Sep 2008, Volume 2 Issue 3

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  • LI Zhikun, WANG Xingfen, ZHANG Yan, ZHANG Guiyin, WU Liqiang, CHI Jina, Zhiying MA
    Seventy-one glandless cotton germplasm resources were firstly evaluated genetically by using nine agronomic traits, 33 simple sequence repeat (SSR) primers and ten amplified fragment length polymorphism (AFLP) primer combinations. Principal component analysis (PCA) of the agronomic traits showed that the first six principal components (PCs) explained a total of 86.352% of the phenotypic variation. A total of 329 alleles were amplified for 33 SSR primers, and 232 polymorphic bands in a total of 389 bands were obtained by using ten AFLP primer combinations. The average polymorphic information content (PIC) value was 0.80 and 0.18 for SSR primers and AFLP primer combinations, respectively. The DIST (average taxonomic distance) and DICE (Nei and Li’s pairwise distance) coefficients ranged from 0.373 to 3.164 and 0.786 to 0.948, respectively, for agronomic traits and SSR&AFLP data based on UPGMA analysis. This suggested that there was a higher diversity in the evaluated population for both agronomic traits and molecular markers. The Mantel’s test showed that the correlation between the dendrograms based on agronomic traits and SSR&AFLP data was non-significant.
  • CAO Yunfei, WANG Jiaojiao, GUO Li, XIAO Kai
    The acclimation of plants to cold, salt and dehydration is involved in the action of the transcription factor (CBF) cold-response pathway. In this paper, nineteen rice CBF genes, including seven previously released and twelve unpublished novels, were identified and characterized. The multi-members of rice CBFs (OsCBF1 to OsCBF12) were divergent at the nucleotide and amino acid level. Expression analysis shows that five novel rice CBF genes (OsCBF1, OsCBF2, OsCBF3, OsCBF8, and OsCBF9) responded to short-term (1 h or 3 h) stresses of low temperature, salt stress and dehydration. The transcripts of OsCBF2, OsCBF8 and OsCBF9 in the roots were rapidly elevated when the plants were exposed to low temperatures, suggesting that they were possibly involved in low temperature responses in rice plants. Meanwhile, the expression level of OsCBF2 in leaves was enhanced when exposed to salt stress of 1–3 h, implying that OsCBF2 functioned as a transduction component in the salt stress signal cascade. Various expression patterns in OsCBF1, OsCBF2, OsCBF3, OsCBF8, and OsCBF9 under low temperature, salt and drought conditions, together with the different expression patterns between roots and leaves for each of these indicated that every rice CBF gene has unique and non-redundant functions in the response to the abiotic stresses.
  • BI Ruiming, WANG Honggang
    Using mature embryos (MEs) as the explants, the callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency in diploid and tetraploid wheat of four genotypes were studied. The tested four genotypes wheat included cultivable emmer wheat (Triticum dicoccum Schuble), durum wheat (Triticum durum Desf.) and the common wheat progenitors Triticum dicoccoides and Triticum aegilopides. Results indicated that there were significant differences in the efficiency of callus induction, callus differentiation and plant regeneration among the tested genotypes. The efficiency of differentiation and regeneration shows strong genotype dependence. The rates of callus induction, embryogenic callus differentiation, plantlet regeneration and culture efficiency respectively were 95.00%, 90.00%, 32.40%, and 27.70% in cultivable emmer wheat, which were significantly higher than other tested genotypes. Therefore, this study has provided a basis for genetic transformation, gene cloning and molecular plant breeding in wheat and other related species.
