农业生物技术学报

Analysis of Chromosome Karyotypes of Oreochromis aurea(♀), Siniperca chuatsi(♂) and Their Offspring

CAO Li-ping;DING Wei-dong;JIA Yong-yi;XIA De-quan;WU Ting-ting

2006, 14(2): 187-190 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The progenies of the distant hybridization between Oreochromis aurea (♀) and Siniperca ahuasti (♂) were successfully obtained through years of research. And their karyotypes were studied by half-micro blood lymphocytic cell cultivation. The result indicated that the diploid chromosome number of O. aurea (♀) was 2n =44, and karyotype formula was 6sm+24st+14t, arm number (NF) =50; the diploid chromosome number of S. chuatsi (♂) was 2n =48, and karyotype formula was 8sm+4st+36t, NF=56; the diploid chromosome number of their progenies was 2n =44, chromosome formula was 6sm+26st+12t, NF=50. The results showed the karyotypes between O. aurea (♀) and progenies were similar by comparison.

SCAR Molecular Markers of the B Biotype and Two Non-B Populations of the Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

ZANG Lian-sheng;IANG Tong;U Jing;IU Shu-sheng;HANG You-jun

2006, 14(2): 208-212 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

RAPD was performed with random primer H16 for B biotype and two non-B populations of Bemisia tabaci collected from Zhejiang, China. The results revealed that the specific sequence fragments containing 446, 390 and 1317 nt were amplified for the B biotype, non-B ZHJ-1 and ZHJ-2 populations, respectively. The three specific fragments were cloned and sequenced, and three pairs of SCAR primers were designed according to the sequences. With improvement of the conditions of PCR, the specific fragment of B biotype, non-B ZHJ-1 and non-B ZHJ-2 populations, namely, 439, 366 and 1238 nt, respectively, could be amplified with the SCAR primer of the corresponding population, while the specific fragments of the other populations of B. tabaci or Trialeurodes vaporariorum could not be amplified.

Cloning and Sequence Analysis of Myo Inositol-1-phosphate Synthase Gene in Sweetpotato

LIU Zhe-sheng;LIU Qing-chang;ZHAI Hong;WANG Yu-ping

2006, 14(2): 219-225 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Nongda 603, a mutant resistant to sweetpotato stem nematode (Ditylenchus destructor), was obtained from the gamma-irradiated progenies of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Xushu 18 susceptible to the stem nematode. Primers were designed from the NBS (nucleotide-binding site) conserved amino acid sequence of plant nematode resistance genes. The mRNAs of storage roots of Nongda 603 and Xushu 18 were used as temple. RT-PCR analysis indicated that the mRNA abundance of Myo inositol-1-phosphate synthase (MIPS) gene in Nongda 603 was higher than that in Xushu 18. The 3' cDNA of MIPS gene was amplified using 3' RACE and the 5' cDNA of MIPS gene was amplified by PCR using the specific primer of 3' cDNA and the degenerate primer designed based on the conserved amino acid sequence of the 5' end of plant MIPS genes. The DNA sequence alignment showed that sweetpotato MIPS gene had high homology to MIPS genes of soybean (Glycine max) and tomato (Lycopersicon esculentum ) with the identities of 83.63 % and 83.89 %, respectively. The cloning of sweetpotato MIPS gene is useful for the further research of the relationship between sweetpotato MIPS gene and sweetpotato stem nematode resistance.

Expression and Bioactivity Assay of Fusion Protein of
Cowpea Trypsin Inhibitor(CpTI) and Thioredoxin

CHANG Wen-jun;WANG Yu-gong;LEI Lu-wang

2006, 14(2): 226-230 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

To construct fusion protein expression vector, cowpea trypsin inhibitor (CpTI) gene was linked with thioredoxin gene in the pTrxFus through a flexible linker designed with the software Chou-Fasman. The recombinant vector pTrxFus-CpTI was identified and transformed into Escherichia coli GI724 to overexpress the fusion protein. The expression was induced with tryptophan. The fusion protein was confirmed to be soluble by SDS-PAGE analysis of the supernatant and precipitate of lysates. SDS-PAGE also showed that expressed protein accounted for about 20% of the total bacterium protein. With Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride(BApNA) as substrate, the bioactivity and heat resistance of fusion protein were assayed and the result showed, like soybean trypsin inhibitor, fusion protein performed relatively high thermal resistance. SDS-PAGE showed most fusion protein remained soluble after treated at 80 ℃, which greatly facilitates its purification and application. Thioredoxin-CpTI could be digested into thioredoxin and CpTI by incubating at 37 ℃ for 16 h with enterokinase. The CpTI inhibition activity was about 80% of thioredoxin-CpTI, indicating that the CpTI stability was increased via the fusion with thioredoxin. This work has laid a foundation for application of thioredoxin-CpTI as a biological pesticide.

