农业生物技术学报

Construction of Recombinant CVI988 Virus Expressing GFP and Escherichia coli gpt Genes

2009, 17(2): 183-188 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Two fragments in the non-essential region of Marek's disease virus (MDV) were amplified by PCR from the genome of MDV CVI988 strain, and cloned into pUC19 for generating the 5.5 kb homologous recombinant arms. A transfer vector pUS-gpt-GFP was constructed by inserting the expressing cassettes of the CMV-gpt-ployA or CMV-GFP-polyA, respectively, into the unique BglⅡsite of US2 region in the recombinant arms. The transfer vector was transiently transfected into CHO cells and the expression of the green fluorescence protein was observed under the fluorescence microscope. This transfer vector was then transfected into CEF infected with MDV CVI988 strain. The recombinant CVI988 viruses expressing foreign genes, named rMDVgptGFP, were cloned by purifying the plagues expressing GFP in the MX-HAT selection medium. The purified recombinant viruses were further confirmed by PCR detection and growth of recombinant virus in selective and nonselective media.

Segmental Distribution and Ontogenetic Regulation of Peptide Transporter 1(PepT1) mRNA Expression in Intestine of Pigs

2009, 17(2): 229-239 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Segmental distribution and ontogenetic regulation of peptide transporter 1 (PepT1) mRNA expression were evaluated in pigs along the horizontal axis of the intestine. A total of 35 littermate purebred Lantang gilts and 35 littermate purebred Landrace gilts were divided into seven groups at the ages of day 1, 7, 26, 30, 60, 90 and 150, respectively. Intestinal segments (duodenum, jejunum, ileum and colon) were collected. The PepT1 mRNA abundance was determined by Real-time RT-PCR using SYBR Green Ⅰ RT-PCR mix Kit. Results showed that the abundance of PepT1 mRNA in duodenum was higher than in jejunum and ileum (P <0.05). There was no PepT1 mRNA expression in colon. The highest expression of PepT1 mRNA was on 7 and 90 d of Lantang and 60 d of Landrace in duodenum, 60 d of Lantang and 90 d of Landrace in jejunum and ileum, respectively (P <0.05). The ontogenetic expression of PepT1 mRNA in intestine of Lantang and Landrace pigs was similarly during post-weaning (weaning on 28 d) period. The expression patterns of PepT1 mRNA in jejunum and ileum were similar after post-weaning. Abundance of PepT1 mRNA in duodenum and jejunum of Lantang pigs on 7 and 90 d in duodenum, 7 and 60 d in jejunum and 30 d in ileum was higher than in corresponding segment of Landrace pigs. The results indicated that PepT1 mRNA expression can be regulated by developmental stage, breed and segment of intestine in pigs.

Polymorphism Analysis on Partial Exons of Estrogen Receptor (ESR) Gene in Goats

2009, 17(2): 237-242 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The estrogen receptor (ESR) plays an important role in animal reproduction through binding specifically with estrogen and interacting with estrogen to regulate the expression of related genes of reproductive function, and was studied as a candidate gene for prolificacy in some goat (Capra hircus) breeds. Single nucleotide polymorphisms (SNPs) of exons 1 and 4 of ESR gene were detected in high prolificacy breed (Jining Grey goat), moderate prolificacy breed (Boer goat) and low prolificacy breeds (Inner Mongolia Cashmere and Angora goats) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Results showed that no polymorphism was detected in the amplified region in the four goat breeds tested. The nucleotide sequences of these two fragments were detected and the corresponding amino acid sequences were deduced. The comparison of the fragments of exons 1 and 4 of goat ESR gene with those of human, cattle, pig, horse, sheep, rat, mouse and chicken ESR gene showed that the homologies of the nucleotide sequences and amino acid sequences of the fragments with these eight species were from 51.3% to 97.3% and from 64.5% to 98.3% respectively. Two special variations were found in amino acid sequence of goats, which were 81st (proline insertion) in exon 1 and 4th (D4N) in exon 4. The results indicated that the ESR gene was highly conserved among mammals, and exons 1 and 4 may not be the functional domains that affect the high prolificacy in goats.

