农业生物技术学报

Effects of Conjugating Conditions on Goat Anti-rabbit Antibody Immobilization onto Bacterial Magnetic Particles

CHEN Ji-feng;LI Ying;JIANG Wei;WANG Zheng-fang;SUN Jian-bo;Li Jian;LI Ji-lun;LI Shao-hua

2006, 14(3): 423-428 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Goat anti-rabbit immunoglobulin G (antibody) was applied as a sample for studying antibody conjugation onto bacterial magnetic particles (BMPs). The conditions for this study were different pre-treatment BMPs (-196 ℃, -70 ℃ and -20 ℃ for 5 h) for freeze-dry, temperatures with varied conjugation durations, amount of BMPs and antibody, conjugation buffers, and buffer pH and concentrations. The results showed that different pre-treatments for freeze-dry BMPs had different linkage rate of antibody (LRA). Liquid nitrogen(-196 ℃) as a pre-treatment for 5 h, the LRA was significantly higher than that of -70 ℃ and -20 ℃ pre-treatments. Any kinds of pre-treatments and freeze-dry BMPs, the LRAs were decreased 36%~55% compared with no freeze-dry BMPs. Room temperature (about 15 ℃), and incubation for 18 h resulted in the highest LRA (64.06 μg/mg) in the applied temperatures and conjugation durations. LRAs depended upon the amount of antibody and BMPs, when BMPs decreased from 5.0 mg to 0.1 mg, and antibody increased from 10 μg to 300 μg, the LRAs increased gradually. When BMPs at 0.1 mg /mL and antibody at 300 μg/mL were used, the LRA reached to 762.37 μg/mg. Many buffers could be used for antibody conjugation onto BMPs, and under the normal buffer pH and concentration, the LRAs of 8 buffers were 46.28±0.58 ~ 90.83±1.64 μg/mg. The different buffer had different pH and concentration to reach the peak LRA. When pH 3.5 and 20 mmol / L for HEPES, pH 5.6 and 5 mmol / L for Na3PO4, the LRAs were 108.01 and 76.78 μg/mg respectively. SDS-PAGE showed that antibody was conjugated onto BMPs.

Genetic Diversity and Molecular Markers of 5 Species of Snappers

LIU Li;LIU Chu-wu

2006, 14(3): 349-355 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to protect and develop the valuable fishes of snappers, genetic diversity and molecular markers of 5 species of snappers, Lutjanus vitta, L. fulvus, L. fulviflamma, L. sebae and L. stellatus，were detected using RAPD and SSR technique. The amplified products of genomic DNA in the 5 species of snappers by RAPD technique indicated that the polymorphic loci ratio (P ) were 89.30%，86.70%，92.11%,86.47% and 86.00%, the mean genetic distances of intra-species (D) were 0.3431，0.2130，0.3121, 0.1825 and 0.1775, and the genetic diversity indexes of intra-species (Hi ) were 0.1634，0.1095，0.1353, 0.1022 and 0.1024 respectively. Nine molecular markers, OPA8-413bp, OPA8-140bp, OPP10-418bp, OPA8-697bp, OPP10-526bp, OPA8-361bp, OPP10-449bp, OPA8-311bp and OPP10-599b , which could be used for identifying 5 species of snappers were found in the amplified products by primer of OPA8 and OPP10. The amplified products of genomic DNA in 5 species of snappers by SSR technique showed that effective numbers of allelic was 1.9610，3.3793，3.2957，1.7893 and 3.6591 respectively, average heterozygosities were 0.332，0.462，0.593，0.367 and 0.676, polymorphism information contents (PIC) were 0.302，0.438，0.554，0.332 and 0.641 respectively. Six molecular markers, 13 Prs229-115 bp, 4 Lca43-212 bp, 4 Lca43-240 bp, 13 Prs229-288 bp, 19 Prs275-156bp and 7Lca91-118 bp, which could be used for identifying 5 species of snappers were found in 11 loci of micrrosatellite.

Analysis of Disease Resistance of nsLTPs-like Antimicrobial Protein Gene Transgenic Tobacco against Brown Leaf Spot and Granville Blast

WANG Xiao-wen;YANG Xing-yong;LI De-mou;SANG Xian-chun;SHE Rong;PEI Yan

2006, 14(3): 365-370 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Both Lj-L gene and its modified version Lj-S gene from Leonurus japonicus Houtt were transformed into tobacco (Nicotiana tabacum cv. Xanthi) mediated by Agrobacterium tumefaciens. GUS and PCR identification showed that foreign gene was integrated into tobacco genome. RT-PCR analysis revealed that foreign gene could transcript normally. Analysis of resistance to pathogenic fungus (Alternaria alternata) using detatched leaves indicated that disease indexes (DI) of T0-transgenic tobacco was remarkably decreased from 100% for wild type to 27.66% for Lj-L gene and 40.77% for Lj-S gene respectively. Similar results were obtained in the in vivo analyses of resistance to pathogenic bacterium Pseudomanas solanacearum. The results suggested that the nsLTPs-like protein gene Lj-L might have great potential in generating transgenic plants with broad-spectrum resistance to both fungal and bacterial pathogens.