Transgenic Plants of Brassica campestris with Resveratrol Synthase Gene

HU Xiao-ping;SHANG Wen-jing;CAI Wen-qi;HAN Qing-mei;KANG Zhen-sheng

2006, 14(2): 231-234 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A plant constitute expression vector EHA105/pBinRS, which contained NPTⅡ and resveratrol synthase (RS) gene, was constructed through plasmid pBin438. RS gene was then transferred into Brassica campestris ssp. chinensis using an Agrobacteriume tume faciens mediated method. The regeneration plants were screened on MSBk medium and assayed by PCR and RT-PCR. The results showed that RS gene had been integrated into genome of B. campestris ssp. chinensis and normally expressed. And seven transgenic plants were obtained.

Breeding of Allium tuberosum via Male-sterile Clone

LI Chun-ling;JIANG Zhong-ren;TONG Xi-ran

2006, 14(2): 235-240 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

A single plant of Allium tuberosum without pollen was discovered in the field and proved to be a male-sterile plant with female fertility. Tissue culture technique was used to multiply the male-sterile plant forming male-sterile clones which were used as female parent crossing with other lines. From the F1 plants, new strain J-54 and new cultivar Haijiu 1 have been bred. The male-sterile clones could be kept in test tube for a long time. New types of male-sterile plants were also found in self-crossed plants of 3 F1 hybrids and 2 cultivars and 5 new male-sterile clones have been established from them by tissue culture.

RAPD Analysis of Genetic Diversity among Zingiber officinale Cultivars

GAO De-min;LIU Zhen-wei;FAN Shou-jin

2006, 14(2): 245-249 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The genetic diversity of 20 Zingiber officinale cultivars was studied by RAPD. Twenty five primers were screened from over 200 arbitrarily primers, and a total of 171 bands were amplified, DNA bands were amplified from100～3 500 bp, among which 111 were polymorphics (64.91%). The average number of DNA bands produced by each primer were 6.84. The J coefficient was worked out by NTSYS-pc softwarefrom amplified bands and were clustered. Based on UPGMA, a DNA molecular dendrogram was established for 20 Z. officinale cultivars in China. When the coefficient was 0.67, these 20 cultivals were divided into four groups, GroupⅠ, Ⅱ, Ⅲ and Ⅳ. GroupⅠwas divided into section A, B and C, and GroupⅣ was divided into Section D and E. Meanwhile, Section A was divided into Subsection A1 and A2, while Section B was divided into Subsection B1 and B2. The coefficient in each subsection was very high, which suggested that the relationship among subsections was fairy close, but some genetic difference existed.

Enzyme Linked Immunosorbent Assay for PAT Protein Detection in Genetically Modifed Rape

XU Wen-tao;HUANG Kun-lun;DENG Ai-ke;LUO Yun-bo

2006, 14(2): 250-254 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The immunoassay method to detect genetically modified (GM) rape(Brassica campestris ) containing the phosphinothricin acetyltransferase (PAT) were developed and applied. The purified PAT was identified by Western blot and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both saturated ammonium sulfate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of polyclonal antibody has been demonstrated in ELISA test. The result of the ELISA for antiserum sensitivity was about 2×10-5 mg/mL and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1 %. The detections of transgenic plants were evaluated with two transgenic rapes (MS1/RF1 and MS8/RF3) and the transgenic rapes (MS1/RF1 and MS8/RF3) could be easily distinguished by ELISA.