  • DU Xiong, BIAN Xiuju, YANG Fucun, ZHANG Lifeng, ZHANG Weihong
    To counter the actual problems of forage shortage and low quality existing in the agriculture–animal husbandry ecotone in North China, a research was conducted to study the effects of plastic-film mulching and nitrogen application on the production of forage-oriented maize with the aim of producing water-saving forage with high-yield and good quality. Field experiments combined with laboratory experimental estimation and analysis was adopted. Plastic-film mulching increased the dry biomass of forage-oriented maize by 23.8% with effectively improving the maize’s nitrogen absorption so that the apparent utilization ratio and output-input ratio of nitrogen were enhanced. The content of crude protein in maize plant was increased and thus, forage nutritive quality was improved. Plastic-film mulching remodeled the maize field water consumption scheduling pattern and increased the water use efficiency by over 10%. Nitrogen application to forage-oriented maize co-improved the biomass and the nutritive quality with the nutritive matter (percentage and yield) several times of the biomass. Nitrogen application increased maize biomass production by 36.1%–39.5% and it increased the contents of crude protein and crude fat in maize plant by 109% and 145%, respectively. The yields of the two nutritive matters increased by 160% and 210%. Nitrogen application at the rate of about 200 kg·hm-2 to the uncovered field and the rate less than 300 kg·hm-2 to the field with film mulching were considered as the most proper rates to guarantee high yield and good quality of forage-oriented maize and were the rates to keep the available nitrogen balanced in the soil. Plastic-film mulching and nitrogen fertilizer application to forage-oriented maize was an effective way of producing forage with high yield and good quality, relieving the shortage of animal forage and accelerating ecological recovery and economic development in this ecotone in North China.
  • WANG Xiaoying, HE Mingrong, LI Fei, LIU Yonghuan, ZHANG Honghua, LIU Chungang
    Effects of irrigation and nitrogen fertilization on the grain yield, protein composition, protein quality, starch composition and starch pasting properties of a strong-gluten winter wheat were investigated in a high fertility field. Compared with non-irrigation treatment, grain yields under irrigation treatments were significantly increased, but the content of grain protein, monomeric protein and flour wet gluten was reduced. There were no significant differences in the above parameters between the irrigation treatments. Nitrogen application could significantly increase grain yield under low irrigation frequency (W0 and W1), while the neglected effect on yield was observed with high irrigation frequency (W2 and W3). With the increase of irrigation frequency, the glutenin content leveled off, but the changes of glutenin composition were not uniform, in which the soluble glutenin content was increased, while the insoluble glutenin content and polymerization index (the ratio of insoluble glutenin to total glutenin) were reduced. Both dough development time and stability time became shorter with the increased irrigation frequency. Nitrogen application improved the content of all grain protein fractions and grain quality, in which the increased degree in non-gluten protein (albumin and globulin) was higher than gluten protein (gliadin and glutenin), and the increased degree in soluble glutenin was found higher than that of insoluble glutenin. The interactive effects of irrigation and nitrogen on starch composition were significant. Starch content and amylopectin content was increased as irrigation frequency added in non-nitrogen treatment. Compared to non-irrigation treatment, irrigation significantly increased the starch content and the amylopectin content in nitrogen application treatment, but the starch and amylopectin content had no significant difference between irrigation treatments. Amylose content and the ratio of amylose to amylopectin were reduced while RVA indexes (peak viscosity, breakdown, final viscosity and setback) were increased as irrigation frequency was increased. Nitrogen application significantly improved the amylopectin content and decreased the amylose content in lower frequency irrigation, while the amylopectin content was decreased and the amylose content was enhanced by nitrogen application in higher frequency irrigations.
  • MEN Mingxin, XU Hao, YU Zhenrong
    The objective of this article was to study the spatial distribution pattern of rainfall erosivity. The precipitation data at each climatological station in Hebei Province, China were collected and analyzed and modeled with SPSS and ArcGIS. A simple model of estimating rainfall erosivity was developed based on the weather station data. Also, the annual average rainfall erosivity was calculated with this model. The predicted errors, statistical feature values and prediction maps obtained by using different interpolation methods were compared. The result indicated that second-order ordinary Kriging method performed better than both zero and first-order ordinary Kriging methods. Within the method of second-order trend, Gaussian semi-variogram model performed better than other interpolation methods with the spherical or exponential models. Applying geostatistics to study rainfall erosivity spatial pattern will help to accurately and quantitatively evaluate soil erosion risk. Our research also provides digital maps that can assist in policy making in the regional soil and water conservation planning and management strategies.