Analysis of a Rice OsWRKY19 Gene Promoter

HAO Zhong-na;WANG Hai-hua;BAI Yang;GUO Ze-jian

2006, 14(3): 371-375 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A 1404 bp promoter fragment of rice(Oryza sativa) transcription factor WRKY19 gene was amplified by PCR from rice Xiushui11 and named as OsW19p. The full length of OsW19p(p1404) and its 5' end deletions with a length of 105, 378, 9 77, 1 106, 1 205 and 1 306 bp were fused with gus gene, respectively. All of the contructed vectors were transformed into rice calli by Agrobacterium-mediated method. The results of histochemical GUS staining and fluorometric measurements showed: (1) Expression of gus gene was intensively inhibited by 2,4-D in the culture medium at callus stage; (2) Expression of gus gene was observed in roots, stems，leaves, flowers, and the embryos of germinating seeds. GUS staining were observed in adventititious, primary and lateral roots of rice seedlings, however, only lateral roots were stained at maturation phase; (3) GUS activity of all of deletions except p105 were detected in leaves of transformed plants, the level of p1205 was the highest, while expression was reduced for p977 and p1306. It suggests that some regulatory elements exist in -1404～-1306 bp and -1205～-977 bp, and fragments of -1306～-1205 bp and -977～-378 bp may contain negative elements.

Cloning and Identification of DREB-like Transcription Factor in Nicotiana benthamiana

LIU Wei-qun;WANG Yong-liang;ZHAO Tong-jin;ZHOU Hai-meng;GUO Ai-guang

2006, 14(3): 376-380 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Five EST (expressed sequence tag) sequences were obtained in NCBI web BLAST according to the AP2 domain sequence of DREB(dehydration-responsive binding protein)1a gene from Arabidopsis. Specific primers were designed based on the EST sequences, and two DREB-like genes were amplified from tobacco (Nicotiana benthamiana) cDNA by PCR， and named NbDREB1 and NbDREB2 respectively and could be expressed in Escherichia coli. Comparing with other dehydration-responsive element binding related protein found that NbDREB1 and NbDREB2 possessed typical structural characteristics of A type of EREBP subgroup in AP2/EDREBP transcription factor family . The yeast one-hybrid (expression vector YepGap) showed that the two genes had no active function. Anti-activator yeast one-hybrid (expression vector pGADT7containning GAL4 domain) assay showed that NbDREB1 could bind to DRE cis-element in yeast, but NbDREB2 could not. The difference of AP2 domain between NbDREB1 and NbDREB2 was only in the 2nd and 49th amino acid, which was Y and K in NbDREB1 and N and R in NbDREB2.

Cloning and Expression Pattern of a Cold Stress-responding Gene from Lentinula edodes

FENG Zhi-yong;MI Shuo-fu;ZHAO Ming-wen;CHEN Ming-jie;LI Jun-hui;TAN Qi;PAN Ying-jie

2006, 14(3): 381-386 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Fruiting body development in Lentinula edodes ((Berk.) Pegler) depends on temperature alterations, and cold stress leads to differential expression of related genes. Differential display PCR and rapid amplification of cDNA ends (RACE) were used to clone the full-length cDNA of a cold stress-responding gene. The cDNA contained an open reading frame of 2432 bp encoding 654 amino acids, and the deduced amino acid sequence showed overall homologies of 46% and 40% with polyketide reductase from Penicillium nordicum and α-aminoadipate reductase from Saccharomyces cerevisiae, respectively. Comparison of the genomic and cDNA sequences indicated the presence of 12 extrons and 11 introns. Significant levels of gene expression were detected at 20 ℃ but transcription increased after mycelium was subjected to cold stress and changed colour from white to brown at 15 ℃. The cloned gene, tentatively designated ICS (induced under cold stress, GenBank accession No. DQ211659), may play a role in transmitting signals related to cold stress. The above results suggest that its role in fruiting initiation may be linked to changes in nitrogen metabolism and provide a new candidate gene for hypothesis-based investigations of the cold stress response and the onset of fruiting.

