农业生物技术学报

Pathogenic Studies on the Diseases of Avian Embryo in AA Broiler Breeder and the Detection of Its Main Pathogens by PCR

LIU Hong-qin;ZHU Rui-liang;SUN Qiu-yan;HU Xiao-na;BI Jian-min;LIU Hong-Zhen;LI Xin-cang, WANG Wei

2006, 14(5): 646-651 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A kind of disease appeared in one large-scale AA broiler breeder farm in Shandong Province with the symptom of embryo death, increase of weak chicken, ineffective immunity, stunted growth of 30 days chicken since June, 2004. Based on the epidemiology investigation and the pathogenic studies, it was showed that the co-infection of Bordetella avium, Salmonella, Escherichia coli, mycoplasma, Chicken infectious anemia virus(CIAV) and Viral arthritis virus(VAV) was the major cause of the disease. These pathogens spread vertically from the eggs to the embryos. The 16S rRNA gene of Bordetella avium, CIAV VP3 gene and VAV S1 gene were amplified by PCR and 783 582 and 532 bp specific bands were acquired respectively. The PCR products of CIAV VP3 gene and VAV S1 gene could hybridize with the corresponding probe and appear positive reaction.

Polymorphism of Porcine Uncoupling Protein 3 (UCP3) and Its Association
with Fat Deposition and Meat Quality

REN Liang;ZHU Bao-qin;HAN Dan;ZHANG Yi-bo;SONG Hui-juan;ZENG Rui-xia;SU Yu-hong

2006, 14(5): 652-656 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Based on quantitative traits loci (QTL) mapping, porcine uncoupling protein 3 (UCP3) gene was chosen as a candidate gene for pig fat deposition and meat quality traits. Partial coding region of UCP3 gene was sequenced and there was one single nucleotide polymorphisms (cSNP) at 395 bp. This mutation was G→A and it resulted in the amino acid change from glycine to arginine. This site was also recognized by restriction endonuclease SmaⅠ. The polymorphism of the UCP3 SmaⅠwas analyzed with PCR-RFLP among 186 Large white × Meishan F2 progeny. The genotypes of UCP3 SmaⅠwere AA, AB and BB. The frequency of A allele was 0.56 and B was 0.44. With statistical analysis, it was found that the SmaⅠ polymorphism in the F2 population was significant associated with backfat thickness at thorax-waist and at buttock, intramuscular fat, drip loss rate and water holding capacity. The function of UCP3 SmaⅠmainly showed additive effect. The genotype AA reduced back-fat thickness and water drip loss rate, and increased water holding capacity. Meanwhile, it also decreased the intramuscular fat content .

Establishment of Real-time Fluorescence PCR to Detect Streptococcus suis Serotype 2

LUO Bao-zheng;BO Qing-ru;CHEN Jing-fan;XU Hai-nie;YANG Su;YANG Jian-yun; SHA Cai-hua;LIAO Xiu-yun

2006, 14(5): 783-787 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A real-time fluorescence polymerase chain reaction(PCR) method to detect Streptococcus suis serotype 2 was established. Primers and Taqman probe were designed according to cps2I(capsular polysaccharide 2I) gene with software Primer Express 2.0 and Oligo6.0. Eighty-one base pair DNA fragment was amplified from S. suis serotype 2 genome DNA, and the PCR product was cloned into pMD18-T vector. Sequencing result confirmed that DNA fragment was target segment. Real-time fluorescence PCR amplification curve on Lightcycler showed that this method could successfully amplify S. suis serotype 2, while reference S. suis strain, E. coli, Salmonella, Staphylococcus aureu, Shigella, Listeria monocytoge and blank control were all negative, so it had good specificity. Ten-fold dilution of S. suis serotype 2 was used to measure the sensitivity of real-time fluorescence PCR. Ten bacteria could be detected in one PCR reaction and the reaction could be completed within 30 min. To examine the stability of the real-time fluorescence PCR, positive control was detected at two different times with 20 repeats. Result showed that Ct value of two times had no statistic difference(P > 0.05), thus this method has a reliable stability. The newly-built real-time fluorescence PCR has potential to apply in entry-exit inspection and quarantine.

Expression of vp1 Gene Fragment of Goose parvovirus in Escherichia coli and Preparation of Its Antiserum

WANG Jing;WANG Dong-shuai;HAN Zong-xi;SHAO Yu-hao;RAN Duo-liang;LIU Sheng-wang;KONG Xian-gang

2006, 14(5): 788-792 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A 662 bp fragment at 5'- end of vp1 gene of Goose parvovirus(GPV) isolate HG5/82 was amplified by PCR. The PCR product was cloned into pMD18-T Simple vector and the recombinant plasmid was transformed into Escherichia coli DH5α. After digesting with BamHⅠand Hind Ⅲ, the inserted fragment was subcloned into prokaryotic expression vector pPROEXTMHTb and the recombinant plasmid was verified by DNA sequencing and transformed into E.coli DH5α. SDS-PAGE results indicated that engineering bacteria could express a 32 kD product after inducing with 0.6 mmol/L IPTG. Densitometric scanning showed the expressed fusion protein could account as much as 22.8% of total bacterial protein of DH5α. The induced engineering bacteria were lysed by 6 mol/L guanidine hydrochloric acid and ultrasound, then the fusion protein was purified with ProBondTM resin from the suspension centrifuged. Antiserum against the fusion protein was obtained by immunizing rabbits. Western blotting analysis showed that the antiserum could specifically recognize the fusion protein as well as VP1 and VP2 of GPV HG5/82 strain.