Analysis and Sequencing of the Capsid Protein Gene of Rabbit hemorrhagic disease virus (RHDV) JX/97 Strain

LIU Guang-qing;NI Zheng;ZHANG Yu-ying;YUN Tao;LIANG Hua-li;HUA Jiong-gang; LI Shuang-mao

2006, 14(2): 191-196 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The capsid protein gene of Rabbit hemorrhagic disease virus (RHDV) JX/97 strain was cloned and sequenced .The results showed that the nucleotide sequence of the gene was 1 739 nt in length encoding a protein of 580 aa. Variation analysis of the capsid protein gene of JX/97 strains and other 21 published RHDV isolates showed that RHDV had a high degree of conservation, the homology of the amino acids among different isolates exceeded 90%. The results of computer analysis showed that the antigenic region hadn’t changed remarkably in past 20 years. The results of phylogenetic tree analysis showed that all the reference RHDV isolates could be divided into 4 genetic groups and the distant between them appeared to have evolved to different extents, that means during adapting to the special environment, RHDV may form its only biology characteristic gradually.

Sequence Analysis of the σC Gene of Muscovy duck reovirus ZJ99 Strain and Its Expression in Escherichia coli

YU Xu-ping;HE Shi-cheng;ZHU Jun-li;JIN Jun-jie;ZHENG Xin-tian

2006, 14(2): 197-202 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The σC gene (GenBank: AY619690) of Muscovy duck reovirus (mDRV) ZJ99 strain was amplified and sequenced with RT-PCR. BLASTn analysis results showed that the σC gene of mDRV ZJ99 had 98% homology to mDRV MW9710 isolated in Fujian Province of China, 92% and 93% homology to mDRV 89026 and mDRV 89330 reported in France. BLASTp analysis showed that the deduced amino acid sequence of ZJ99 had 94%, 89%, 89% identities and 95％, 92％, 93％ similarities to that of MW9710, 89026 and 89330, respectively. The σC gene was further subcloned into pET-28a and transformed into Escherichia coli BL21(DE3). SDS-PAGE analysis showed that the σC protein was successfully expressed in E. coli, which existed as inclusion body and accumulated up to 22.9% of total cellular proteins.

Development of Diagnostic Gene Chip for Detecting Five Kinds of Virus in Horses

ZHU Lai-hua;LIANG Cheng-zhu;LU Cheng-ping;WU Hua;YANG Yuan-jie;MA Feng-zhong;WANG Shu-feng;YU Hong-guang

2006, 14(2): 203-207 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The highly conserved DNAs of Equine herpesvirus 1(EHV1), Equine arteritis virus(EAV), Equine influenza virus(EIV), Equine infectious anaemia virus (EIAV) and Eastern equine encephalomyelitis virus (EEEV) were acquired by molecular cloning, and then spotted on the treated glass slides as the diagnostic gene-chip. And the cDNAs reverse-transcripted from RNAs of samples were labeled with Cy5 fluorescence as probes. Following specific hybridization of deposited gene chip and labeled probes, fluorescence signals were scanned by laser scanner and the obtained image was analyzed by QiamtArray software with the digital computer. The results showed that the prepared gene chip could detect and distinguish the five equine viruses. And its sensitivity was about 25 copies of viral genomes. The hybridization specificity was confirmed by the presence of red fluorescence signals on the corresponding sites with samples from the five relevant viruses in horses and by the absence of positive signals with the specimens from irrelevant viruses in other animals. Peripheral blood leucocyte (PBL) from some seropositive horses of post-arrival quarantine was negative by virus isolation but positive for EHV1 and EAV by gene chip. The evidence suggests that gene chip, which is quick, specific, sensitive, and reliable, can provide a practical alternative to screen and quarantine a large number of samples within a very short period of time.

Effect of 60Co-γ Rays on In vitro Mutagenesis of Chrysanthemum morifolium Ramat

WANG Jing;LIU Lu-xiang;ZHAO Shi-rong;GUO Hui-jun;ZHAO Lin-shu;CHEN Wen-hua

2006, 14(2): 241-244 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The microshoots of in vitro-cultured Chrysanthemum morifolium cv. Qiuzhishan were irradiated with 5~30 Gy 60Co-γ rays. After irradiation the microcuttings were transferred to root-inducing medium and subculture medium respectively to study the inductive effects of γ rays on rooting, shoot growth and multiplication on media. The plant growth and flower characters were investigated after the regenerated plantlets were transplanted out of the protected aseptic environment. The results showed that 20 Gy was the lethal dose of 60Co-γ rays to irradiate the microshoots of in vitro -cultured Chrysanthemum morifolium cv. Qiuzhishan, while 10 Gy was the optimum dose to induce mutations. After bloom, 8.2% of the plants in the treatment of 10 Gy were selected for their flower color mutations. In contrast with the bright yellow flowers of the control, different shades of red color were observed in those mutant flowers. All the mutant plants were homogeneous and stable mutants.

