type(O1 strain and O2 strain) FMDV but failed to amplify AsiaⅠ (AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV. AsiaⅠtype-specificity Real-time RT-PCR could successfully amplify AsiaⅠ(AsiaⅠ1 strain and AsiaⅠ2 strain) FMDV but failed to amplify O type (O1 strain and O2 strain) FMDV. All methods could detect 10 copies of DNA template, closely reach the detection limit. The whole Real-time RT-PCR reaction could be finished in 70 min. These methods have potential to apply in FMDV screen and differential detection in laboratory.

Stress Physiological Roles of Diacylglycerol Kinase (AtDGK7) Gene in Arabidopsis thaliana

2009, 17(1): 90-97 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Transgenic AtDGK7 plants were obtained by constructing PBI121-AtDGK7 expression vector and transformating it into Arabidopsis thaliana. And the stress-resistant function of diacylglycerol kinase (AtDGK7) gene in Arabidopsis thaliana was researched by stress resistance determination, ABA, NaCl and mannitol resistance experiment and RT-PCR. The results showed that the AtDGK7-overexpressed Arabidopsis thaliana plants were less sensitive to high concentration ABA and displayed normal germination of seeds. Under salt stress, the AtDGK7-overexpressed Arabidopsis thaliana plants grew more robust than that of the wild-type Arabidopsis thaliana plants , and could resist the salt stress. Meanwhile, the free proline and malonaldehyde contents, and superoxide dismutase activities were determined. And the results showed that the low temperature-resistant of the AtDGK7-overexpressed Arabidopsis thaliana plants were proved.

Construct RNAi Structare in Plant Gene by Recombiant PCR

2009, 17(1): 176-177 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

RAPD Analysis of Genetic Diversity among Five Species of Dendrolimus (Lepidoptera: Lasiocampidae )

2009, 17(1): 178-179 | doi: | Full text (HTML) (1 KB) | PDF

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PCR Ampification and Sequence Analysis of rDNA-ITS of Corynespora cassiicola from Rubber

2009, 17(1): 180-181 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Application of RNA Interference in Plant Parasitic Nematodes

2009, 17(1): 170-175 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

RNA interference (RNAi) has been demonstrated in plant parasitic nematodes. Genes expressed in a range of cell types are silenced when pre-parasitic juvenile nematodes take up double-stranded (ds) RNA that elicits a systemic RNAi response. It is a potentially powerful investigative tool for the genome-wide identification of gene function that should help understanding plant parasitic nematodes. Recent studies have described its application to the mechanism, route, characterization, efficacy and durability in plant parasitic nematodes. Important developments show that plant expression of a dsRNA targeting a nematode gene can successfully induce silencing in parasitizing nematodes.