  • WANG Lianfeng, SUN Xin, CAI Yanjiang, XIE Hongtu, ZHANG Xudong
    Freeze-thaw event often occurs in regions at mid-high latitude and high altitude. This event can affect soil physical and biological properties, such as soil water status, aggregate stability, and microbial biomass and community structure. Under its effects, the bio-indicators of soil microbes including the kinds and quantities of some specific amino sugars may vary, and the process and intensity of soil nitrogen transformation may change, which can result in an increase in nitrous oxide (N2O) production and emission, making the soil as the major source of N2O emission. This paper summarizes the research progress on the aspects mentioned above, and suggests further research directions on the theoretical problems of soil N2O production and emission under the effects of freeze-thaw event.
  • LI Juan, ZHOU Jianmin
    A growth chamber experiment was conducted to investigate the influence of NH4+/NO3- ratio and elevated CO2 concentration on the pH in nutrient solution, growth and root vigor system of tomato seedling roots, which attempts to understand whether the elevated CO2 concentration can alleviate the harmful effects of higher NH4+–N concentration in nutrient solutions on the tomato root system. Tomato (Lycopersicon esculentum Mill. var. Hezuo 906) was grown in pots with nutrient solutions varying in NH4+/NO3- ratio (0:1, 1:3, 1:1, 3:1 and 1:0) and the growth chambers were supplied with ambient (360 ?L·L-1) or elevated CO2 concentration (720 ?L·L-1). The results showed that the pH changed with the growth process and CO2 concentration increased. At both CO2 levels, pH increased when 100% NO3-–N was supplied and decreased in other treatments. The pH decrease in the nutrient solution was directly correlated to the NH4+-N proportion. The pH value was more reduced in 100% NH4+–N nutrient solution than increased in the 100% NO3-–N nutrient solution. CO2 enrichment increased the dry weight of shoots and roots, root vigor system, total absorbing area and active absorbing area of tomato seedlings. All the measurement indexes above were increased in the elevated CO2 concentration treatment with the NO3- proportion increase in the nutrient solutions. Thus, under the elevated CO2 concentration, the dry weights of shoots and roots, root vigor system, total root absorbing area and active absorbing area were found to be inversely correlated to NH4+/NO3- ratio, leading to about 65.8%, 78.0%, 18.9%, 12.9% and 18.9% increase, respectively, compared with that under the ambient CO2 concentration. Our results indicated that tomato seedling roots may benefit mostly from CO2 enrichment when 100% NO3-–N nutrient solutions was supplied, but the CO2 concentration elevation did not alleviate the harmful effects when 100% NH4+–N was supplied.
  • LIAO Wenhua, LIU Jianling, Wang Xinjun, JIA Ke, MENG Na
    The yield response of Chinese cabbage to phosphate fertilizer and manure was studied. The effect of over-application of phosphate fertilizer and manure on plant total phosphorus content and phosphorus accumulation in soil was also investigated. The experiment was arranged in a plastic barrel in the field for two years. Application of phosphate fertilizer at the rates of 150–600 mg·kg-1 gave a yield increase of 14.9%–21.5% of Chinese cabbage. Application of manure at the rates of 33.3–133.2 g·kg-1 gave a yield increase of 18.2%–25.9% of the crop. There was no significant difference of yield response at the rates of 150, 300 and 600 mg·kg-1 phosphate fertilizer, and no significant yield response to the application of phosphate fertilizer after applying manure. The total P content in Chinese cabbage was increased gradually with the rate increase of phosphate fertilizer and manure. Phosphorus was absorbed luxuriously by the plant with over-application phosphate fertilizer and manure. The content of total-P, Olsen-P, water-soluble P, biological available P in the soil was increased with the rate of phosphate fertilizer and manure. Organic phosphorus in the soil was increased by the application of manure. Olsen-P had high correlations with water-soluble-P and biological available-P, but there was a poor relationship between Olsen-P and organic-P.