Effects of Diet Protein Level on the Growth, Body Composition and Digestive
Enzyme Activities of the Barbudes caldwell Juvenile

2009, 17(2): 276-281 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Seven isoenergic semi-purified test diets containing graded levels of protein ranging from 20% to 50% were formulated using fish meal and casein as the protein sources. Triplicate groups of Barbudes caldwell juveniles with initial body weight of 1.26±0.02 g respectively were fed by the test diets for 8 weeks. The results indicated that there was no significant effect of dietary protein levels on the survival rate, relative weight of viscera and relative weight of liver of the juvenile fish (P >0.05). The weight gain and specific growth rate of the fish were increased with dietary protein level increasing from 20% to 35% (P >0.05), but not affected significantly as dietary protein level increased from 35% to 50% (P >0.05). Feed efficiencies were not significantly different when fish fed diets with protein levels from 30% to 50%, but significantly higher than those of the other two treatments when fish fed diets with protein levels of 20% and 25% (P <0.05). The protein efficiency ratio(PER) was negatively correlated to the diet protein level(x) (PER = 3.006－0.03251x, R =0.9366). There was no significant effect of dietary protein levels on carcass moisture, crude protein and ash of the juveniles (P >0.05). While carcass lipid was decreased with the increase of dietary protein level(x) (L =8.2169－0.0458x, R =0.8551). There was no significant variation of hepato-pancreas protease activity among tests (P >0.05). Intestine protease activity and hepato-pancreas amylase activity were increased to some extent, and then decreased with continued increasing dietary protein level. The intestine amylase activity (IAA) of the juvenile was negatively correlated to dietary protein level (x) (IAA = 84.625－0.9147x, R =0.8463). It was estimated that the suitable protein level for the B. caldwell juvenile was 34% of dry diet when the broken-line model was introduced to regress the relationship of weight gain of the juvenile and dietary protein level.

Screening Differentially-expressed Genes of Haemophilus parasuis from the Selective Capture of Transcribed Sequences (SCOTS) by Reverse Dot-blot Hybridization

2009, 17(2): 189-194 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A reverse Dot-blot hybridization (RDH) method was established for screening the differentially-expressed genes of Haemophilus parasuis in vivo and in vitro from the selective capture of transcribed sequences (SCOTS) pools by optimizing the hybridization conditions including the kinds and concentration of target DNA, probe concentration, and UV irradiation time. Six samples were verified to be differentially-expressed genes in vivo of H. parasuis from ten samples of cDNA libraries (SCOTS pools) by RDH. The results showed that SCOTS was an effective approach to establish cDNA libraries containing differentially-expressed genes and RDH could be used to effectively pick up the differentially-expressed genes from the SCOTS pools.

Expression and Identification of Peste des petits ruminants virus N Gene in Escherichia coli

2009, 17(2): 195-200 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A pair of primers added EcoRⅠand NotⅠsites was designed and synthesized based on the sequence of the nucleocapsid（N）protein gene of Peste des petits ruminants virus (PPRV), which was for amplifying the full-length N gene fragment of pCI-neo -PPRN plasmid from Austria. The fragment was cloned into pGM-T vector and transformed into Escherichia coli TOP10. Then the cloned fragment was subcloned into prokaryotic expression vector pET-32a(+), and the recombinant expression plasmid pET-PPRN was expressed in E.coli BL21 (DE3) with inducement. The size of the recombinant N fusion protein was 80 kD. The recombinant PPRV N fusion protein could be detected with Histidine monoclonal antibody or goat anti-PPRV sera by Western blot.

Prokaryotic Expression of Nucleoprotein Gene of Transmissible gastroenteritis
virus and Development of ELISA Based on the Expressed Protein

2009, 17(2): 201-205 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The recombinant PET-N plasmid, which includes the N gene of Transmissible gastroenteritis virus(TGEV), was transformed into Escherichia coli BL21(DE3) and expressed by 1.0 mmol/L IPTG at 37 ℃. The indirect ELISA for detecting TGEV nucleocapsid protein antibody was established after the reactionogenicity of the recombinant protein was proved by Western blot. The optional working circumstances for the ELISA are as follows: antigen concentration 15 μg/mL, serum dilution 1∶40, blocking solution 0.5% FBS, serum sample incubated for 90 min, concentration of HRP-spa 1∶5 000 incubated for 60 min, the substrate incubated at room temperature for 10 min, and the threshold value of ELISA OD450≥0.35 tried out with chess titration. Sensitivity , specificity and concordance of this method were 93.5%, 93.8% and 93.5%, respectively compared to Svanova TGEV/PRCV antibody diagnosis kit．