Construction and Purification of Recombinant Goatpox virus
Expressing Exotic Green Fluorescence Protein

CHENG Xiao-wen;HU Sen;WANG Xi-jun;Bao En-Dong;BU Zhi-gao

2006, 14(5): 798-802 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Vaccine strain of Goatpox virus (GPV) cultivated and widely used in domestic was employed as the mother virus. The tk gene of GPV, the nonessential region during virus replication, was chosen as target site and homologous arm when inserting foreign gene. The stability of lacZ and gfp genes expression in recombinant rGPV-lacZ-gfp downstream of back to back promoter 11 and promoter 7.5 was testified after recombination of the transfer vector pTK-lacZ-gfp with the mother GPV, then lacZ gene in pTK-lacZ-gfp was replaced by gpt gene in order to obtain a purified recombinant GPV expressing foreign gene stably (rGPV-gpt-gfp) by using MPA blocking off the nucleic acid metabolize. Further more recombinant GPV construction system and optimized the purification methods were established, which supply a technique platform for investigation of live vector vaccine and relative basal research.

Detection of Roundup Ready Soybean in Highly Processed Products by Triplex Nested PCR

ZHANG Ming-hui;GAO Xue-jun;YU Yan-bo;QIN Jun;LI Qing-zhang

2006, 14(5): 752-756 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Seven representative highly processed product samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, crude soybean oil, soybean refine oil, soybean salad oil) were detected by the triplex nested PCR. The first triplex PCR could not detect the insert signals in the processed products, and the sensitivity was 0.5％. However, the second triplex PCR could simultaneously detect RR soybean elements, lectin, 35S-CTP and EPSPS-NOS, and a sensitivity was 0.005%. The results indicate that triplex nested PCR is suited to qualitative detection for Roundup ready soybean in highly processed products.

Variations and Biological Characterization of K88ac Fimbria of Escherichia coli of Late Years

CHEN Xiang;MIAO Xiao-qing;LIU Jing;HUANG Li-li;Gao Song;JIAO Xin-an;LIU Xiu-fan

2006, 14(5): 757-762 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Some Escherichia coli strains were isolated from swine colibacillosis in China during 2000 to 2005. They reacted with K88 a specific monoclonal antibody while without K88 b, c and d specific monoclonal antibodies in the slide agglutination test, respectively. O149 was common O serogroup of E. coli and 100.0%(16/16) strains possessed the genes of estII in this study. These isolates were identified with K88ac variant by cross-absorbed rabbit antisera to K88ac fimbria. The apparent molecular weight of K88ac antigens purified from these isolates was about 26 kD in SDS-PAGE, and experienced a positive reaction with the rabbit antisera absorbed with K88ac reference strain by Western blot. The faeG gene was amplified from the genomic DNA of SEC464, SEC525, SEC586, SEC799 and SEC910 by PCR. Alignment analysis of major FaeG subunits of the K88ac fimbria of these isolates showed some differences in amino acid composition comparing with those of K88ac reference strains, implying that the antigenic differences between these isolates and reference strains were due to the mutation of major subunits of the fimbria. Comparison of the sequences of amino acids of the major units showed that the percent identity was 97.7% ~99.6% among those isolates in amino acid sequences of major subunit FaeG, and 90.0%~91.6% comparing with K88ab reference strains, 94.6%~96.6% with K88ac reference strains, 87.0%~88.9% with K88ad reference strains, respectively. These results revealed that K88ac fimbrial antigens experienced some variations comparing with those of K88ac reference strains.

Molecular Characterization and Overexpression of Erwinia chrysanthemi Strain CSCL006 hrpNCSCL006 Gene, which Encodes An Elicitor of the Hyperdensitive Reaction

TANG Cheng;CUI Ya-ya;WU Bo-ji ;LI Ming-yang

2006, 14(5): 763-767 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The DNA library of Erwinia chrysanthemi isolation CSCL006 was constructed. The hrpNCSCL006 gene was cloned from the DNA library and the nucleotide sequence was determined. The nucleotide sequence of hrpNCSCL006 revealed a coding region of 1 020 bp which matched well with the size of hrpNCSCL006 transcript determined by Northern blot. Alignment of the deduced amino acid sequence of harpin CSCL006 with harpin sequences of other soft-rot Erwinia species in database demonstrated that harpin CSCL006 shares high homology with harpins of Erwinia chrysanthemi strains EC16 and 3937, but relatively lower homology with harpins of Erwinia carotovora subspcorotovora. hrpNCSCL006 was cloned into a expression vector pET28a(+) and overexpressed in Escherichia coli. The ca. 34 kD overexpressed polypeptide matched well with the size of predicted harpinCSCL006. Using anti-harpinEcc antibodies as probe, Western blotting analysis confirmed that the overexpressed protein was harpinCSCL006. The harpinCSCL006 fractionated from E. coli could elicite the hypersensative reaction in tobacco (Nicotiana tabacum) leaves.