  • YAO Yuxin, HAO Yujin, LI Ming, PANG Mingli, LIU Zhi, ZHAI Heng
    Malate dehydrogenase (MDH) ubiquitously exists in animals, plants and microoganisms, and catalyzes the interconversion from oxaloacetate to malate. Cytosolic NAD-dependent MDH gene (cyMDH) encodes a key enzyme crucial for malic acid synthesis in the cytosol which has not been extensively characterized in plants. In this study, a full-length cDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3′ and 5′ rapid amplification of cDNA ends, and designated as Mal-cyMDH (GenBank accession No. DQ221207). It contained a 996-bp ORF and its sequence analysis shows a high similarity to other plant cyMDHs. Phylogenetic analysis indicated that almost all the cyMDHs could be clustered into the same group and it was likely to represent the original MDH. A roughly 37-kDa fused protein was obtained by the recombinant prokaryotic expression and its enzyme activity assay showed that it mainly catalyzed oxaloacetate to malate. It was also discovered that the enzyme activity of cyMDH exhibited remarkable difference between the high- and low-acid apple germplasm.
  • LAN Tianying, LIU Bo, DONG Fengping, CHEN Ruiyang, LI Xiulan, CHEN Chengbin
    In this study, multicolor fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes of spinach with biotin-labeled 25S rDNA, DIG-labeled telomere sequences and biotin-labeled and DIG-labeled 5S rDNA was performed. There were six 25S rDNA loci located on the satellites of the third, the fifth and the sixth chromosomes, and four 5S rDNA loci located on the long arms of the third and the fifth chromosomes. The telomere loci were located on the end of the sixth chromosome and also on both the end and centromeric regions of other chromosomes. This study is an important complement to both traditional karyotype analysis and FISH karyotype analysis in spinach.
  • YANG Cuiyun, DAI Guanghui, WANG Zhezhi, WANG Na, MA Yajun
    To determine the pathogens of cucumber downy mildew and cucumber powdery mildew by molecular marker, we amplified and sequenced the rDNA-ITS region of the pathogens of cucumber downy mildew and cucumber powdery mildew collected from the Shanghai region. The intra-/interspecific sequence difference was analyzed by rDNA-ITS sequence. The results show that the length of rDNA-ITS1 and rDNA-ITS2 of cucumber downy mildew’s pathogen was 141 bp and 406 bp, respectively, with GC contents of 41.13% in ITS1 and 46.8% (Minhang and Jinshan District, sm1 and sm2) or 46.55% (Pudong District, sm3) in ITS2. The rDNA-ITS sequence was intraspecific conservation. The interspecific difference was related with their kin relationship. The pathogen of cucumber downy mildew was identified as Pseudoperonospora cubensis by molecular marker. The length of rDNA-ITS1 and rDNA-ITS2 of cucumber powdery mildew’s pathogen was 136 bp and 89 bp, respectively, with GC contents being 59.56% and 66.29%, and rDNA-ITS sequence being highly conservative in this study that was the same as Sphaerotheca cucurbitae. But the sequence difference between the strains in the Shanghai region in this study with S. fuliginea was 4.5%, which was identified by morphology. It is suggested that the pathogen of cucumber powdery mildew should be further clarified and determined.