Density-increasing of Peach Genetic Map by AFLP Technique and Comparative Analysis between the Density-increased and the origin Peach Genetic Maps

XIN Cui-hua;ZHU Mei-yun;WU Jun;ZHANG Kai-chun;JIANG Li-jie;ZHOU Xiao-hang; ZHANG Xiao-ming

2006, 14(3): 387-390 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

AFLP markers were used to increase density of the peach (Pruns persica (L.) Batsch) genetic map based on a F2 of 109 progenies derived from the cross of cultivars Okubo and Xingjin. Thirty and five new markers were obtained and 28 markers were added to the genetic map constructed previously. A total of 144 markers were assigned to 11 linkage groups (LOD 3.0), the total length of linkage map was 1167.6 covering 97.3% of the peach genome. The average length of 11 groups and the average interval between markers was 106.15 cM and 12.04 cM respectively. Compared with the previous linkage map, 9 new markers were assigned to group 9, but 5 markers previously located at this group were lost. At group 6, not only the order of the loci was changed, but also the average interval distance of them increased. Furthermore, locations of some markers were changed between group 3 and group 11, between group 1 and group 10. These inconsistencies were also discussed.

RAPD Analysis on Main Cultivars of Litchi (Litchi chinensis Sonn.) in Hainan

WANG Jia-bao;DENG Sui-sheng;LIU Zhi-yuan;LIU Ling-zhi;DU Zhong-jun; XU Bi-yu;CHEN Ye-yuan

2006, 14(3): 391-396 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To detect the genetic polymorphisms of litchi (Litchi chinensis Sonn.) cultivars, twenty-two accessions of litchi germplasms from Hainan, including fifteen cultivars, were analyzed with RAPD markers. Twelve random primers from eighty ones were selected because they could amplify well reproducible bands. Their percentage of polymorphic bands (PPB) was 64.1%. The average PPB of all tested litchi germplasms was 31.1%. Genetic distances among tested litchi germplasms ranged from 0.0000 to 0.6180, with an average of 0.3732. UPGMA cluster analysis showed that 22 germplasms could be mainly divided into 2 groups. and some tested litchi cultivars had obvious homonym.

Safety and Immunogenicity of the Recombinant Adenovirus Expressing the GP5 and M Protein of PRRSV in Piglets

ZHANG Zhi-tao;LI Yu-feng;JIANG Ping; TANG Jing-yuan;ENG Yu-xiu;DONG Xin-tian;LI Jun-xing

2006, 14(3): 312-318 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to study the safety and immunogenicity of recombinant Adenoviruses expressing GP5(rAd-GP5) and M protein(rAd-M) of Porcine reproductive and respiratory syndrome virus (PRRSV), Forty-seven 30-day-old post-weaning PRRSV-negative piglets were vaccinated with the viruses and bred respectively for clinical observation, virus shedding test and immune examination. The results were as following: (1) The piglets vaccinated with rAd-GP5 and rAd-M showed no significant clinical syndrome, and blood, nasal scraping, faecal samples and pen environment were all negative against PRRSV and parental Adenovirus by RT-PCR. (2) The PRRSV specific antibody titers in piglets, which vaccinated with rAd-GP5, rAd-M, rAd-GP5+M (rAd-GP5/rAd-M, V/V=1), PRRSV-Resp strain, and no inoculation, respectively, were examined at different time post immunization. The results indicated that PRRSV specific antibody could be detected by ELISA and neutralization assay 14 and 45 days post vaccination. In groups of rAd-GP5 and rAd-M, the titer of ELISA antibody were more than 1∶2400 at day 60 and 90 post vaccination, which were similar to that of piglets inoculated with PRRSV-Resp vaccine. However, the neutralizing antibody titers in the two groups of rAd-GP5 and rAd-M were less than 1∶6, which were lower than that of PRRS-Resp group, from 60 to 90 days post vaccination. Furthermore the level of ELISA antibody to PRRSV in piglets vaccinated with both rAd-GP5 and rAd-M were more than that of piglets vaccinated with singlerAd-GP5 or rAd-M. (3) Following challenge with virulent strain of PRRSV-S1 at 28 days post vaccination, no significantly temperature variation of the immunized pigs was observed. The time of viremia and virus shedding in piglets vaccinated with recombinant Adenoviruses were only 2 weeks, which were shorter than that of control piglets with no vaccine. And the titers of PRRSV specific ELISA antibody and neutralizing antibody were enhanced in piglets vaccinated with rAd-GP5 or rAd-M post challenge. The results suggest that rAd-GP5 and rAd-M are safe to piglets and can elicit good protective immune responses corporately.