Genetic Diversity of In situ Conserved Dongxiang Wild Rice Detected by SSR

KONG De-li;XIE Jian-kun;ZHUANG Jie-yun;FAN Ye-yang;WAN Yong

2006, 14(5): 742-746 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The genetic diversity analysis is important precondition for germ plasm resource's collection and utilization. The genetic diversity of 113 Dongxiang wild rice(Oryza rufipogon Griff.) was evaluated by 24 pairs of simple sequence repeat primers. A total of 114 alleles were generated with average allele per primer pair of 4.75, indicating that the Dongxiang wild rice had high genetic diversity. Clustering result showed that some individuals in the same colony were similar, the minimal genetic distances of the individuals in two colonies were 0 and in the other one was 0.018, the other individuals in the same colony were highly variant and the maximal genetic distance was 0.851. Genetic distance of intrinsic-populations were 0.171~0.435, being smaller than the values of inter-population which were 0.432~0.652. Consequently, the present populations of Dongxiang wild rice must be preserved strictly in original place.

Cloning of Heart-type Fatty Acid-binding Protein (H-FABP ) Gene of Swine and
Difference of Its Expression at Different Stages

HUANG Yu-hai;WANG Yi-zhen;SHAN Ti-zhong;LIU Jian-xin;FENG Jie

2006, 14(5): 657-661 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Heart-type fatty acid-binding protein(H-FABP) gene mRNA extracted from muscle of pig(Duroc×Landrace×Yorkshire) was amplified using RT-PCR. And a DNA fragment of 211 bp in length was obtained and the PCR product was cloned into PGEM-T vector. The H-FABP gene was isolated and sequenced from the positive clones screened. Sequence analysis suggested that this fragment was partial sequence of H-FABP cDNA. The gene homology of fragment obtained in this study compared with that of reported H-FABP sequence in muscle of porcine was 99%. Based on the H-FABP gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using 18S rRNA as inner control, the difference of H-FABP gene expression at different stages of swine was researched. The result indicated that the H-FABP gene expression level decreased from 1 to 50 kg, and increased from 50 to 90 kg of body weight.

Cloning, Expression and Purification of Chicken MHC I α-BirA Substrate Peptide (BSP) Sequence in Escherichia coli

LIU Guang-liang;TONG Tie-gang;WANG Qun;XIAO Yi-hong;MENG Qing-wen;WU Dong-lai

2006, 14(5): 662-668 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The gene of chicken MHC I α (BF2 ) was amplified from total RNA of peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T. And then the PCR product which deleted the signal sequence at the 5' end in the BF2 gene and included BirA substrate peptide(BSP ) sequence at the 3' end was amplified from pMD18-T including BF2 gene by PCR amplification and subcloned into an expression vector pET-28a(+) named pET-BF2-BSP. The recombinant vector pET-BF2-BSP successfully expressed in the Escherichia coli strain BL21(DE3). The results of SDS-PAGE and Westernblot showed that the target protein was about 40 kD containing 6×His tag. The overexpressed recombinant protein could be gained when the bacteria was at OD600nm of 0.8 to 1.2 induced for 2 to 4 h and 1.1 mmol/L IPTG. Additionally, the recombinant target protein was purified with a Ni2+NTA column including His-Bind Resin and high quality of recombinant protein was recovered from the inclusion body. All these results indicated that high quality of recombinant protein of BF2-BSP was obtained, which will be contributed to the construction of chicken MHC peptide tetramer.

SCAR Conversion of Two RAPD Markers Linked to An Avirulence Gene Cluster in Magnaporthe grisea

LIN Yan;WANG Bao-hua;YANG Jie;LEI Cai-ling;LU Guo-dong;WANG Zong-hua

2006, 14(5): 768-773 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To clone and characterize the avirulence gene cluster of Avr-Pi1, Avr-Pi2 and Avr-Pi4a, genomic DNAs of Magnaporthe grisea strains FJ81278ZB15, GUY11 and their F1 progenies were isolated and used as templates to screen for tightly linked random amplified polymorphic DNA(RAPD) markers . Two RAPD markers, P1414700 and P1389420 were identified using bulk segregant analysis, then cloned and sequenced. To overcome difficulty in reproducibility of RAPD markers, conversion of sequence characterised amplified region(SCAR) was further conducted. Two pairs of primers were designed based on sequences of P1414700 and P1389420 and their amplification polymorphism was validated with templates from FJ81278ZB15, GUY11 and their F1 progenies. The PCR results indicated that P1414700 was converted into a SCAR marker SC1414 successfully, but not for P1389420 . The genetic distances of molecular markers, including SC1414, P1389420, RPF1.2 and the avirulence gene cluster of Avr-Pi1,Avr-Pi2 and Avr-Pi4a, were then calculated by using program Mapmaker. The genetic distances were 5.8, 2.2 and 4.4 cM respectively from SC1414, RPF1.2 and P1389420 to Avr-Pi2; 15.9,7.9 and 5.7 cM to Avr-Pi1; 20.7, 12.7 and 10.5 cM to Avr-Pi4a. These markers will facilitate the map-based cloning of the avirulence gene cluster.