  • POUDYAL Bharat Kumar, DU Guoqiang, ZHANG Yuxing, LIU Jie, SHI Qingchun
    Yali is one of the best pear cultivars cultivated extensively in China and other countries. However, mortality rates of explants during the initial phase of tissue culture were found to be very high during the summer particularly from the shoot tip explants. A thorough investigation on the browning problem of Yali Aikansui and Abbe Fetel pears was done and their control measures are suggested in this paper. Emphasis has been given to the Yali pear. Shoot explants were collected from the field as well as from the plantlets grown in vitro during different months and observed for browning. The explants were excised and cultured in Murashige and Skoog (MS) solid and liquid medium as needed. The cultures were maintained in the dark as well as in a 16/8 hours light/dark photoperiod regime as needed and were kept in a growth room at (25 ± 2)°C temperature. According to the experimental results, Yali was found more severely infected by browning than the other two cultivars. Similarly, it was also found that, the intensity of browning was less during spring and increased with time and reached the maximum during summer. Shoot tips of Yali were found more severely infected by browning than the second node and other nodes. This accelerated the mortality rate up to 81% of shoot explants during the summer months of July and August. Browning was greatly influenced by water-soluble polyphenols, more of which were found in the shoot tip of explants collected from the field (significantly higher at P ≤ 0.05 and P ≤ 0.01). Total polyphenol contents were found less in the explants from plantlets grown in vitro, and less browning of medium appeared resulting in a lower mortality rate of explants. Collections of explants during early spring (P ≤ 0.05 and P ≤ 0.01) and use of other nodes rather shoot tip because explants (P ≤ 0.05 and P ≤ 0.01) were found to be better in preventing the browning problem. Finally, for curative measures to control browning, use of ascorbic acid at the rate of 100 mg·L-1 (P ≤ 0.05 and P ≤ 0.01), 0.02% polyvinyl pyrrolidone (PVP) in the culture medium (P ≤ 0.05), 96 hours dark treatment of other nodes (P ≤ 0.05), and 12 hours cold treatments of explants at 4°C (P ≤ 0.05 and P ≤ 0.01) prior to sterilization of explants, were found to be the best methods to control browning and therefore to increase the survival rate of cultured explants of the Yali pear.
  • TANG Zhonglin, LI Kui, PENG Zhongzhen, LIU Bang, FAN Bin, ZHAO Shuhong, LI Xiaoping, XU Sanping
    The effects of breed, sex and birth parity on the traits of growth, carcass and meat quality were investigated in three different breeds including Swedish Landrace, British Large White and an indigenous Chinese breed – Tongcheng pigs. The results indicated that the Swedish Landrace and the British Large White had advantages over the Tongcheng pigs in growth rate, feed conversion rate (FCR) and carcass traits, but had disadvantages in meat quality. In contrast to the British Large White, the Swedish Landrace had higher average daily gain during the trial (ADG2) (877.04 gday-1 vs. 813.95 gday-1, P < 0.05), and marbling score (MS) (2.48 vs. 2.02, P < 0.01), larger eye muscle area (EMA) (41.80 cm2 vs. 35.14 cm, P < 0.01) and more efficient feed conversion rate (FCR) (3.06 vs. 3.29, P < 0.05), lower muscle shear force (MSF) (4.20 kgf (1 kgf = 9.80665 N) vs. 4.93 kgf, P < 0.05) and backfat thickness (BF) (all P < 0.01) including live backfat thickness (LBF), average backfat thickness at 3 points in the carcass (ABF), backfat thickness between 6th and 7th ribs (6–7 th BF) and backfat thickness at 10th rib (10 th BF). There was a difference (P < 0.05) only in MS between castrated females (CF) and castrated males (CM). Birth parity affected ADG2 (P < 0.05), some carcass characteristics and meat quality, including CL1 (P < 0.001), CL2 (P < 0.05), BF (ABF, 6–7th BF and 10th BF, all P < 0.001), EMD (P < 0.001), EMA (P < 0.001), percentage of leaf and caul fat (PLC) (P < 0.05), proportion of lean and bone of the ham (PLBH) (P < 0.05), muscle drip loss percentage (DL) (P < 0.05) and intramuscular fat content (IMF) (P < 0.05). The breed-sex interaction only impacted the average daily gain from birth to marketing (ADG1) and DL (both P < 0.05). However, there was no significant effect of sex-parity interaction on all the traits tested.