Tissue Specificity of Somatostatin(SS) mRNA Expression
in Gastrointestinal Tracts of Chicken

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

2006, 14(2): 170-173 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The objective of this study is to investigate the the tissue specificity of somatostatin (SS ) mRNA expression in gastrointestinal tracts by relative quantitative RT-PCR . Arbor Acre(AA) broilers at age of 30 days were used and the gastrointestinal tracts were sampled. The result showed that SS mRNA expression in glandular stomach was higher than that in intestine segment(P＜0.01)；Duodenum and jejunum expressed SS mRNA similarly and their mRNA abundance was higher than that of ileum(P＜0.05); Qualitative analysis showed that muscular stomach and colorectum could express SS mRNA, but the abundance was significantly lower than that of glandular stomach, duodenum, jejuneum and ileum. The abundant expression of SS mRNA in digestion organ (glandular stomach) and absorption organ (duodenum and proximal jejunum) indicate that SS has important function on regulating the digestion and absorption of nutrients, but the mechanism needs further research.

Cloning of Lipoprotein Lipase(LPL)Gene of Swine and the Difference of LPL Gene Expression at Different Avoirdupois Stages

SHAN Ti-zhong;WANG Yi-zhen;LI Min

2006, 14(2): 151-155 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Genome RNA was extracted from mesentery adipose of pig (Duroc Landrace Yorkshire) and lipoprotein lipase (LPL) mRNA was amplified using RT-PCR. A DNA fragment about 689 bp in length was obtained and the PCR product was cloned into pGEM-T vector. The LPL gene was isolated and sequenced from the screened positive clones. Result of sequence analysis showed that this fragment was the partial sequence of LPL cDNA and coded 279 amino acid residues. The gene homology of the obtained fragment compared with that of reported LPL gene sequence in adipose of porcine was up to 98%, and amino acid homology was 98.94%. Based on the LPL gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using β-actin as inner control, the difference of LPL gene expression at different avoirdupois of swine was researched, which increased from new born to 30 kg, decreased from 30 to 50 kg and rose again at the stage of 50 to 90 kg.

Analysis of Calpastatin Gene Polymorphism and Its Associations with Pork Quality and Backfat in Five Pig Populations

SUN Li-bin;MENG He;LI JING;LI Chang-long;BAI Chun-yan;PAN Yu-chun

2006, 14(2): 156-161 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Calpastatin (CAST) is the endogenous inhibitor of calpain system, which has been found to affect the postmortem tenderization of skeletal muscle. Using PCR-RFLP, the polymorphism of CAST gene in five pig populations (total of 110 pigs), were analysed, and the relationship between the CAST genotypes and pork quality and backfat was evaluated. The results showed that there were 4 polymorphism sites (A, B, C and D) in CAST gene which presented a significant differences in the five populations. In A and B sites, the genotype effects were significant on pork tenderness only(P<0.05); the gene additive effects were significant (P <0.05) but the dominant effects were not. In C site, the genotype effects were not significant on all the traits analysed(P >0.05). In D site, the genotype effects were significant on backfat only(P <0.05); the gene dominant effects were significant(P <0.05), but the additive effects were not. It is concluded that CAST gene can be used as a candidate gene for pork quality and carcass quality traits.