Fusion Expression of ORF5 gene of Porcine reproductive and respiratory syndrome virus

CHEN Yong-jun;SU Xin-ming;GAO Xiao-fei;ZHENG Qi-sheng;YU Chun-mei;CAO Rui-bing;ZHOU Bin;CHEN Pu-yan

2006, 14(3): 319-322 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Three pairs of specific primers were designed according to the sequence of Porcine reproductive and respiratory syndrome virus ORF5 gene published on GenBank(accession No.AY737282). Two modified ORF5 gene fragments ORF5-1 and ORF5-2 which deleted the N-terminal transmembrane region (28 residues, including signal peptides) sequence and the middle tansmembrane region (60 residues) sequence of ORF5 gene respectively were obtained from recombinant plasmid pMD-ORF5 by PCR amplification using these primers. ORF5-1 and ORF5-2 gene fragments were inserted into prokaryotic expression vector pET-32 a(+), resulting in the recombinant plasmids pET-ORF5-1 and pET-ORF5-2. After transformed into Escherichia coli BL21(DE3) cells and induced with IPTG, SDS-PAGE analysis revealed that ORF5-1 and ORF5-2 recombinant proteins with a expression level amounted to 12.2% and 39% of the total bacterial proteins respectively . Expression of ORF5 gene has been improved by removing the dual transmembrane regions. Western blotting analysis showed that the recombinant proteins had immunoreactivity and proved that the deleting of transmembrane regions had no significant effect on the antigenicity of the recombinant proteins.

Isolation, Identification and Expression Profile of Fourteen Differentially Expressed Sequence Tags in the Longissimus Dorsi Muscle Tissues from
Meishan, Large White and Meishan×Large White Cross Pigs(English)

LIU Yong-gang;XIONG Yuan-zhu;ZUO Bo;JIANG Si-wen;DENG Chang-yan;LI Jia-lian;LI Feng-e;ZHENG Rong

2006, 14(3): 323-328 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to detect the molecular mechanism of the heterosis in pigs, mRNA differential display (DD) technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Meishan, Meishan×Large White cross and Large White pigs. Fourteen expressed sequence tags (ESTs), differentially expressed between the hybrids and purebreds, were isolated and identified through semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The nucleotide sequence analysis revealed that the 14 ESTs were not homologous to any of the known genes or ESTs. These novel ESTs were then deposited into GenBank database. Tissue expression profile analysis showed that the ESTs were expressed in most of tissues including heart, spleen, liver, kidney, small intestine, ovary and lung, and this also implied that these genes might be important for the life process. Our results indicate the diversity of differential display directions of genes between the hybrids and purebreds in the Meishan×Large White cross combination. Results also suggest that the heterosis in pigs might be derived from the differential expression of many indispensable genes towards different directions in the specific phase of life

Construction and Over-expression of the Activator Gene from Comamonas testosteroni

DAI Yi-min;PAN Da-ren;Guangming Xiong;WU Ming-xia;ZHOU Yi-fei

2006, 14(3): 416-422 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The activator gene from Comamonas testosteroni was amplified by PCR from the genomic DNA of C. testosteroni. The PCR products were cloned into plasmid pKtac1 (containing tac promoter), and the recombinant plasmids pKtac1-act1 and pKtac1-act2. were obtained and identified by the analysis of restriction enzymes and DNA sequencing and then respectively transformed into Escherichia coli HB101. The total cell lysate of the host bacteria was extracted to detect the quantity of activator using ELISA. Moreover, the recombinant plasmids were also co-transformed with plasmid pAX1 (containing 3α-HSD/CR gene) into E.coli HB101 respectively. The total cell lysate of the host bacteria was also extracted to detect the quantity of activator and 3α-HSD/CR using ELISA. Moreover, the results indicated that the recombinant plasmids can improve the expression of activator; and the expressions of activator and 3α-HSD/CR can be also improved by plasmid pAX1 co-transformed with recombinant plasmids.

Biosynthesis of Alky Gallates Catalyzed by Tannase in Reverse Micelle

WANG Zheng;TIAN Yun;LU Xiang-yang;YU Hua-zhong;LUO Ze-min

2006, 14(3): 429-433 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Alky gallate is a kind of antioxidant and processes patient therapy value. However, it is synthesized by chemical method, which brings serious circumstance containment and low productivity. To avoid the defects, the method of biosynthesis for alky gallates was established in reversed micelles reaction system, in which propyl gallate (PG), butyl gallate (BG) and amyl gallate (AG) were successfully synthesized. The reverse micelle consisted of docusate sodium salt (AOT), isooctane and water; the tannase concentration was 0.6 mg/mL. In the reverse micelle reaction systems for PG, BG and AG, when W0 (the mole ratio of water to AOT) was respectively 15,15 and 12.5; reaction temperatures were 30, 40 and 45 ℃, respectively; AOT concentrations were 0.1, 0.2 and 0.2 mol/L respectively; pH values all were 6, conversion of gallic acid reached the highest in the different systems. The highest conversion rates of gallic acid reached 87.4%, 90.3% and 92.5% respectively. Various factors had different effects on tannase catalytic activity due to three different alky alcohol isomers.