QTL Analysis of 5 Important Traits in Brassica napus

LIU Lie-zhao;LIN Na;CHEN Li;TANG Zhang-lin;ZHANG Xue-kun;LI Jia-na

2006, 14(5): 747-751 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Protein content in meal, hull content, oil content in hull, 1000-seed weight and seed oil content were analyzed using 132 F2:3 families which derived from across between yellow-seeded parent GH06 and black-seeded parent P174 of Brassica napus. The results showed that these five traits were typical quantitative trait with the phenotype continual distribution. Using the method of composite interval mapping(CIM), a total of 13 QTL(quantitative trait locus) affecting these five traits were detected in linkage group 7, 8, 13, 14, 19, 20 and 21. As for seed oil content and 1000-seed weight, only one QTL was obtained with 14.1% and 18.7% contribution for the phenotype variation respectively. Five QTL, 3 QTL and 3 QTL were detected for the protein content in meal, hull rate and oil content in hull and the contribution for each trait ranged from 7.8%~14.4%, 26.5%~32.8% and 8.3%~12.1% respectively. Furthermore, overlapping region was observed in linkage 20 among the QTL of protein content in meal, oil content in hull and 1000-seed weight.

Expression of Xylanase Gene xynB from Aspergillus niger in Escherichia coli and Characterization of Its Recombinant Xylanase

DENG Ping;CAO Yun-he;LU Wen-qing;GUO Xiao-hua

2006, 14(5): 774-778 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The gene xynB encoding β-1, 4-xylanase was cloned from Aspergillus niger mafic-005. The sequencing results showed that the full-length gene contained 745 bp, including an intron of 67 bp, and encoding a protein of 225 amino acids which had a presumed molecular mass of 24 kD(GenBank accession No. DQ174549). Compared this xylanase xynB gene with others from A. niger in GenBank, the homology was high. The xynB gene was inserted into the expression vector of pET-28a(+) and transformed into Escherichia coli BL21. The recombinant protein was expressed successfully in E. coli by inducing with IPTG, and purified by Ni-NTA Sepharose FF. The molecular mass of the recombinant protein was about 30 kD according to SDS-PAGE. Characterization of the recombinant enzyme indicated that the optimum temperature and pH of the recombinant xylanase were 40℃ and 5.0, respectively. The recombinant protein showed an extreme stability in the acidic solution and ambient temperature.

Real-time Quantitative PCR Detection of Escherichia coli O157:H7

CHEN Si;HUANG Kun-lun;XU Wen-tao;LI Yuan;LUO Yun-bo

2006, 14(5): 779-782 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A rapid and accurate real-time quantitative polymerase chain reaction (Real-time PCR) method with SYBR GreenⅠfor detecting Escherichia coli O157:H7 was established. A pair of amplify primers were designed to amplify eae gene. The dissociation curves showed that the amplified product was very specific. The optimal reaction program and standard curve were founded. The result indicated that Real-time PCR was sensitive 1000 times more than ordinarily PCR.

Isolation of Kunming Mice Embryonic Germ Cells Derived From Primordial Germ Cells and Their Passage Culture

GUO Zhi-lin; WANG Xin-zhuang;ZHANG Yong;WANG Mu;WANG Yi-min;HU Wen-ju

2006, 14(5): 688-691 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Four isolation methods: co-culture back 1/3 position of embryonic tissue, co-culture genital ridges, different speed attaching and puncture the genital ridges were compared to improve the efficiency for establishing the embryonic germ cells (EG cells) line from Kunming mice. Two passage ways, hand and digestion together with fibroblast, were used to clone EG cells. Results showed that co-culture the genital ridges and different speed attaching were more effective methods than that of the others. And passage by hand could improved passage efficiency of EG cells compared with digestion together with fibroblast. When isolated EG cells were examined by morphology observation, AKP strain and in vitro differentiation ability, they were convinced to have a series of characters of mice EG cells including cloning grow way, undifferentiation state and pluripotency of cells.

Effects of Cytochalasin-B and Cytoplasm Removal Treatment on In vitro Parthenogenetic Embryos of Goat

LIU Feng-jun;ZHAO Ming-tao;WANG Guo-hua;WANG Yong-sheng;ZHENG Yue-mao;ZHANG Yong;ZHANG Yu-ling

