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。     

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。     

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.     

SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f. sp. lolii) in perennial ryegrass (Lolium perenne)

Crown rust (caused by Puccinia coronata f. sp. lolii) is a serious foliar disease of the pasture and turfgrass perennial ryegrass (Lolium perenne). Previous genetic studies have detected both qualitative and quantitative resistance mechanisms, and interpretation of the genetic system is complicated by variation within the sexually reproducing pathogen. Resistant and susceptible parental genotypes of ryegrass were identified using a composite urediniospore population collected from three geographically distinct locations. A two-way pseudo-testcross mapping population was obtained as the F₁ progeny of the pair-cross between ryegrass parental genotypes Vedette₆ and Victorian₉. Both parents showed intermediate resistance against a pathogen population collected in a single geographical zone (Hamilton, Victoria), but in the F₁ population, significant variation for a range of resistance-associated characters was detected. Statistical analysis of phenotypic data suggested a major gene effect, hence bulked segregant analysis with map-assigned simple sequence repeat (SSR) markers was used to scan the genome. A marker showing strong association with resistance was assigned to linkage group (LG) 2 of perennial ryegrass. Analysis of 11 LG2 SSR markers defined an interval between loci xlpssrh03f03 and xlpssrk02e02 as containing the gene or genes (LpPc1) conferring crown rust resistance. Resistance gene determinants were inherited from both parents, with up to 80% of the total phenotypic variation explained by markers segregating from Vedette₆ and up to 26% of the variation explained by markers segregating from Victorian₉. The two contributions together resulted in an additive increase in effect, with fully resistant individuals requiring determinants from both parents. A conserved syntenic relationship was observed with linkage group B of Avena strigosa, which is the location of a cluster of resistance genes to the oat form of crown rust. The implications of this study for marker-assisted selection of disease resistance in perennial ryegrass are discussed.     

Functional analysis of the 3′ end of avrBs3/pthA genes from two Xanthomonas species

The phytopathogens Xanthomonas oryzae pathovar (pv.) oryzae and Xanthomonas axonopodis pv. citri each contain several avrBs3/pthA family genes. Structural features of these genes important for avirulence and/or virulence functions include a central region of multiple direct repeats and three nuclear localization signals (NLSs) and an acidic activation domain (AAD) at the 3′ end. To identify other regions critical to function in the 3′ ends of these genes, we constructed several chimeras using apl1 and apl2 from X. axonopodis pv. citri and avrXa10 and avrXa7 from X. oryzae pv. oryzae and evaluated their functions by inoculation to citrus and rice. The apl1 and avrXa7 genes are major virulence determinants in citrus and rice, respectively, while the contributions of apl2 and avrXa10 to virulence are negligible or not measurable. Constructs that contained a 417 bp HincII-SphI fragment from the 3′ end of apl1 in combination with the repeats from avrXa7, avrXa10, and apl1 caused a canker phenotype on citrus. Interchange of the HincII-SphI fragment between avrXa7 and avrXa10 abolishes avrXa7 avirulence function and reduces its virulence but it does not affect avrXa10 avirulence function in rice. avrXa7 caused a hypersensitive response (HR) in citrus and replacement of it's 3′ end with that of apl1 resulted in loss of canker and induction of HR. Thus, the HincII-SphI fragment of the avrBs3/pthA gene family is important for avirulence and virulence functions in two different plant species, Oryza sativa and Citrus natsudaidai HAYATA.     

Evidence that a Leaf-Disk Test Allows Assessment of Isolate-Specific Resistance in Brassica oleracea Crops Against Downy Mildew (Peronospora parasitica)

A rapid resistance/susceptibility test for Peronospora parasitica (downy mildew) was established by inoculating leaf-disks of four Brassica oleracea accessions. Several conditions were tested: disk disinfection or not, agar medium with or without nutrients and with 50 or 100 ppm of benzimidazole. Using disinfected disks placed on agar (no nutrient and benzimidazole at 50 or 100 ppm), the responses of leaf-disks to four isolates were similar to those obtained using the classical cotyledon test, whereas undesired contaminations occurred in all other conditions. The possible effect of the particular leaf used for obtaining the disks was also studied. In each incompatible interaction tested, disks were resistant whatever the leaf used. In compatible interactions, susceptible phenotypes were observed on disks derived from the six lowest leaves, but disks from upper leaves were resistant. The genetic basis of resistance in a F1 hybrid broccoli was assessed, by testing six isolates on an F2 population derived from this hybrid. The cotyledon test only allows inoculation of two isolates per seedling, whereas many isolates can be tested on each plant by using leaf-disks. The segregation of the resistance to each of the six isolates was analysed: two dominant genes (tightly linked) control resistance to all isolates (one to five isolates; the other to only one isolate).