Effects of 5 Microsatellite DNA Markers Linked with PPAR Gene on Meat Quality Traits in Pig

SHAO Gen-bao;JIA Chao;LIU Hong-lin;ZHAO Ru-qian;DING Hong-mei;CHEN Jie; HUANG Rui-hua;XU Yin-xue

2006, 14(2): 162-165 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The 5 microsatellite DNA loci linked with PPAR (peroxisome proliferator activated receptor) gene were selected from the published genetic map of chromosome 13 in pig, and polymorphisms of these microsatellites in 100 samples from Sutai pigs (Duroc × Erhualian) were detected. The results revealed that the number of alleles were 6~9, heterozygosity 0.59~0.81, polymorphism information content 0.51~0.76, the correlation coefficients between back fat thickness(BFT) and tenderness, water-holding capacity(WHC), and intramuscular(IMF) were –0.358, 0.020 and 0.311, respectively, and there existed significant correlations between BFT and tenderness, IMF (P<0.01). Moreover, effects of S0021, SW937, SW482, S0222, and S0281 on meat quality traits were analyzed with PROC GLM of SAS. Results showed that the effect of SW482 on BFT was significant (P<0.05) and SW937 on WHC reached a significant level at P<0.01. S0222, S0281 and S0021 had no significant effect on the two selected meat quality traits(P>0.05).

Cloning and Sequence Analysis of Exon 2 of MTNR1A Gene in Small Tail Han Sheep

CHENG Du-xue;CHU Ming-xing;LIU Wen-zhong;FANG Li;YE Su-cheng

2006, 14(2): 166-169 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The 824 bp fragment of exon 2 of melatonin receptor 1A(MTNR1A ) gene was amplified successfully by PCR in both 10 ewes of nonseasonal estrous breed (Small Tail Han sheep) and 10 ewes of seasonal estrous breed (Dorset sheep), and then cloned into pGEM-T Easy vector. The positive clones were further identified by PCR and sequenced. The nucleotide sequence of exon 2 of MTNR1A gene in Small Tail Han sheep was the same as that published in GenBank (U14109). The differences in exon 2 of MTNR1A gene between Small Tail Han sheep and Dorset sheep consisted of five nucleotide changes (C329T, G355T, C566T, C580A and A675G), and homology of nucleotide sequence was 99.4%. Homologies of nucleotide sequence and amino acid sequence of exon 2 of MTNR1A gene between Small Tail Han sheep and goat, cow, pig, human, house mouse, Norway rat, Siberian hamster and chicken were 73.2% to 98.5% and 78.5% to 98.5%, respectively.

Stable Transformation of phaC2 Gene in Tobacco Chloroplast Genome

WANG Yu-hua;WU Zhong-yi;ZHANG Xiu-hai;HUANG Cong-lin;YANG Qing

2006, 14(2): 213-218 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHAs biosynthesis is type Ⅱ PHA synthase. The gene phaC2 encoding type Ⅱ PHA synthase was placed under the control of psbA-pro and psbA-ter of rice (Oryza sativa ) to construct phaC2 cassette, which was ligated with the screening marker gene aadA cassette (prrn-aadA-TpsbA-ter) . These recombined fragments were cloned between the plastid rbcL and accD genes for targeting to the large single copy region of chloroplast genome. Chloroplast transformation vector of pTC2 was constructed and introduced into tobacco(Nicotiana tobacum ) chloroplast genome through particle bombardment. PCR and Southern blotting analysis confirmed stable integration of phaC2 into the chloroplast genomes of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homoplasmy. The expression of phaC2 at transcription level was detected by RT-PCR. Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation of phaC2 encoding type Ⅱ PHA synthase in tobacco via chloroplast genetic engineering.

Analysis of Sequence Variability in CP Gene of Citrus tristeza virus

ZHANG Jian-kun;HONG Ni;WANG Guo-ping

2006, 14(2): 259-264 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Using total RNA as template extracted from three citrus (Citrus reticulata Blanco) samples (HN, JX-1 and JX-2) collected from Daoxian, Hunan and Chongyi, Jiangxi, CP gene of Citrus tristeza virus (CTV) was amplified by RT-PCR. The results of cloning and sequencing of amplified products demonstrated that three samples were affected with CTV. The results for single-strand conformation polymorphism (SSCP) analysis showed that genetic variation existed among the CP genes of CTV from three samples. The CP genes of CTV from HN and JX-1 contained a unique sequence type, but that from JX-2 had a population of sequence types, two of which were predominant. Sequence analysis results showed that clone HN-K had 98% nucleotide sequence identity with Portuguese isolate 28C and Israeli stem pitting isolate VT; clone JX-1-1 98%~99% with Indian isolates Pune and Bangalore; clone JX-2-7 and JX-2-17 97%~98% with Japanese seedling yellows isolate NUagA and Californian severe stem pitting isolate SY568. All four clones shared only 91%~93% identity with Floridian quick decline isolate T36, mild isolate T30 and Spain mild isolate T385. Phylogenetic tree analysis revealed that four Chinese clones appeared in three different clusters, shared high homology with seedling yellows or stem pitting isolates from different geographical origins but low homology with quick decline and mild isolates, and these suggested that there had no correlation between geographic origin of CTV and their nucleotide distances