Preparation of Rubisco Antibody and Determination of Rubisco Content
of Flag Leaves in Wheat by ELISA

LI Wei-fang ;WANG Xiu-hai;HE Wen-juan

2006, 14(3): 397-400 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) is crucial to the overall modulation of photosynthesis. Rubisco was purified by (NH4)2SO4, DEAE-Sepharose F F and Sephacryl S 300 from wheat (Triticum aestivum L. cv.) leaves. The rabbit polyclonal antibody was prepared by vaccinating rabbit with purified Rubisco. The Rubisco antibody was showed specificity to Rubisco by Western blot. And using Rubisco antibody，contents variation of Rubisco in flag leaves was determined by ELISA. The result showed that Rubisco contents were positive relative with photosynthetic rates during development stages of flag leaves. This method is quicker and more accurate than that of immune precipitation.

Screening and Characterization of Monoclonal Antibody to Bursaphelenchus xylophilus

JIANG Li-qin;ZHENG Jing-wu;CHEN Zheng-xian;WU Jian-xiang

2006, 14(3): 412-415 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The pine wood nematode, Bursaphelenchus xylophilus, is one of the most important pathogens in pine trees. Exact, quick and sensitive method for identification and diagnosis of the nematode is the precondition for managing the nematode transmission. One hybrid cell lines, named as 1D3, secreting monoclonal antibody ( Mab ) to Bursaphelenchus xylophilus were developed by fusing splenocytes of BALB/c mice to mouse mycloma cell line SP2/0. The ELISA titer was 1∶5.12×105. This specific Mab was identified as IgG1.Western blotting analysis showed that the Mab could react with B. xylophilus. The production of monoclonal antibody to B.xylophilus provides a rapid and sensitive method for identification and diagnosis of the nematode.

Expression of Mouse Parotid Secretory Protein (PSP) in Postnatal Development of Parotid Glands

TIAN Xing-hua;ZHANG Biao;FAN Bao-liang;LI Ning;WU Chang-xin

2006, 14(3): 345-348 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Expression features of mouse parotid secretory protein (PSP) in postnatal development of parotid glands have been researched in mRNA level. The copies of PSP mRNA were 4.79E+06～1.32E+08, which were102 times higher than that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1.32E+04 copies) in different developmental stages. Messager RNA levels of PSP shifted with developmental stages: increased from birth to 3 weeks of postnatal, reached its peak at 3 weeks, then decreased; at last it kept at a even level at 12 weeks

Associations between Genetic Polymorphisms at Five Loci and Milk Production Traits in Xi'an Population of Chinese Holstein

ZHANG Run-feng;CHEN Hong;LEI Chu-zhao;FANG Xing-tang;SUN Wei-bin;HU Shen-rong;SU Li-hong

2006, 14(3): 329-333 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The genotypes' effects of four polymorphic loci on milk production traits of the Holstein cattles were analyzed using insulin-like growth factor binding protein 3 (IGFBP-3) gene as a candidate gene of milk production traits in Chinese Holstein cattle firstly. The effects of genotypes of κ-cn locus on the traits were not statistically significantly(P>0.05). At IGFBP-3 locus, 305 days milk yield of individuals with genotype BB was higher than that of individuals with genotype AA and AB for 1054.17 kg and 838.15 kg respectively(P <0.05), the protein percent of individuals with genotype AB was higher than that of individuals with genotype BB (P <0.05). Genotype AA of β-lg locus had significantly positive effect on fat/protein ratio, Genotype AB had significantly negative effect on the protein percent. At β-lg gene 5′flanking region, 305 days yield of individuals with genotype AA was higher than that of individuals with genotype AB(P <0.05), the fat percent of individuals with genotype AA was higher than that of individuals with genotype BB(P <0.05), but individuals with genotype BB had higher protein percent than individuals with genotyupe AB(P <0.05)

Ontogenetic Expression of SGLT1 and GLUT2 mRNA in Duodenum of Different Genotype Broiler Chicken