2006, 14(5): 692-696 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To optimize the protocols of nuclear transfer, effects of cytochalasin-B(CB) treatment and cytoplasm removal on parthenogenetic embryos of goat were observed. MetaphaseⅡ(MⅡ)stage goat oocytes were treated with 7.5 μg/mL CB for 5、10、15、20 and 30 min, respectively (groupⅠ), removal of cytoplasm from 1/4 to 1/2(group Ⅱ)and with nothing(control). Differential staining of inner cell mass (ICM) and trophectoderm (TE) cells of blastocysts (Day 5~7) was performed with propidium iodide and Hoechst 33258, and the numbers of ICM and TE cells were recorded respectively. The groupⅠand control showed higher cleavage rate(83%~ 85.50%,86.50%)and blastocyst rate(57.40%~62.20%,59.80%)than that of group Ⅱ, the numbers of total cells and ratio of ICM cells had no significantly difference between various groups(P＞0.05). However, the cleavage rate(75.76%~16.07%), blastocyst rate(38.67%~6.67%), numbers of total cells(107.15~63.67)and the ratio of ICM cells(26.32%~19.37%)decreased with increasing removal proportion of cytoplasm, and embryonic developmental ability significantly decreased when the removal proportion went beyond 1/3. These results suggest that(1)The treatment of 7.5 μg/mL CB for 30 min has no significant effect on oocyte development of goat embryo in vitro;(2)The volume of removal of cytoplasm under 1/3 has no significant effect on development of goat embryo in vitro.

Role of Tobacco Vacuolar Invertase Regulated by patatin Promoter in Resistance of Potato Tubers to Cold-sweetening

CHENG Shan-han;LIU Jun;XIE Cong-hua;SONG Bo-tao;LI Jing-cai

2006, 14(5): 716-720 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To slow down reducing sugar accumulation of potato tubers exposed to low temperature storage, an expression vector pBICNI of Nt-VIF gene, a vacuolar invertase inhibitor from Nicotiana tabacum, regulated by the potato tuber specific promoter CIPP was contructed and transformed into potato(Solanum tuberosum) cv. E-potato 3 (E3). Detections by the PCR, Northern and Southern hybridizations indicated that the full longth Nt-VIF Nt-VIF cDNA was successfully transformed into cv. E3. After storing potato tubers of 14 transgenic lines in 4 ℃ or 20 ℃ for 30 d, the activities of vaculoar invertase (VI) and reducing sugar(RS) content were analysed. The results showed that there were no significant differences in RS content between transgenic and untransformed(control) tubers stored in 20℃. However, compared to the control, RS content of transgenic lines reduced obviously in 4 ℃ from 34.0% (line B-13) to 76.8% (line B-1), implying inhibition of VI activity by Nt-VIF cDNA expression resulted in RS content reduction. Further analysis revealed a positive linear relationship between IV activity and RS content (VI = 0.308RS + 0.067). Lines B-1, B-2, B-6, B-9 and B-14 can meet the requirements of potato fries in terms of their low RS content after the cold-storage.

Genetic Variations of Gliadin and HMW Glutenins Subunits in Durum Wheat

WANG Han-yan;WEI Yu-ming;YAN Ze-hong;ZHENG You-liang

2006, 14(5): 721-727 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The wheat baking quality is controlled by the content and composition of wheat endosperm proteins. To exploit new genetic resources and provide fundamental materials for the improvement of bread wheat quality, the genetic variations of the storage proteins in 142 durum wheat (Triticum durum Desf.) accessions from 22 countries were characterized by A-PAGE and SDS-PAGE. A total of 37 gliadin bands were detected, among which all bands(100%) were polymorphic. Furthermore, 142 accessions could be divided into three major groups using the clustering analysis, indicating that the gliadin variations among durum wheat were associated with their geographic origin. Fourteen high-molecular-weight (HMW) glutenin subunits alleles and fifteen subunits combinations were identified. Glu-A1-c(null) was observed at the highest frequency among the 4 Glu-A1 alleles, and Glu-B1-b and Glu-B1-e were the major subunits encoded by Glu-B1.

Comparison of Allelic Variations at High-molecular-weight Glutenin
Subunit Loci in Wheat Cultivars Released in Different Periods in China

SONG Jian-min;LIU Ai-feng;JIANG Guo-hua;LI Hao-sheng;LIU Jian-jun;ZHAO Zhen-dong

2006, 14(5): 728-35 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

High-molecular-weight glutenin subunit (HMW-GS) compositions of 171 hexaploid wheat cultivars and breeding lines released or bred recently from ten major wheat growing provinces and municipalities of China were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Eighteen alleles and 40 different glutenin subunit patterns were identified, and 20 subunit patterns were observed only in one genotype. The most common alleles were 1 and Null at Glu-A1, 7+8 and 7+9 at Glu-B1, 2+12 at Glu-D1, and the prevalent subunit composition patterns were Null, 7+9, 2+12; Null, 7+8, 2+12; 1, 7+8, 2+12 and 1, 7+9, 2+12, accounting for 49.71% of cultivars analyzed. Compared with previous studies on old Chinese cultivars released or planted before, the frequencies of high breadmaking quality related subunits, such as subunit 1 and 5+10, together with allelic variations and subunit composition patterns, increased significantly, resulting Glu-1 quality score above seven. Further improvement of breadmaking quality of Chinese wheat cultivars should be focused on increasing the frequencies of subunit 5+10, 17+18 and other high quality subunits.