Cloning of Porcine Granulocyte-macrophage Colony Stimulating Factor （pGM-CSF） Gene and Its Prokaryotic Expression

2009, 17(2): 206-211 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The porcine granulocyte-macrophage colony stimulating factor (pGM-CSF) full-length gene was cloned from porcine peripheral blood lymphocyte using RT-PCR and ligated into pMD19-T vector. Sequence analysis showed that the cDNA of pGM-CSF was 435 bp in length and encodes 144 amino acids. The gene of mature pGM-CSF deleting a 17 aa signal sequence at N-terminus was cloned by PCR and inserted into pET-32a(+). The recombinant plasmid pET-GM was transformed into Escherichia coli BL21(DE3) and induced by IPTG. The results of SDS-PAGE and Western blot showed that the recombinant protein was about 31 kD, which existed mainly in inclusion body, accounts 30.4% of total bacterium proteins. After dilution renaturation and purification procedure in Ni2+-NTA affinity chromatography column, the biological activity of purified protein was analyzed by MTT method using TF-1 cells. The result indicated that the recombinant protein could effectively stimulate the proliferation of TF-1 cells, with a specific bioactivity of 3.43×105 IU/mg.

Effects of Doses of Bacillus Calmette-guerin (BCG) and Ages of Inoculated Mice on Immune Protection against Tuberculosis and IFN-γ/IL-4 Expressions

2009, 17(2): 212-217 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Seven-day-age and 35-day-age mice (BalB/C) were inoculated with 2×103 and 4×104　cfu of bacillus calmette-guerin (BCG), respectively, and challenged with 105 cfu of BCG at 28 days post inoculation. The ratios of protection against tuberculosis (TB) were observed and the expressions of interferon-γ（IFN-γ）/ interleukin-4（IL-4） were detected with enzyme-linked immunospot (ELISPOT). The results indicated that 100% of protection could be induced with 2×103 cfu of BCG and 75% of protection with 4×104 cfu of BCG in 7-day-age mice, but 67 % of protection could be induced with 2×103 cfu of BCG in 35-day-age mice. In 7-day-age mice inoculated with 2×103 cfu of BCG, the expressed quantity of Th1 type cytokines mainly produced IFN-γin spleen were improved significantly post inoculation and Th2 type cytokines mainly produced IL-4 were reduced; However, whether in 7-day-mice inoculated with 4×104 cfu of BCG or in 35-day-age mice inoculated with 2×103 cfu of BCG,both IFN-γ and IL-4 were significantly improved. It can be concluded that the protection against TB induced with BCG may be correlated to the doses of BCG and the ages of inoculated animals and Th1/Th2 type cell immune responses induced with BCG may cause these differences.

Polymorphism of Gonadotropin Releasing Hormaone Receptor (GnRHR) Gene and Its Relationship with Prolificacy of Jining Grey Goat

2009, 17(2): 218-223 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The gonadotropin releasing hormone receptor (GnRHR) gene was studied as a candidate gene for the high prolificacy of Jining grey goat(Caprar hircus). Eight pairs of primers were designed to detect single nucleotide polymorphisms of exon 1, exon 2 and exon 3 of GnRHR gene in high fecundity breed(Jining grey goat) and low fecundity breeds(Angora, Boer and Inner Mongolia Cashmere goats) by PCR-SSCP. Only the products amplified by primer P4 displayed polymorphisms. For primer P4, three genotypes (AA, AB and BB) were detected in Jining grey, Boer and Inner Mongolia Cashmere goats, only AA and AB genotypes were in Angora goats. Sequencing revealed one mutation (757G→A) of exon 1 in genotype BB compared with genotype AA, which did not cause amino acid changes. In Jining Grey goats, frequency of AA, AB and BB genotypes was 0.623, 0.300 and 0.077, respectively. The Jining grey with genotype BB had 0.69(P < 0.05) or 0.82(P < 0.05) kids, more than those with AB or AA, respectively. The difference of the least squares means for litter size between AA and AB was nonsignificant (P > 0.05) in Jining grey goat.