  • GENG Yan, YANG Zhangping, CHANG Hong, MAO Yongjiang, SUN Wei, GUO Xiaoya, QU Dongyan
    The level of genetic differentiation, gene flow and the relationship between geographical distance and genetic differentiation among six sheep populations of Mongolian group in China (Tong sheep, small-tailed Han sheep, Hu sheep, Tan sheep, Ujumuqin sheep and Bayinbuluk sheep) were analyzed using seven microsatellites. The trees were constructed from diversity coefficient (DC) distances among the six sheep populations. The overall heterozygote deficit across all the populations (Fit) was between 0.167 (OarAE101) and 0.044 (MAF33). The overall significant deficit of heterozygote, because of inbreeding within breeds, (Fis) was between 0.089 (OarFCB304) and 0.005 (MAF33). The coefficient of genetic differentiation (Fst) was between 0.100 (OarAE101) and 0.022 (OarFCB48). It indicated that 3.9% of the total genetic variation could be explained by breed differences and the remaining 96.1% by differences among individuals for each population. This illustrated that most variations existed within breeds and genetic differentiation level were very low among sheep breeds of the Mongolian Group in China. The average number of effective migrants exchanged per generation (Nem) ranged from 2.7369 (Tan sheep and Bayinbuluk sheep) to 44.3928 (Tong sheep and Hu sheep), and the mean value was 11.25213. Significantly positive relationships between the level of genetic differentiation and geographical distance and genetic distances were detected. It is concluded that genetic differentiation of sheep breeds of Mongolian group in China is mainly the result of natural selection (different living conditions).
  • CHENG Zhiping, CHENG Anchun, WANG Mingshu, CHEN Bin, LIU Chuang, DUAN Kun, ZHOU Xue, CHEN Xiaoyue
    In order to study the effect of cell mediated immunity regulation of duck IFN-? eukaryon expression plasmid (pcDNA-SDIFN-?) on duck plague virus (DPV) attenuated vaccine in ducks, pcDNA-SDIFN-? was administered to 28-day-old ducks at doses of 1, 3 and 6 ?g per duck, respectively, by gene-gun. PBS and empty vector pcDNA were used as control. Fifteen days later, all ducks were injected with DPV attenuated vaccine and blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63 and 84 days after injection. T-lymphocyte proliferation tests (MTT) were used to detect the T-lymphocyte proliferation in the peripheral blood (PBL) of ducks. Blood samples collected at 7, 14, 21, 28, 35 and 49 days after injection were detected by fluorescence-activated cell sorter (FACS) for recording the number of CD3+ T-lymphocytes of ducks. Results were as follows: (1) Reaction of T-lymphocytes in PBL to ConA (OD value) of ducks treated with pcDNA-SDIFN-? was higher than that of PBS and pcDNA control groups in 3–84 days. There were highly significant differences between the 1 ?g per duck group and the two control groups in 3–84 days (P ≤ 0.01), between the 3 ?g per duck group and the two control groups in 3–84 days (P ≤ 0.01, P ≤ 0.05), and between the 6 ?g per duck group and the two control groups in 7–49 days (P ≤ 0.01, P ≤ 0.05). The significant difference was also present between the groups of 1, 3 and 6 ?g per duck in 3–35 days (P ≤ 0.05). However, there was no significant difference between the 3 and 6 ?g per duck groups (P ≥ 0.05). The pcDNA control group was higher than PBS control group, but no difference was detected (P ≥ 0.05). (2) Change of the number of CD3+ T-lymphocytes in ducks administered with different doses of pcDNA-SDIFN-? was higher than that of PBS and pcDNA control groups in 7–49 days. The change in the 1 ?g per duck group was significantly higher than that in PBS and pcDNA control groups in 14–49 days (P ≤ 0.01). There were significant differences between the 3 ?g per duck group and the two control groups in 21–49 days (P ≤ 0.01, P ≤ 0.05) and between the 6 ?g per duck group and the two control groups in 7–49 days (P ≤ 0.01, P ≤ 0.05). However, no significant differences among the groups of 1, 3, and 6 ?g per duck groups (P ≥ 0.05) and between the two control groups (P ≥ 0.05) were found. The results indicated that pcDNA-SDIFN-? administered 15 days before injection of DPV-attenuated vaccine could significantly enhance cellular immunity induced by DPV-attenuated vaccine. pcDNA-SDIFN-? is an excellent DPV-attenuated vaccine molecular adjuvant and the best result can be obtained with the dose of 1 ?g per duck of pcDNA-SDIFN-? inoculated by gene-gun.
