Quick detection of sex determining region protein gene in mouse fetuses

AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.     

Gene Flow Analysis of Phytophthora porri Reveals a New Species: Phytophthora brassicae Sp. Nov.

Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2 ⁸³, Ldh-2 ¹⁰⁴, Idh-1 ¹⁰⁸, Idh-1 ¹¹², and Idh-2 ⁹⁸), whereas six different alleles were observed at four isozyme loci (Ldh-2 ⁸⁵, Ldh-2 ¹⁰⁰, Ldh-2 ¹¹⁴, Idh-1 ¹⁰⁰, Idh-2 ¹⁰⁰, and Mdh-2 ¹¹¹) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow.     

苏云金芽孢杆菌cry基因在大肠杆菌中表达产物和生物活性

研究了几种Bt cry基因于大肠杆菌(Escherichia coli)中表达产物在pH 10.0的50mmol/L碳酸钠和20mmol/L乙醇胺溶解液中的溶解性 ,发现同样的Cry蛋白在碳酸钠中的溶解度大于乙醇胺。通过胰蛋白酶消化 ,明确Cry1Ca7、Cry1Ia8酶解产物为 38kD多肽 ;Cry1Ie1、Cry1Cb2、Cry2Ab4酶解产物为 41kD多肽 ;Cry1Ac酶解产物为60kD多肽。采用FPLC层析方法对 6种原毒素及其酶解后得到的毒素多肽进行了分离纯化 ,比较了原毒素和毒素的杀虫活性的差异。其结果表明 ,Cry1Ac的原毒素和毒素对棉铃虫初孵幼虫的校正死亡率均为 100% ,Cry2Ab4的原毒素的毒力高于其酶解毒素。     

东北西部沙地近20年地下水变化动态及其成因分析

本文以奈曼旗为例，分析研究了我国东北西部沙地1979-1997年地下水位时空变化规律及其与降水量和沙漠化的关系。结果表明：（1）沙地多年平均地下水位为2.19m，其中农区为2.85m，牧区1.16m;(2)地下水位是1981-1983年最低为2.49-2.56m,1991-1992年最高为1.90-1.92m，1997年为2.22m，高于1979年的2.41m；（3）该区季节最高水位主要发生于春季（3-5月），发生频率占50.1% ,最低水位主要发生于冬季（12-2月），发生频率为41.2%，平均4月最高为2.07m，10月最低为2.23m;(4)地下水位的年际变化动态和降水量密切相关，但有滞后1-2年的现象；（5）地下水位空间分布上的差异与土地沙漠化及土地利用方式密切相关，沙漠化土地主要用于放牧，由于人为利用少和蒸散少，有利于地下水抬升；（6）由于耕地面积占总土地面积比例低，水浇地和粮食产量的增加尚未对地下水位造成严重影响。     

苹果柱型基因Co的一个AFLP 标记的SCAR转换

 将苹果柱型基因的一个AFLP 标记成功地转换成了简单实用的SCAR 标记。首先对AFLP 标记片段进行序列测定, 然后根据序列特点设计了两对特异引物CoA1/ CoA2 和CoA1/ CoA3 , 每条引物长20 bp。PCR 结果表明CoA1/ CoA2 可以扩增出216 bp 和148 bp 两条带, 其中216 bp 的为柱型性状的特征带; CoA1/CoA3 可以扩增出273 bp 和205 bp 的两条带, 其中273 bp 的为柱型性状的特征带。两对引物在杂交后代中扩增出的特征带与柱型性状的分离重组率都很低(CoA1/ CoA2 为6. 3 % ±2. 5 %; CoA1/ CoA3 为7. 3 % ±2. 6 %) , 所以它们都可以作为该SCAR 标记的特异引物所用。     

根癌农杆菌介导绿色荧光蛋白基因转化印度酸桔的研究

 通过根癌农杆菌介导将绿色荧光蛋白基因转入印度酸桔的胚性愈伤组织中, 经潮霉素筛选,获得抗性愈伤组织, 并再生植株。对这些植株进行GUS 染色、PCR 分析、绿色荧光检测和Sourthern 杂交验证, 结果表明绿色荧光蛋白已经在转基因植株中表达。     

Influences of chronic hypoxia on the gene expression of Kv1.3,Kv2.1 and Kv3.1 induced by acute hypoxia in PASMC of rats

AIM and METHODS: Total RNA was extracted from 6th rat subcultured pulmonary artery smooth muscle cells(PASMC) exposed to continual chronic hypoxia or normoxia and the effects of chronic hypoxia on the changes of Kv1.3,Kv2.1,Kv3.1 mRNA in cultured PASMC induced by acute hypoxia were studied by semiquantitative RT-PCR in vitro. RESULTS:①Kv1.3,Kv2.1,Kv3.1 genes were found to be expressed in PASMC of rats exposed either to hypoxia or normxia.②The expression of Kv2.1 and Kv3.1 in 6th subcultured of PASMC in normaxia group could be upregulated by exposure to acute hypoxia,the levels of Kv2.1 and Kv3.1 mRNA were significantly increased from 0.646±0.092, 0.782±0.104 to 1.059±0.134, 0.985±0.116,respectively (P<0.01,n=5). ③PASMC cultured continuously in chronic hypoxia for 6 subcultures and then exposed to normoxia for 12 h,thereafter the expression of Kv2.1 and Kv3.1 were downregulated by acute hypoxia for 6 hours.The level of Kv2.1 mRNA was significantly decreased from 1.008±0.117 to 0.649±0.097 (P<0.01,n=5). CONCLUSION:Kv2.1,Kv3.1 genes might be oxygen sensitive genes.Chronic hypoxia might change the response of these Kv genes of PASMC to acute hypoxia and down-regulate its expression,which might probably decrease the role of Kv in HPV.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.     

Phenotypic and genetic characterization of Erwinia carotovora from mulberry (Morus spp.)

Phenotypic and genetic characteristics of nine bacterial strains isolated from mulberry ( Morus spp.), which were originally described as Erwinia carotovora ssp. carotovora (Ecc), were investigated. Based on the results of biochemical tests, these bacterial strains were divided into two different types, type 1 and type 2. Two strains of type 1 were similar to Ecc, whereas seven strains of type 2 were distinct from Ecc. A polyphasic study that included serological assay, specific PCR assay for E. carotovora ssp. atroseptica (Eca), PCR-RFLP of a pectate lyase ( pel ) gene and RAPD-PCR was performed on the type 2 strains, and the data were compared with those of related E. carotovora subspecies. The results of serological and specific PCR assays for Eca showed that the type 2 strains were distinct from Eca. In RFLP analysis of the pel gene using Sau 3AI, the type 2 strains showed a unique RFLP pattern. On the basis of RAPD analysis, similarity of RAPD patterns within the type 2 strains was very high. A unique RAPD fragment was isolated from the type 2 strains and used as a probe for Southern hybridization. This probe hybridized only with PCR products from the type 2 strains. Based on phenotypic, serological and genetic characteristics, the type 2 strains isolated from mulberry may belong to a distinct E. carotovora subspecies other than Eca or Ecc.