Identification of Polymorphism of the Callipyge　Mutation Gene（CLPG） in
Goats and Its Associations with Production Traits

2009, 17(2): 224-228 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The Dorset ram with Callipyge phenotype shows muscular hypertrophy at buttock, and its inheritance is polar overdominance. A partial DNA fragment of 250 bp in goat (Capra hircas) Callipyge mutation gene(CLPG) (GenBank Accession No.EU753362) was obtained, and had a 96.04% and 88.65% identity with the corresponding region of ovine(Ovis aries) and porcine CLPG, respectively. And a polymorphism in the DNA fragment was detected by PCR-SSCP. Sequencing results indicated that no A→C mutation was corresponding to ovine CLPG, whereas a A→C transversion located 147 bp downstream from the CLPG site was identified. The polymorphism was named after its position as SNP216 was investigated in Boer (n = 63), Laiwu Black (n = 70), Lubei White × Boer Hybrid (n = 40), Lubei White (n = 29) and Inner Mongolia Alashan white cashmere (n = 115) goat populations. The results indicated that allele A was predominant in the four goat populations of Boer, Laiwu Black, Lubei White × Boer Hybrid and Inner Mongolia Alashan white cashmere, and except for the Inner Mongolia Alashan White cashmere goat, the other four populations were in the state of Hardy-Weinberg equilibrium (P > 0.05). In Inner Mongolia Alashan white cashmere goats, least square means of birth weight, production of cashmere and body weight gain from birth to weaning between AA and AC were not significantly different (P >0.5).

Genetic Effects of Prolactin (PRL) and Growth Hormone (GH) Genes on Egg Performance in Gaoyou Duck

2009, 17(2): 263-268 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The polymorphisms of prolactin (PRL) and growth hormone (GH ) genes were detected using PCR-RFLP and PCR-SSCP, respectively. The association between the two genes (PRL and GH ) and egg performance of Gaoyou duck were analyzed. The results showed: ①Three genotypes AA, AB and BB were found in PRL gene by PCR-RFLP. Frequencies of allele A and B were 0.226 and 0.774. A mutation T→C at the position of 1 326 bp (PRL intron 1) of the PRL gene in Gaoyou duck was found by DNA sequencing. Three genotypes CC, CD and DD were found in GH gene with the preponderant gene D frequency of 0.778 and the gene C frequency of 0.222. The mutations C→T at the position of 3 701 bp (GH extron 4) was found by DNA sequencing. ②The highly significant association between PRL polymorphism and egg weight (30-weeks) was observed (P <0.01). Three significant associations were found: for PRL and double-yolk percentage(P <0.05), GH and the highest clutch days (P <0.05), GH and double-yolk percentage (P <0.05), respectively.

A Facile, Rapid and Effective Method for RNA Quality Determination

2009, 17(2): 282-287 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A series of comparative test analysis were conducted and results showed that the effect of detection of RNA by gel electrophoresis was not only associated with comb size and RNA concentration, but also associated with migration time in gel electrophoresis and the amount of RNA to be loaded. Finally, a facile, rapid and effective method for RNA quality determination was established, namely using 1.2% agarose gel with the comb of breadth 7 mm, loaded 3~5 μg RNA, mixed with 6×RNA loading buffer and diluted with RNase-free water, followed by electrophoresis for 15~30 min and the reality of RNA quality could be determined. This method had the characteristic of reduplicate in practice, and can be used as a good reference for researcher.

Cloning of Bovine　Interleukin-1 Receptor-associated Kinase 2(IRAK2) Gene and Its Bio-information Analysis

2009, 17(2): 243-248 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Toll-like receptors (TLRs) can recognize the pathogen associated molecular pattern and mediate signaling for initiate innate and adaptive immune responses. Interleukin-1 receptor-associated kinase 2 (IRAK2) is one of the important downstream signal molecules in TLRs signal transduction pathways. In order to further study the function and signal transduction of TLRs on the diseases induced by pathogen infection, bovine IRAK2 gene isolated using RT-PCR and RACE experiments was analyzed by bio-informatics and tissues expression profiles. The results showed that bovine IRAK2 gene could be expressed in many tissues including mammary gland, liver, duodenum, fat, womb, kidney, heart, lung, pancreas and ovary. Its cDNA sequence was 2 148 bp (GenBank accession No. EU528620), including 36 bp 5'-UTR, 1 866 bp CDS and 246 bp 3'-UTR. IRAK2 gene codes 622 aa, molecular weight and isoelectric point are 68 628.31 D and 5.3, respectively. IRAK2 protein contained the death domain(DD) and S_TKc domain and located at lots of cell organs such as cell nucleus, cytoplasm, mitochondrium and protoplasmic membrane. It is inferred that IRAK2 may be very important for signaling and regulating on TLRs signal transduction.