WANG Xiu-qi;TAN Hui-ze;SHU Gang;SU Hai-lin;DAI Fa-wen;FENG Ding-yuan

2006, 14(3): 334-340 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to investigate the ontogenetic expression of sodium/glucose cotransporter1(SGLT1) and glucose transporter2 (GLUT2) mRNA in duodenum of different genotype broiler chickens, 120 1-day-old parental male Arbor Acre (AA) broiler chickens and 120 parental male yellow cover chicks were randomly divided into 4 replicates, respectively. The productive performance was recorded from day 1 to 58 d. On 2,16,30,44 and 58 d, duodenum samples were collected. The mRNA expression abundances of SGLT1 and GLUT2 at different developmental stage were determined by relative quantitative RT-PCR. It was found that: (1) The average daily feed intake (ADFI) and average daily gain (ADG) of every week of AA chicken were higher than that of yellow cover chicken (P <0.05) , the change curve of every week ADFI and ADG were similar between the two genotypes. (2) Ontogenetic expression model of SGLT1 mRNA were consistent between AA broiler and yellow cover chicken. The SGLT1 mRNA abundance in the two genotypes were increased from day 2 to day 30, then decreased on day 44 and increased again on day 58. The SGLT1 mRNA expression level of AA chicken was higher than that of yellow cover chicken and the differences were significant on day 16 and 30 (P<0.05). (3) GLUT2 mRNA expression of the two genotypes trended to increase from day 2 to day 30, and the abundance of day 16 and 30 were higher than that of day 2 at significant level (P <0.05). On day 44 and day 58, the GLUT2 mRNA expression of AA broiler were lower than that on day 16 and 30 (P <0.05), while the expression of yellow cover chicken was going on increasing from day 44 to day 58 and GLUT2 mRNA expression abundance of day 58 was higher than that on day 2,16 and 30; Compared GLUT2 mRNA expression with yellow cover chicken, AA broiler was higher on day 16 and 30 (P <0.05), while on day 58 yellow cover chicken GLUT2 mRNA expression was higher than that of AA chicken. These results demonstrate that: 1) Productive performance of AA broiler chicken excells yellow cover chicken. The alteration of nutritional level has great effect on the performance and the AA broiler chicken is more sensitive than yellow cover chicken. 2) SGLT1 mRNA expression of the two genotype has the same ontogenetic model in duodenum and paralleled with the productive performance. To change diet can decrease the expression of SGLT1 mRNA, indicating that the productive performance can be improved by up-regulating of SGLT1 expression. 3) GLUT2 mRNA expression ontogenetic model of late-finishing yellow cover chicken is distinct with AA chicken, and its mechanism and effect on production needed further study.

Regulation of Docosahexaenoic Acid (DHA) on Adipocytes Cycle and COX-2 Expression

LI Hui-xia;YANG Gong-she;SUN Shi-duo

2006, 14(3): 341-344 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A cells culture system of rat primary preadipocytes was established in vitro to investigate the regulation of docosahexaenoic acid(DHA) on preadipocytes cycle and cyclooxygenases-2(COX-2 ), mRNA expression by treated preadipocytes (cultured day 1) and adipocytes (differentiated day 1) with 0 μmol/L (control group), 40 μmol/L (lower dose group) and 160 μmol /L (higher dose group) DHA. Preadipocytes cycles were analyzed by flow cytometry(FCM), and the expression of (COX-2 ) mRNA in adipocytes were detected by reverse transcriptase -polymerase chain reaction (RT-PCR). It was demonstrated that the percent of preadipocytes in G1 phase were increased after treated by DHA at 48 h, but no remarkable difference compared with control. Expression of COX-2 mRNA were markedly decreased after treated with 40 μmol/L DHA at 24 h, but increased in 160 μmol/L group. It can be concluded that DHA had no obvious effect on preadipocytes cycle but regulated the expression of COX-2 mRNA in a concentration-dependent manner.

Study on Normalization Method for cDNA Microarray Data

ZHANG Ji-Gang;ZHANG Qin;YIN Zong-Jun

2006, 14(3): 356-359 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In view of the limitations of the currently widely used method for the cDNA microarray data standardization (often called log-ratio transformation method), a new method was proposed which named as non-transformation method. It does not transform the data but directly estimates the gene transcription level after correction of the background effect based on Huber's model and principle of least square estimation through an iteration process. Using computer simulation, the new method was compared with the log-ratio method under various conditions (different number of repeated probes on a chip, different background variation, and different ratio of up- and dawn-regulated expressed genes). The results showed that in all conditions the non-transformation method performed better than the log-ratio method. The relative biases from the non-transformation method were less than 5%, while those from the log-ratio method more than 20%. Furthermore, the log-ratio method was very sensitive to the background variation and the ratio of up- and dawn-regulated expressed genes. With the increase of the background variation or the unbalance level of the ratio of up- and dawn-regulated expressed genes, the biases from the log-ratio method increased rapidly, while the non-transformation method was almost not influenced by these factors. Therefore, the non-transformation method could be used as an alternative method for cDNA microarray data standardization