Analysis of Sec-type Signal Peptides in the Ti and AT Plasmids of Agrobacterium tumefaciens C58 Cereon

LUO Deng-tao;FAN Ji-ying;FAN Cheng-ming;ZHAO Ming-fu;HE Yue-qiu

2006, 14(2): 265-268 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The 745 ORFs of the Ti and AT plasmids of Agrobacterium tumefaciens C58 Cereon were used for signal peptide prediction by comprehensive analyses with Signal P3.0 and PrediSi and screening based on L value and TMHMM. Total 35 sec-type signal peptides including 6 RR-motif and 2 bacteriocins-pheromones signal peptides were found and among them, 24 peptides were annotated newly as signal peptides, 1 for plasmid Ti and 23 for plasmid AT, respectively. Among the 35 signal peptides, the N-domain has 2~19aa with average 6.8aa, and the H-domain had 8~34aa with average 17.1aa.

Constitutive Expression of α-galactosidase Mel1 Gene in Pichia pastoris

ZHANGXue-wen;TANGXiang-shan;ZHANGJin-chen;ZHANGHuai-yun

2006, 14(2): 269-272 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Mel1 gene was amplified from yeast(Saccharomyces cerevisiae ) AH109 with PCR, and cloned into integrated vector pGAPZαA, constitutive and secreted expression α-galactosidase plasmid pGAPZα-Mel1 was constructed. The linearized recombinant plasmid pGAPZ α-Mel1 was transformed into Pichia pastoris KM71 by electropotation, and the blue positive colonies were screened out on YPDS plate with X-α-gal and 100 mg/mL zeocin. There was a special 53 kD band by SDS-PAGE from supernatant of yeast culture and a color band was observed by soak the native PAGE gel in X-α-gal to illustrate the α-galactosidase activity; The α-galactosidase enzyme activity was 12 U/mL after fermenting pGAPZα-Mel1 /KM71 for 6 days.

Rapid Determination of Ploidy Level of Chromosome in Tobacco(Nicotiana tabacum)

ZHU Hui-qin, ZHANG Xian-yin, XUE Qing-zhong

2006, 14(2): 255-258 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The ploidy level of 763 anthers deirived from both crosses (G28×NC2326 and K326×Coker176) of tobbaco (Nicotiana tabacum) were determined. The result of identification by counting the number of stoma chloroplast in guard cell was consistent with those observations of flower and seed fertillity of the plants, with the average accurate rate of 93.52%, showing that measurement of the number of stoma chloroplast in guard cell could be considered as a fast and accurate method to determine haploid, diploid or poly-ploid of tobacco in the seedling stage. In order to reduce the waste of haploid plants and speed up establishment DH population, it was feasible to select out undoubted haploid plants with <14 chloroplast number in stoma treated with colchicine when young plantlets just transplanted in the field.

Purification and Properties of Tibetan Sheep Lactate Dehydrogenase-A

SI Xiao-hui;ZHAO Xing-bo;ZHENG Yu-cai;WEN Yong-li;XU Yuan;JIN Su-yu;YANG Xue

2006, 14(2): 178-182 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

HiTrapTM Blue HP affinity chromatography and DEAE-Sephadex ion-exchange chromatography were used to purify lactate dehydrogenase-A (LDH-A) from skeletal muscle of a male Tibetan sheep (Ovis aries) that lived in plateau. The relative activity of purified LDH-A was 100.94 U/mg protein, with purification times of 12.0. Only one band was observed when the purified LDH-A was separated with SDS-PAGE or native PAGE. Kinetic analysis showed that Michaelis constants (Km) value for reduced nicotinamide adenine denucleotide (NADH) was 0.022, and Km value for pyruvate was 0.444, which were significantly higher than those of rabbit. The optimal pH for the reduction of pyruvate was approximately 5.8. Oxalate and Hg2+ inhibited LDH-A activity, and Hg2+ was a reversible inhibitor of LDH-A.