Identification of a Differentially Expressed Gene in Fatty Liver of Overfeeding Landes

2009, 17(2): 256-262 | doi: | Full text (HTML) (1 KB) | PDF

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To obtain understanding of its fattening mechanism, mRNA differential display reverse transcription PCR (DDRT-PCR) was applied to study the differences of gene expression in French Landes grey goose in conditions of overfeeding and normal feeding. One gene was found to be significantly up-regulated in fatty liver（P < 0.01）, which had a cds length of 1 248 bp (GenBank accession No.EF541127) and showed 94% identity to chicken transforming growth factor beta 2 (TGFB2). The sequence analysis revealed that its 1 239 bp-in-length open reading frame (ORF) encoded a 412-amino-acid protein containing two putative conserved domains of TGFb-propeptide and TGF-beta， and had high homology with its homologues. The function of this protein had been briefly reviewed based on published information. The tissue expression analysis indicated that goose TGFB2 mRNA was expressed higher in liver, muscular stomach, spleen and ovary than that in other tested tissues. The present results suggested that overfeeding could increase the mRNA expression level of goose TGFB2.

Cloning of MxYSL1 Gene in Malus xiaojinensis and Its Expression Analysis

2009, 17(2): 288-293 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Malus xiaojinensis is an iron-efficient apple genotype. Through the analysis of apple’s EST, a 791 bp YSL (yellow stripe 1-like protein) gene fragments were obtained, and their specific primers were designed to obtainYSL gene fragment in Malus xiaojinensis . The complete 3' sequence of YSL gene was obtained by RACE method, which were composed of 1 114 nucleotides. The predicted amino-acid sequence included 7 putative membrane-spanning domains and its homology with AtYSL1 was up to 74%, and was named as MxYSL1. The results of semi-quantitative RT-PCR and Northern blotting analysis showed that MxYSL1 was expressed in all organs of M. xiaojinensis. The expressions were down-regulated in roots, stems and mature leaves with Fe deficiency treatments(EDTA-NaFe,4 μmol/L), but up-regulated with Fe excess treatments(EDTA-NaFe,320 μmol/L). The young leaves had opposite expression model.

Genetic Polymorphism of Six Genetic Loci and Their Correlation with Reproductive Traits

2009, 17(2): 249-255 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

ESR(estrogen receptor), PRLR(prolactin receptor), FSHβ(follicular -stimulating hormone beta subunit), NCOA1(nuclear receptor coactivator 1), OPN (osteopontin) and PRL (prolactin) genes related with reproductive traits of Large White pig were regarded as candidate genes and the polymorphisms of these genes were detected by electrophoresis method and PCR-RFLP with restriction endomuclease PvuⅡ, AluⅠ, RsaⅠand BsaⅠ, respectively. The results showed: allele B was the dominant gene of ESR, PRLR, FSHβ and OPN. And the allele A (frequency = 0.87) and C (frequency = 0.79) were the dominant genes of NCOA1 and PRL. Among first parity sow group, the total number born (TNB), number born alive (NBA) and birth litter weight (BLW) of AB genotype for NCOA1 were 1.12 piglets, 1.08 piglets and 1.71 kg less than that of AA genotype（P < 0.05）; TNB, NBA and BLW of AA/BC genotypes for PRL were 3.37 piglets, 3.84 piglets, and 4.75 kg more than that of AC genotype（P < 0.05）. In multi-parity sow group, the heterozygote of PRLR had significant difference （P < 0.05） with homozygote in TNB and BLW, and the differences were 1.0~1.5 piglets and 1.0~1.4 kg respectively. The BLW of PRL AA and BC genotypes were 1.08 and 1.05 kg heavier than that of AC genotype. The TNB of BB genotype for OPN was 2.13 　　piglets more than that of AA genotype. The combined genotypes analysis indicated that allele A was positive to TNB for ESR and PRLR., and allele B was positive to TNB, NBA and BLW for OPN. And in combined genotypes, the AA homozygote had positive effect on litter size.