Divergence and Mapping of SNPs of the FbL2A Gene for Cotton Fiber Development

HAN Zhi-guo;CHU Ying;GUO Wang-zhen;ZHANG Tian-zhen

2006, 14(3): 360-364 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Using PCR-based method, FbL2A gene was isolated from seven allotetraploid cotton (Gossypium hirsutum L. and G. barbadense L.) materials with different fiber quality and two diploid progenitors (G.herbaceum L. and G. richmondii U.). Based on single nucleotide polymorphisms (SNPs) of the FbL2A gene sequences in TM-1 and H-7124, the amplified products were digested by BstUⅠ through NEBcutter software online. And the gene was mapped on the chromosome 14 by Mapmaker v3.0b mapping software. In the accessions of upland cotton (G. hirsutum) with high fiber quality, the coding region SNPs with non-synonymous substitute were found, which may have phenotype effect.

cDNA Cloning of the Cellobiohydrolase Gene cbh1 from hermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris
　

CHEN Jing;LI Duo-chuan;ZHANG Yu-qin;DU Xin-ke

2006, 14(3): 406-411 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The cellobiohydrolase gene cbh1 fragment (GenBank Accession No. AY840982) was amplified by RT-PCR from thermophilic fungus Thermoascus aurantiacus var. levisporus. RACE was used to obtain its full-length cDNA (1710 bp), encoding 457 amino acids. The first 19 amino acids of the deduced amino acid sequence were presumed to be a signal peptide. The fragment encoding mature cellobiohydrolase was inserted into Pichia pastoris vector pPIC9K to construct recombinant plasmid pPIC9K/cbh1. The pPIC9K/cbh1 was then introduced into Pichia pastoris GS115 and 61 transformants were obtained, After comfirmed by G418 risistance and PCR, and induced expression, one clone GSp-15 was selected from the 61 transformants. The expression level of GSp-15 was 1.17 mg/mL after induction for 144 h in methanol, and its activity was 20.3 U/mL with p-NPC as substrate.

Construction of Recombinant DNA Plasmid Co-expressing Capsid Protein of
Porcine circovirus Type 2 and Porcine Granulocyte-macrophage Colony-stimulating Factor and Its Expression In vitro

SONG Qin-ye;YANG Han-chun;GUO Xin;CHEN Yan-hong;CHA Zhen-lin

2006, 14(3): 307-311 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The orf2 gene of Porcine circovirus type 2 (PCV2) and porcine granulocyte-macrophage colony-stimulating factor (gm-csf) were inserted into pIRESneo to construct recombinant plasmid pIRES-orf2/gm-csf. The recombinant plasmid pIRES-orf2/gm-csf was then transfected transiently into COS7 cells, the expressions of orf2 and gm-csf genes were confirmed by indrect immunofluorscence assay (IFA) and colony-stimulation forming assay. The results showed that the transfected cells could display specific immunofluorscence and the supernatant of cell culture had the activity for stimulating the formation of porcine bone marrow cell colonies. The successful construction of pIRES-orf2/gm-csf co-expressing PCV2 orf2 and gm-csf genes provides foundation for the further study of the PCV2 DNA vaccine.

Comparation of Protection Effects of H5 Subtype Avian Influenza DNA Vaccine Optimized with HA Gene and Expressive Vector

JIANG Yong-ping;ZHANG Hong-bo;LI Cheng-jun;BU Zhi-gao;DENG Guo-hua;YU Kang-zhen;CHEN Hua-lan

2006, 14(3): 301-306 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To evaluate the effect of H5 subtype avian influenza DNA vaccine optimized with HA gene codon and expressive vectors, groups of 3-week-old SPF chickens were intramuscular inoculated singly with 100 and 10μg of pCIHA5, pCAGGHA5, pCIoptiHA5 and pCAGGoptiHA5 in 200 μL volume, respectively. A group of chickens were injected with 200 μL PBS as controls. Sera were collected every week after vaccination for detecting the HI and AGP antibodies. After four weeks the all chickens were challenged with 100 LD50 of highly pathogenic A/Goose/GuangDong/1/96(H5N1)[Gs/GD/1/96(H5N1)], Oropharyngeal and cloacal swab specimens were collected from all the chickens 3, 5 and 7 days after inoculation for titration of virus in eggs, respectively, and they were observed daily for disease signs and deaths for 2 weeks. Results showed that in the 100 μg groups, pCAGGoptiHA5 and pCAGGHA5 vaccinated chickens were completely protected from virus challenge (no disease signs, no virus shedding and no deaths), while only partial protection were obtained in pCIoptiHA5 (75%) and pCIHA5(50%). And in the 10 μg groups, pCAGGoptiHA5 and pCAGGHA5 vaccinated chickens were protected from virus challenge (no disease signs and no deaths), and pCIoptiHA5(75%) vaccinated chickens were partially protected from virus challenge , while pCIHA5(50%) vaccinated chickens were all died after challenge. Results indicated that codon optimization of HA gene and β-actin promoter expression vector PCAGGS could observably enhance the protection effciency of H5 subtype avian influenza DNA vaccine. And the construction pCAGGoptiHA5 could protect chickens from lethal H5N1 virus challenge even at the low dose of 10 μg, implies the potential commercialization of avian influenza DNA vaccine in the future.