Ultraviolet-B Induced Chalcone Synthase (CHS) Gene Expression and Its Signaling Pathways in Arabidopsis thaliana

WU Ying;LIANG Yue-rong;DONG Jun-jie;LU Jian-liang

2006, 14(5): 736-741 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Transcripts of chalcone synthase(CHS) gene in six lines of Arabidopsis thaliana in response to UV-B radiation and treatment of hydroxyl peroxide(H2O2), D/L-NG-monomethyl-arginine(D-NAME, L-NAME) and 2-phenyl-4,4,4,4-tetramethylimidazo-
lin-1-oxyl-3-oxide(PTIO) were studied by RT-PCR method. Mutant Fah1-2 was the most sensitive and wild WS2 was the most tolerant to UV-B damage in the six lines of Arabidopsis . Accumulation of transcripts of CHS gene was markedly elevated at second hour but disappeared after 24 h of 15 μmol/m2/s UV-B. H2O2 had not effect on the transcription of CHS gene. D-NAME and L-NAME suppressed the increase of CHS transcription in response to UV-B radiation while the effect of PTIO on CHS was very weak. It appears that CHS transcription in response to UV-B radiation is not regulated by nitric oxide(NO) and H2O2.

Preparation of Bioactive Recombinant Chicken Leptin Fusion Protein

LIU Ying;LIU Zhi;SHI Zhen-dan;LI Xiao-wei;ZHONG Zi-sui

2006, 14(5): 641-645 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The cDNA sequence of chicken Leptin mature peptide was cloned into Nhe Ⅰ and Hind Ⅲ sites of expression vector pRSET A to construct recombinant plasmid pLepSCAU2 which was transfected the Escherichia coli BL21(DE3). After OD600 of the recombinant plasmid which grow in LB medium supplemented ampicillin 100 μg/mL was more than 0.6, the recombinant chicken Leptin fusion protein (including a 6×His tag) of molecular mass of 17.5 kD was expressed with the induction of IPTG 0.05 mol/L and the maximal expression which was 25% of the total protein in bacteria was achieved after 4 h induction and existed in inclusion body. The soluble part was achieved after purified by 50% Ni-NTA agrose and renaturated and folded by dialyze with changes of urea solutions of specific concentrations and pH. Such obtained soluble Leptin fusion protein was characterized of its biological activities in 35 d old growth yellow broilers. Following intraperitoneal injection of 2 mg/kg body weight of Leptin, feed intakes between 80 to 120 min post-injection were all significantly depressed in both pullets (P <0.01, n =10) and cockerels (P <0.05, n =10) compared respectively with PBS treated control pullets (n =10) and cockerels (n =10). Thereafter the accumulative feed intake in Leptin treated cockerels increased to the same level of the PBS treated control cockerels at 330 min post-injection. However in pullets, the depression of accumulative feed intake by Leptin fusion protein was maintained from 120 min to 330 min post-injection. The above results demonstrated that soluble and biologically active recombinant chicken Leptin was obtained, and there was a sex difference in depression of appetite by Leptin treatment.

Analysis of a 412 insertion mutation at scarlet locus of outcross progeny of Bar fly

YANG Jun;LIAN Lin-sheng;ZHAO Chun-jiang;BAI Li-hua;WU Chang-xin

2006, 14(5): 669-672 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Six pairs of oligonucleotide primers were designed to amplify and sequence scarlet gene of mutant stbr flies which were obtained by outcross between Bar and wild type flies. A 7.5-kb insertion identified as 412 element was detected at 4th exon of scarlet gene. Heterozygous 412 insertion and homozygous 412 insertion were also detected at scarlet gene of Bar and st1 flies imported from Japan respectively. The 412 insertion site of stbr was at 1612thbp-1613thbp of scalet gene with a 450-bp loss of 5´ LTR but the insertion of st1 was at 1722ndbp-1723rdbp

Analysis of SNP Markers for Blue-shelled Gene in Chicken by PCR-SSCP

ZHAO Rui;LIU Zhen-zhen;XU Gui-yun;YANG Ning

2006, 14(5): 673-676 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Oocyan(O) gene determines blue shell pigmentation of domesticated chicken. O locus is closely linked to ALEV1 on chromosome1 with a genetic distance of 2.3 cM. Blue eggshells contain protoporphyrin, biliverdin and zink biliverdin chelatate, which belong to the porphyrin metabolic pathway. A bioinformatic approach was first used to blast the genes for enzymes of porphyrin pathway and no gene was detected to specify O locus. Due to limited number of SSR markers around O locus, PCR-SSCP was then employed to search for candidate SNP markers. The sequence bearing a marker was found to be consisted of two transversions at Chr1:61754089T->A and Chr1:61754175A->T, respectively. Population screening showed that 81% of homozygous blue-shelled layers(OO) were classified as AA genotype at the SNP loci, while 89% of heterozygous blue-shelled layers(Oo) were AB genotype and 93% of tinted layers(oo) were BB genotype. The results indicated that there was a close association between O locus and SNP locus, which suggests that the marker locus can be used as a molecular marker in breeding for blue-shelled chickens.