Effects of Culturing Time before Electrical Fusion and Chemical Activation on Constructed Sheep Embryos

2009, 17(2): 269-275 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

In order to compare the effects of culturing time before electrical fusion and chemical activation on the fusion and development of the constructed embryo, embryos were reconstructed by subzonal injecting the sheep (Bos gaurus) somatic cells into matured sheep oocytes in vitro. Results showed that culturing for 1 h before electrical fusion, there were no significant differences in cleavage, morulas and blastulas rates among the groups(P >0.05), however, the integrity rate of couplets cultured for 2 h before activation (95.81%) was significantly higher than control (74.64%)(P < 0.05); under the condition of culturing for 2 h before chemical activation, there were no remarkable differences in the rate of fusion, integrity and the blastulas(P > 0.05), but the rate of cleavage and morula which were cultured for 1h before activation (76.88% and 40.63%) were dramatically higher than control(64.48%and 21.50%)(P <0.05). When developed into morula or blastulas in commercial G1/G2 sequential medium, the embryos were transferred into 41 recipients, only one finally was developed into a fetus but aborted when it was 104 day old in vivo. Then microsatellites DNA analysis with 5 pairs of primers was carried out on the fibroblast of aborted fetus, donor cells, the fibroblast of recipient sheep and one control chosen occasionally. Results indicated that the bands had polymorphism after 10% Ago-Gel electrophoresis of PCR products and silver staining, and the fingerprint of cloned fetus were completely the same as those of donor cells, while definitely different from those of recipient sheep and the control, and the gene of cloned fetus came from donor cells and the fetus was a cloned sheep.

Construction of cDNA Library and Analysis of Expressed Sequence Tags of
Chinese Wild Vitis pseudoreticulata Clone Baihe-35-1 Resistance to Downy Mildew

2009, 17(2): 294-300 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A cDNA library was constructed with Vitis pseudoreticulata clone Baihe-35-1 leaves inoculated by Plasmopara viticola using SMARTTM cDNA Library Construction Kit. The titer of primary library and amplified library was about 0.92×106 pfu/mL and 5×109 pfu/mL, respectively. In the primary library, 97% clones were recombinant and the length of insert cDNAs were from 500 to 2 200 bp. Two hundred and fifty-four ESTs (GenBank accession No. FG269201~FG269449 and FG396006~FG396010) were obtained by random sequencing of the cDNA library. BlastX search showed that 56.3% ESTs had higher homology with functional genes, 14.6% ESTs with hypothetical putative or unknown protein, and 20.9% ESTs had no-hits.

Characterization and Mutation of Soybean MYB Transcription
Factor GmMYBZ2 and Its Prokaryotic Expression

2009, 17(2): 301-306 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Soybean (Glycine max) GmMYBZ2 is one of typical plant R2R3-MYB family genes. In this study, its protein characterization and transcriptional activity were analyzed by DNAMAN and yeast (Saccharomyces cerevisiae) one-hybrid system, respectively. Moreover, the prokaryotic expression vector was constructed and expressed in Escherichia coli as well. Results showed that GmMYBZ2 not only exhibited typical features of the R2R3-MYB transcription factors, but also had a conserved amino acid motif and a predicted zinc finger region at its C-termini. Genomic DNA sequence analysis showed that it contained an intron in the R3 domain which was located at 263~395 bp. In order to investigate the infections of these motifs in GmMYBZ2’s transcriptional activity, the conserved motif and zinc finger region were detected by PCR. The effect of the altered proteins were monitored using yeast one-hybridize, which showed that GmMYBZ2 had transcriptional activation function. And the intron, conserved motif and zinc finger region might repress the transcriptional activity of GmMYBZ2. Moreover, the encoding sequence of GmMYBZ2 gene was inserted into pET30a to construct prokaryotic expression vector pET-ZD2 which was then transformed into E. coli BL21 (DE3) pLysS and Rosetta2(DE3) pLysS, respectively. Results showed that GmMYBZ2 could be expressed in E. coli Rosetta2(DE3) which contained complementary rare codons successfully.