Expression of vip2A(c) Gene from Bacillus thuringiensis in Insect Cell

SHI Yong-xia;LV Lei;XU Wei;YUAN Mei-jin;PANG Yi

2006, 14(3): 401-405 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The recombinant Bac-gv2 DNA was obtained by inserting a fused gfp gene with Bacillus thuringiensis vip2A(c) gene encoding possible enzymatic component under the control of the polyhedrin gene promoter of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Trichoplusia ni cell line TnHi5 was transfected with Bac-gfp and Bac-gv2 DNAs respectively. Fluorescent cells expressing the fusion protein GV2 were much fewer than those expressing GFP alone, and did not obviously increase in number from 2 to 5 days after transfection. This results show that Vip2A fusion protein may have an ADP-ribosylating activity on cell skeleton actin and exert an influence on insect cell, the production and diffusion of the budded virus.

Cloning of NAD-SDH from Apple Fruit and Transformation to Agrobactrium tumefaciens from Its Antisense Expression Plasmid

GUAN Qing-mei;MA Feng-wang;LIANG Dong;CHENG Shun-chang;JIN Wei-bo

2006, 14(3): 440-441 | doi: | Full text (HTML) (1 KB) | PDF

PDF (246 KB) ( 269 )

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Abstract

Comparison and Improvement of Three Methods for Extracting Total RNA from Adipose

WU Jiang-wei;YANG Gong-she;SUN Chao

2006, 14(3): 444-445 | doi: | Full text (HTML) (1 KB) | PDF

PDF (171 KB) ( 365 )

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Abstract

Sequence Analysis of Cytochrome b Genes from Three Kinds of Labeoninae Family Fishes and Germplasm Identification

ZHANG Dian-chang;JIANG Shi-gui;SHAO Yan-qing;HUANG Yan-qin;LV Jun-lin;ZHU Cai-yan

2006, 14(3): 446-447 | doi: | Full text (HTML) (1 KB) | PDF

PDF (205 KB) ( 348 )

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Abstract

Polymorphism Analysis of the Exon 2 of SLA-DQA Gene of Tibet Pig by PCR- RFLP

JIANG An-an;LI Hua;YU Hui;LI Xue-wei;HE Zhi-ping;ZHAO Hai-quan

2006, 14(3): 442-443 | doi: | Full text (HTML) (1 KB) | PDF

PDF (236 KB) ( 247 )

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Abstract

Isolation and Primer Designing of Microsatellite Marker in the Pacifi
Abalone(Haliotis discus hannai)

CAO Jie;CHANG Ya-qing;DING Jun;SUN Xiao-wen;LU Cui-yun;JIA Zhi-ying

2006, 14(3): 448-449 | doi: | Full text (HTML) (1 KB) | PDF

PDF (288 KB) ( 318 )

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Abstract

Progress on Plant Transposable Elements and Their Molecular Markers

DAI Hong-yan;ZHANG Zhi-hong

2006, 14(3): 434-439 | doi: | Full text (HTML) (1 KB) | PDF

PDF (585 KB) ( 456 )

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Abstract

Transposable elements (TEs), which are DNA segments moving within genomes, play a major role in gene and genome evolution of eukaryotes. Plant transposable elements are classified into two major groups: class Ⅰelements or retrotransposons and class Ⅱelements or DNA transposons. Retrotransposons are divided into two principal groups, the long terminal repeat (LTR) and the non-LTR retrotransposons, while DNA transposons are divided into three types, the autonomous elements, non-autonomous elements and miniature inverted-repeat transposable elements (MITEs). The molecular markers based on plant TEs，including sequence-specific amplification polymorphism (S-SAP), MITE display, transposon display (TD), inter-MITE polymorphism (IMP), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP), were developed. In this review, the principles and applications of above-mentioned markers in genetic biodiversity and phylogeny analyses, gene mapping, and cultivar certification, were summarized.