Identification of the leptin Gene from the Ctenopharyngodon idellus
and Its Expression in Pichia pastoris

DAI Han-chuan;WU Xiao-xiong;NIE Fen;ZHAO Na;ZHANG Wei-min;ZENG Cui-ping;LONG Liang-qi

2006, 14(5): 677-682 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The grass carp (Ctenopharyngodon idellus ) leptin gene was amplified by RT-PCR. PCR product was cloned into Pichia pastoris vector pPIC9K. The recombinant plasmid was transformed to competent cell GS115 by electroporation and expressed by methanol induction. Recombinant protein was analyzed with SDS-PAGE and Western blot. Results revealed that grass carp leptin gene had a length of 438 nucleotides which encoded a 146-amino peptide（GenBank Accession AY551335）. Its nucleotides sequence and deduced amino acid sequence homologues were compared with carp(Cyprinus carpio), humans, pigs and rats, which displayed a fairly high degree of conservation, and homologue of the nucleotides sequence were 99%, 84%, 86% and 95% respectively; homologue of the amino acid sequence were 99%, 84%, 82% and 96% respectively. There was significant difference between the grass carp and pupper(Takifugu rubripes） in amino acid sequence. The homologue was only 9%, which displayed that there was difference among the fish species. Grass carp leptin gene was expressed in Pichia pastoris. Molecular weight of expression product was estimated to be approximately 16 kD. Western blotting analysis showed that Leptin protein took on strong immunology activity.

The Osteoblast Differentiation In vitro of Goat Marrow Mesenchymal Stem Cells(MSCs)

PANG Quan-hai;DOU Zhong-ying

2006, 14(5): 683-687 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

he mesenchymal stem cells (MSCs) derived from bone marrow of the adult Guanzhong dairy goats were used to be induced to differentiate into osteoblast phenotype in vitro. The results showed that, after induced for 4~5 d by dexamethasone, beta-glycerophosphate and ascorbate-2 phosphate, the volume of some cells of the goat MSCs gradually largened and changed their form from spindle/polygon to cuboid/ovoid; With the induce time continuing, the numbers of the cuboid/ovoid cells continuous increased; At 15th day, there were many cuboid cells, and the modality of the cells changed from cuboid to ovoid; At 25th day, the oval and cuboid cells accumulation were observed. And the osteoblasts differentiation rate of the cells was 90.2% ± 2.97%. These indicated that the goat MSCs were induced to differentiate into osteoblast phenotype and the new born cells demonstrated positive staining for alizarin red and alkaline phosphatase (ALP). These results suggested that the MSCs were induced to differentiate into osteoblst phenotype in vitro. Accordingly, the data can provide a scientific foundation with cellular mode and further study for the bone defaults repairing.

Experiment on Cryopreservation of Porcine Oocytes by Using Glass Micropipette

ZHU Liang-cheng;RUI Rong;YANG De-ji;ZHANG De-fu;LIU Dong

2006, 14(5): 697-700 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The self-made glass micropipette(GMP) was used for cryopreservation of pig oocytes, obtaining good results. The post-thaw survival rate of oocytes was assessed by fluorescein diacetate(FDA) staining. And the results showed that the programmed freezing method and GMP vitrification method respectively resulted in the post-thaw survival rate of 34.5% and 63.3%(P <0.05) in cryopreservation of porcine MⅡstage oocytes. Comparing plastic straw with GMP in vitrification of porcine MⅡ stage oocytes, the survival rates of both groups were 45.0% and 65.9% (P < 0.05), respectively. When GV-stage pig oocytes were cryopreserved by straw programmed freezing method and GMP vitrification method, they yielded survival rates of 30.0% and 59.7% respectively and there was significant difference between them(P < 0.05). The present study shows that GMP method is an efficient freezing method for porcine oocyte cryopreservation.

Effect of Mannan-oligosaccharide on Bacterial Community of Piglet after Weaning

HANG Su-qin;MAO Sheng-yong;HUANG Rui-hua;SU Yong;ZHU Wei- yun

2006, 14(5): 701-705 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A total of 34 weaning piglets from six litters were randomly assigned into two groups, offered basal diet(control) and basal diet plus manno-oligosaccharide (MOS), respectively. Faecal samples were collected on day 1(before first feeding), 7, 14, 21 and 28 after weaning. DNA was extracted from the faecal samples and the V6～V8 regions of bacterial 16S rDNA were amplified. Amplicons were analyzed by DGGE to investigate the diversity of the bacterial communities of weaning piglets. Results showed that piglets from the same litter had a higher similarity(81%~88%) of the bacterial community on day 1(before weaning) compared with that after weaning(61%~70%). DGGE similarities of microbial community between neighboring time points, e.g. 1~7, 7~14, 14~21 and 21~28, decreased during the first week for all piglets, but the value in the control remained around 61%, while those in the MOS treatment gradually increased to 70%. Diversity analysis showed that after 7 days the value for the MOS treatment increased while that for the control decreased. On day 28 the diversity index of the bacterial community from the MOS diet was 1.52 and the control group was 1.38. The differences of the diversity between the two groups increased from 0.09 on day 1 to 1.14 on day 28. In agreement with diversity data, DGGE band numbers revealed that during the first week of weaning the average band numbers reduced for both groups. However, after 14 days average band number for piglets fed the MOS diet increased, while that from piglets fed the control diet decreased. The results demonstrated that weaning had caused rapid changes of the bacterial community of piglets and that MOS supplemented in the diet could result in a rapid development and consequently early establishment of a high diverse bacterial community after weaning.