  • ZHU Li, LI Xuewei, SHUAI Surong, LI Mingzhou, LI Fangqiong, CHEN Lei
    Restriction fragment length polymorphism (RFLP) data was applied to analyze the distribution of the MyoD gene in 10 pig breeds and pig breed crosses. The population genetic information about genetic distribution, variation, and heterozygosity of the MyoD gene in different breed populations were analyzed. Based on the allele frequency, genetic distance and evolution distance among each breed populations were calculated and Unweighted Pair Group Method with Arithmetic mean (UPGMA) phylogenetic tree was gained based on the evolution distances between populations. The results indicated that the distribution of the MyoD genotype kept in Hardy-Weinberg equilibrium in most tested groups but not in Duroc (D) and Duroc × (Landrance × Yorkshire) (DLY) population. Generally, the genetic diversity of the MyoD gene was abundant and these tested breed populations had high genetic variations. The evolution of the MyoD gene was under natural selection pressure. On the phylogenetic tree, 10 pig breeds were divided into 4 clusters. The first cluster consisted of four breeds developed from Landrace. The second cluster was two indigenous Chinese pig breeds. The third cluster was three breeds developed from Duroc. The fourth cluster was a Tibetan pig breed. The constitution of the topology of the phylogenetic tree was consistent with the breeding history of each pig breed. From this experiment, we can conclude that some RFLP data obtained from functional gene can be used in the genetic deviation research between some closely related species or between different populations in certain species.
  • GAO Fengshan, WANG Lei, LI Xinsheng, LI Yungang, FANG Qinmei, HAO Huifang, XIA Chun, WANG Huifei
    In order to investigate the prompt conformations of swine major histocompatibility complex (MHC), a swine MHC-II protein complex (SLA-II) was reconstructed in vitro. DRA and DRB were cloned from a crossbred commercial pig (Changbai-Dalan). Subcloned extracellular parts of DRA and DRB were linked together by a linker containing rich glycine/serine (G4S)3, and the whole length of two genes, named DRA-linker-DRB, was amplified by splicing overlap extension PCR (SOE PCR). DRA-linker-DRB was inserted into pMAL-p2X prokaryotic system and expressed. The expressed fusion protein MBP-DRA-(G4S)3-DRB was identified by Western-blot, purified and cleaved to obtain the protein of interest DRA-(G4S)3-DRB. The secondary structure of this protein was determined in circular dichroism (CD) apparatus. The results indicated that the subsequent protein MBP-DRA-(G4S)3-DRB was soluble and its molecule weight was 83.4 ku in consistent with the Western-blot. Cleaved by Factor Xa, the protein of interest was separated with a molecular weight of 40.9 ku. The CD spectrum demonstrated that the protein displayed a favorable ?-Helix structure, and the contents of ?-Helix, ?-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The identical ratios of ?-Helix, ?-sheet, turn, and random coil between MBP-DRA-(G4S)3-DRB and DRA-(G4S)3-DRB were 95%, 96.7%, 91.1% and 93.0%, respectively. The results also suggested that the reconstructed SLA-II complex presented an ideal conformation and can be used for studying its structure and function in vitro.
  • ZHANG Xiaohui, XU Shangzhong, GAO Xue, ZHANG Lupei, REN Hongyan, CHEN Jinbao
    The advantages and disadvantages of asymmetric PCR-SSCP and the traditional PCR-SSCP were compared in this study. The mutations in 3′UTR of Smad4 gene of Luxi cattle and the Holstein cow were analyzed by asymmetric PCR-SSCP and one insert “T” mutation and one G/A mutation in this region were found. The G/A mutation created a HhaI restriction enzyme digestion position and the frequencies studied by asymmetric PCR-SSCP and HhaI-RFLP in 116 Luxi cattle and 75 Holstein cows were all the same. The asymmetric PCR-SSCP had fewer, clearer and more stabile bands than traditional PCR-SSCP. This indicates that the asymmetric PCR-SSCP is suited for mutation detection.