Sequence-characterized Amplified Region(SCAR) Markers Linked to Watermelon mosaic virus Resistant Gene in Cucumber

2009, 17(2): 312-316 | doi: | Full text (HTML) (1 KB) | PDF

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Markers linked to Watermelon mosaic virus ( WMV ) resistant gene in cucumber （Cucumis sativus） were identified using 120 recombinant inbreed lines (RIL6) made between a resistant and susceptible parent (Qiupeng× No.8 European). Genetic analysis based on virus inoculation at the seedling stage showed that the resistant trait in Qiupeng was controlled by a single recessive locus. The AFLP marker E-ACT/M-CTT-427 was linked to the resistant gene with a genetic distance of 8 cM. The polymorphism came from a 6 bp deletion of poly(T) repeat in the susceptible parent. And this AFLP marker was converted into a co-dominant sequence-characterized amplified region(SCAR) marker which could be useful in marker-assisted breeding for WMV resistance.

Establishmeut of Dynamic Models for Keratinase of Bacillus subtilis KD-N2 by Batch Fermentation using Feather andHuman Hair as Carbon and Nitrogen Substrate

2009, 17(2): 328-333 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Keratinase batch cultivation in a 5 L fermentor was studied using Bacillus subtilis KD-N2 as fermentation bacteria, and feather and human hair as carbon and nitrogen substrates. The kinetic models were proposed for cell growth and product formation based on the Logistic and Luedeking-Piret equations. Parameters of the models were established with the experimental data. The models for cell growth and keratinase formation in feather substrate were described by equations of dX/dt =0.1013(1－X/ 0.3742)X and dP/dt =276.69dX/dt－3.6X ,respectively; for human hair substrate, the equations were of dX/dt =0.0728(1－X/ 0.197)X and dP/dt =422.34dX/dt + 5.187X.

Construction of Brassica campestris Representative Library Using Differential Subtractive Chain

2009, 17(2): 307-311 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

High polymorphism genome representative library is the key for an efficient molecular marker technique based on microarray or sequencing. A genome representative libray with relatively high polymorphism ratio was developed by differential substractive chain (DSC). Nine accessions belonging to different Brassica campestris cultivar groups were used, eight of the nine accessions were mixed as tester DNA, while one accession mizuna (B.campestris ssp. nipposinica) was used as driver. The two DNA samples were both digested by EcoRⅠ/MseⅠbefore ligated to different adapters. Three rounds DSC were performed before the product was ligated to the GatewayTM vector pDONR201. Southern Dot blots were proformed by tester or driver probes using two randomly selected batches of clones (20 and 95 clones), respectively. The results showed 9 clones in the first batch and 58 clones in the second batch were polymorphic, with polymorphism rate of 45% and 61% respectively, indicating that the constructed library has a significantly higher polymorphism rate than those in previous reported research (15%～17%) by diversity array technology (DArT).

Evaluations of Trichloroacetic Acid-acetone Method and Phenol Method for Protein Extraction from Cotton Ovule

2009, 17(2): 317-322 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Trichloroacetic acid-acetone precipitation method (TCA method) and phenol isolation method (phenol method) were evaluated by measuring the yield ratio and purity of protein and analysis of the gel electrophoresis map on ovule of DPL971 and DPL972 in Gossypium arboreum during primary cell wall synthesis period. The results were as follows: firstly, the massive soluble proteins could be obtained by the two methods, but the yield ratio, quality and protein types by phenol method were better than those by TCA method; secondly, the types of extracted protein were different by two methods, the phenol method had better performance in acidic region and in high/low molecular weight region; finally, the phenol method could efficiently get rid of the interfering substance and achieve the excellent gels, therefore, the phenol method was suitable for protein extraction from the cotton ovule. As a result, the useful information can be provided for the proteomics research of cotton fiber during the development period and have certain significance for guiding the protein extraction of many other non-model plants containing various interfering elements.

Purification and Characterization of the Recombinant 1-aminocyclopropane-
1-carboxylate (ACC) Oxidase from Sugarcane

2009, 17(2): 323-327 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The plasmid pET30a-SgACO, which contains 1-aminocyclopropane-1-carboxylate (ACC) oxidase （SgACO） gene from sugarcane (Sacharum sinensis Roxb), was transformed into Escherichia coli BL21 (DE3) plysS successfully, and the SgACO protein was expressed in BL21 (DE3). The SgACO was highly expressed in BL21 (DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in an inclusion body form. The final SgACO activity of 132.58 nmolC2H4/mg/h was obtained by one-step renaturation and purification of Ni2+- NTA column to the inclusion body protein. The renaturation and purification protein was relatively stable at the pH ranging from 5.5 to 8.0 and at the temperature ranging from 20 ℃ to 45 ℃. Its optimum pH was 6.7, optimum temperature was 33 ℃, Km was 61.77 μmol/L and Vm was 0.9647 μmol/min. The enzyme activity was activated by certain concentration of CO2 and ACC, and inhibited by Mg2+, Cu2+, Zn2+ and EDTA.