Effect of CpG-DNA on Mastitis Induced by Escherichia coli Infection in Rat Model

ZHU Yu-min;MA Hai-tan;CHEN Wei-hua;ZOU Si-xiang

2006, 14(5): 706-710 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Seventy-two lactating SD rats were randomly divided into control and treatment groups(n =36). The 100 μL of sterile 0.01 mol/L，pH 7.2 PBS(phosphate-buffered saline)(control group) and CpG 200 μg/rat(treatment group) were injected intramuscularly into tibialis anterior of the left leg after parturition 0 h respectively. Then 100 μL /rat of bacterial suspension containing 2×1012 cfu(colony forming unit)/mL(both sides) of Escherichia coli were inoculated into the fourth (abdominal) mammary gland via the teat duct 72 h after parturition respectively. Before defined at o h and after 8,16,24,48 and 72 h (n =6) of inoculation, all the rats euthanatized and the mammary glands and serum were harvested. The histopathologic evaluations showed that polymorphonuclear leukocytes (PMN) accumulation in alveoli peaked 16 h postinfection. The PMN in treatment group influxed more rapidly and no PMN was found in alveoli 72 h after infection. Bacteria counts of E. coli in mammary gland peaked at 16 h postinfection and CpG-DNA induced significant decrease of viable bacteria at 16, 24 and 48 h postinfection. CpG-DNA induced significant increase of IL-2 in mammary gland before infection. Significant increases in IL-6 and TNF-αin mammary gland were observed at different time points in both group. The maximal increases in IL-6 were at 16 h postinfection, the time that was coincident with peak infiltration of PMN. CpG-DNA could induced significant increase of IL-6 in mammary gland at 16 h postinfection. No significant changes of N-acetyl-β-D-glucosaminidase (NAGase) in mammary gland from treatment group were observed. The result showed that CpG-DNA induced more prompt migration of PMN from blood to mammary gland at the initial stage of E. coli infection, stimulated the secretion of IL-2 and IL-6, decreased E.coli counts in mammary gland and attenuated the destroy of inflammation-mediator to cell. The study indicates that CPG DNA protects against mastitis at some extent induced by E. coli infection in rat.

Screening of Hybridoma Lines of Monoclonal Antibody and Development of ciELISA Kit for Rapid Detection of Phenobarbital

WANG Zi-liang;ZHANG Gai-ping;ZHANG Hai-tang;YANG Yan-yan;WANG Yan-rong; CHAI Shu-jun

2006, 14(5): 711-715 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Balb/c mice were immunized with BSA-pAPB and hybridoma lines secreted monoclonal antibody against phenobarbital (PB mAb) were generated with cell fusion and filtered by indirect ELISA and blocking ELISA. A ciELISA kit for detection PB (PB-Kit) was developed with PB mAb and its traits were tested. The results showed that three hybridoma lines were filtered and the best one was 3F6-C4, which secreted PB mAb with indirect ELISA titers of 1∶6.4×105 in ascites. PB mAb had a high affinity constant(Ka) with 1.96×1010 L/mol, a good sensitivity with an IC50 of 5.7 μg/L to PB, 12.4% cross-reactivity to barbiturate sodium and little or no cross-reactivity to other compounds. The PB-Kit had the linear detection range of 1.0 to 81 μg/L, the sensitivity of 0.75 μg/L and the detection limit of 1.0 μg/L. The recoveries of PB spiked in feed and swine urine were 85.8% and 91.3% respectively. The precision and accuracy of the assay determined by inter-assay and intra-assay coefficient variation was both below 15%. The PB-Kit possesses rapidity, sensitivity, specificity and briefness. That is proved to be used for the rapid detection of PB residues in animal food.

Pre-mRNA Alternative Splicing in Plants

GUO Xiao-qin;LI De-bao

2006, 14(5): 809-815 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Alternative splicing is an important cellular mechanism that increases the diversity of gene products. The study of alternatively spliced genes reported so far in plants is far less than that in mammals. But considerable results was also reported in regulating mechanisms， influencing factors，specificities and function of alternative splicing in plant. Here, progresses on alternative splicing in plants are briefly summarized.

Serial Analysis of Gene Expression Technology and Its Application
in the Research of Plant Disease Resistance

LI Xing;LI Ya-ning;YANG Wen-xiang;LIU Da-qun

2006, 14(5): 803-808 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Serial analysis of gene expression (SAGE) can be used not only to analyse gene expression in specific cells or tissues, but also to simultaneously compare the differential genes expressed in different tissues and different conditions for gene mining. In recent years, the application of SAGE in the research of plant disease resistance is enhancing rapidly. The review introduces principle, characteristic, protocol and methodological evolution of SAGE. And the applications of prospect of SAGE technology in the research of plant disease resistance are also discussed.