Isolation, Culture and Characterization of Chicken Primordial Germ Cells

TANG Xin-yan;ZENG Wei-dong;MI Yu-ling;LIU Hong-yun;ZHANG Cai-qiao

2006, 14(2): 174-177 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Primordial germ cells (PGCs) were isolated from the genital ridges of Avian chicken (Gallus domesticus) embryos at 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in primary culture and subculture. Identification of PGCs was carried out by histochemical methods including alkaline phosphatase (AKP) and periodic acid-Schiff (PAS) staining, and proliferating PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating activity of PGCs was compared under different culture conditions among 5% ~20% fetal calf serum (FCS), ITS (M199+10 μg/mL insulin+5 μg/mL transferrin + 3×10-8 mol/L selenite, conditioned medium (CM), 15%FCS+ITS, 15%FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. PGCs grew better without centrifugation than those with centrifugation. PGCs formed larger colonies in media with 5%FCS or ITS than those in other media. The above results indicate that 5%FCS or ITS-supplemented media can be a culture system for PGC proliferation in the somatic cell- PGC coculture, besides the embryonic fibroblast feed layer.

Identification of Bovine and Sheep Derived Materials in Feedstuff by Semi-nested PCR

WEN Wei-gang;CUI Jun-xia;ZHAO Xiu-ling

2006, 14(2): 183-186 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Augment identification of bovine and sheep derived materials in feedstuff is essential for effective control transmission of bovine spongiform encephalopathy and scrapie. This study described a semi-nested PCR assay for the detection of bovine and sheep materials in feedstuff. Bovine- or sheep-specific semi-nested PCR primer had been designed for this purpose based on their published species-specific gene and primer’s sequences. The results of semi-nested PCR showed that 247 and 214 bp bands were obtained from the bovine and sheep materials respectively, the detection limitation was 0.00001%～0.0001% for bovine and sheep derived materials in feedstuff and 10-6 ng～10-5 ng for bovine and sheep genomic DNA respectively, 103 and 105 times higher than that of the detection limitation of PCR. Therefore, the rapid, sensitive and stable semi-nested PCR are the effective methods for detecting trace bovine and sheep derived materials in feedstuff.

Advances in Genetic Mapping of Sorghum Genome

YI Zhi-ben;LIANG Xiao-hong;ZHAO Wei-jun;SUN Yi;YAN Min;CUI Li-xia

2006, 14(2): 279-285 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

The construction of sorghum (Sorghum bicolor L. Moench) molecular genetic linkage map started from early 1990’s. Molecular genetic maps with high-density of markers covering almost whole sorghum genome have been completed and integration of sorghum genetic and physical map is being underway. The correlation between genetic linkage groups and relevant chromosomes was established and the location of the important structures of chromosomes such as centromere, long and short arms, nucleolus organizer region( NOR) etc. have been identified on the linkage groups. With the continuous progress in the field, sequencing of full sorghum genome and studying on sorghum functional genomics will be initiated soon.

Segregation Distortion and Its Effect on Genetic Mapping in Plants

SONG Xian-liang;SUN Xue-zhen;ZHANG Tian-zhen

2006, 14(2): 286-292 | doi: | Full text (HTML) (1 KB) | PDF

PDF (708 KB) ( 612 )

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Abstract

Segregation distortion, which is identified as a strong evolutionary force, is common in genetic mapping. In plants, the percentage, degree, origin and genetic effects of segregation distortion vary significantly with species, population types, crosses and marker types. The exhibition and common features, causes, methods of mapping segregation distortion loci, effects of segregation distortion on map construction and corresponding mapping strategies in plants, mainly in crops, are reviewed.

echnique and Applications of Fusion Gene

LIU Yan;YU Lian

2006, 14(2): 273-278 | doi: | Full text (HTML) (1 KB) | PDF

PDF (541 KB) ( 672 )

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Abstract

The technique of fusion genes has been applied more extensively in the field of biology in recent years. In this paper, the technigue, impact factors and applications of fusion gene were elucidated.