DNA Polymorphism Analysis and a Sequence-characterized Amplified Region (SCAR) Markers of Streptomyces roseoflavus Menmyco-93-63 and Its Mutant Strains

2009, 17(2): 334-340 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Streptomyces roseoflavus Men-myco-93-63 isolated from potato scab decline soil is an antagonistic strain. The strain and its fermentation can inhibit many pathogenic fungi and control the related important plant disease such as Verticillium wilt of cotton, cumber powdery mildew, and so on. The genetic polymorphism of Men-myco-93-63 with its eight antibiotic blocked mutants was analyzed using amplified fragment length polymorphism(AFLP) and 3 142 AFLP bands were amplified using 38 primer pairs, and 2 362(75.2 %) bands of them were polymorphic. Three of the 96 pairs of primers E-CA/M-GA, E-GA/M-AC and E-GA/M-AG were selected for AFLP. The bands with 254, 312 and 209 bp, which amplified by the three pairs of primers respectively, were present only in Men-myco-93-63 and named as EM254, EM312 and EM209. After recovery, cloning and sequencing，the three specific AFLP fragments were successfully converted into single locus PCR marker of a sequence-characterized amplified region (SCAR) which could be more easily to be used to detect mutants of Men-myco-93-63 at the molecular level.

opulation Genetic Diversity of Phytophthora infestans from China Revealed by SSRs and RAPDs

2009, 17(2): 347-354 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

SSR and RAPD molecular markers were used to assess the genetic diversity among 80 isolates of Phytophthora infestans on potato(Solanum tuberosum) from Fujian, Heilongjiang, Hebei and Inner-mongolia. Polymorphism was identified by 13 SSR primers and 14 RAPD primers in the isolates of P. infestans on potato. A total of 76 bands were produced by SSRs with polymorphic bands accounting for 78.9% and similarity coefficient within 0.00 to 0.42. And a total of 189 bands were generated by RAPDs with polymorphic bands accounting for 95.2% and similarity coefficient ranging from 0.04 to 0.66. Analysis of genetic variation showed that there existed higher genetic variation in the Fujian populations in comparison to the populations of Heilongjiang, Hebei and Inner-mongolia. Nei's genetic identity analysis indicated that the populations of Heilongjiang and Inner-mongolia were closer than Fujian and Hebei populations revealed by SSRs and RAPDs. The results of cluster analysis revealed that some isolates from Fujian, the south region of China were distantly related to some isolates from Heilongjiang, Heibei and Inner-mongolia, the north of China and the Fujian populations had a higher genetic diversity than that of the others.

Screening of An Erythromycin-degrading Strain Rhodotorula and Its Degradation Characteristics

2009, 17(2): 341-346 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

An erythromycin-degrading strain was isolated from soil contaminated by erythromycin. Based on analysis of its morphology, 18S rDNA, physiological and biochemical characteristics, the strain was identified to be Rhodotorula mucilaginosa. Its sequence of 18S rDNA was 1 562 bp and the accession number was EU294522 in GenBank. The strain could grow using erythromycin as the carbon source, optimal carbon and nitrogen sources were sucrose and NH4Cl, respectively. The optimal temperature, pH and inoculation for erythromycin degradation were 30 ℃, 5~5.5, and 6% , respectively, and he degradation rate was positively related to aeration, and could be 100% after the strain was cultured for 48 h.

Development of Murine Cloned Embryos Reconstructed by Nuclei of
Cumulus Cell from Self- or Different Individual Donors

2009, 17(2): 361-362 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Prion Protein Gene Phylogeny (PRNP) in Main Chinese Domestic Bovidae Breeds

2009, 17(2): 363-364 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Advances in Fruit-specific Promoter and Its Applications

2009, 17(2): 355-360 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Heterogeneous genes can express specifically in fruits under the control of fruit-specific promoters. Advanced research on fruit-specific promoters, mechanisms of specific-related regulation and its applications were reviewed in this article.