应用PCR-RFLP及PCR-TGGE技术监测农田土壤微生物短期动态变化

在一块农田里于一个月内连续采集5次土样，提取土样微生物总DNA，应用PCR-RFLP及PCR-TGGE技术监测土壤微生物的动态变化。结果表明，细菌16S rDNA和真菌18S rDNA的PCR-RFLP图谱在5个采样时间点上基本一致；细菌的PCR-TGGE图谱平均有40条带，其相似性在90％以上，真菌的平均有20条带，其相似性在70％以上。说明这块农田土壤细菌区系短期内变化不大，而真菌区系存在较小幅度的动态变化，并且细菌多样性远远高于真菌多样性。比较两种分子生物学方法的结果表明，PCR-TGGE技术比PCR-RFLP技术更能精确地反映土壤微生物的动态变化，并且能同时快速地对多个样品进行有效区分。     

PCR-RFLP筛选DNA文库克隆Bt cry基因的研究

 Bt菌株C0 0 2对水稻二化螟、甜菜夜蛾具有高毒力 ,PCR RFLP鉴定含有cry1Aa、cry2Ab、cry1Ca和未知杀虫蛋白基因cryX等 ,其中cry1Ca位于染色体DNA 6～ 9kb的EcoRI片段。染色体和质粒DNA分别经EcoRI完全酶切和Sau3AI部分酶切、电泳回收 6～ 9kb片段。E .coli Bt穿梭载体pHT315分别与目的DNA连接、转化大肠杆菌感受态细胞后获得了相应的DNA文库。约 5 0个转化子合为一个转化子池 ,采用PCR RFLP方法快速检测 ,分别从约 2 0 0 0质粒DNA文库转化子和 4 0 0个染色体文库转化子中筛选获得了cry1Aa、cry2Ab、cry1Ca和未知基因cryX的阳性克隆 ,相应命名为pHT 1Aa、pHT 1Ca、pHT 2Ab和pHT X。限制酶切分析表明 ,含有cry1Aa、cry1Ca和cry2Ab基因的克隆片段均含有相应基因的保守物理图谱。进一步将这些质粒分别导入Bt无晶体突变株CryB-,SDS PAGE分析表明 ,只有cry1Ca表达了约 130ku杀虫晶体蛋白。初步杀虫生测结果显示 ,cry1Ca对甜菜夜蛾具有高毒力 ,7d校正死亡率为 10 0 %。     

Variation and phylogeny of the ribosomal DNA unit types and 5 S DNA inPetunia Jussieu

Summary Seven wildPetunia species including 2n = 18 species (P. parviflora Jussieu,P. linearis Hook.) and those with 2n = 14 (P. parodii Steene,P. axillaris Lam.,P. integrifolia Hook.,P. inflata R.E. Fries,P. violacea Lindl.) and tenPetunia hybrida horticultural lines were compared for polymorphisms in rDNA genes using the four restriction enzymesEcoRI,BamHI,HindIII andXhoI. All the unit types found in the lines pre-existed in the wild forms. There are two different sizes of either 11.45 or 11.6 kb./The 2n = 18 species are closely related to the 2n = 14 species, thus making thePetunia genus homogeneous. Moreover, it is likely thatP. hybrida lines originated in several kinds of crosses between these species. We constructed a dendrogram for all the 15 rDNA unit types found. Two main branches of the tree result from the presence or the absence ofHindIII sites. The main branch is divided according to variability at theEcoRI andBamHI sites. Taking into account the existence of several loci which carry one unit type only, we consider whether or not exchanges might occur between loci. Lines carrying two unit types and lines carrying three unit types support such a hypothesis.XhoI andBamHI fragments enable us to distinguish two types of 5S DNA corresponding to 2n = 18 and 2n = 14 species, respectively.P. hybrida lines and each 2n = 14 wild species carry one of the types only, that corresponds to one 5S DNA locus. The most parsimonious phylogenetic trees whatever the species chosen as the outgroup, do not fit with our knowledge ofPetunia and with taxonomy. This is likely because only few loci formed the basis of these phylogenetic constructions.     

The Presence of a New S-RNase Allele (S10) in Asian Pear (Pyrus pyrifolia (Burm; Nakai))

A PCR (polymerase chain reaction) amplification method using newly designed S-RNase primers was carried out in five Korean-bred pear cultivars and ten Japanese-bred pear cultivars. A new S-RNase allele, designated as S₁₀, was discovered from ‘Chengsilri’, containing a 1513 bp and two exons (213 bp in total) that coded for a peptide of 71 amino acids. The S₁₀-RNase allele contained the three conserved cysteine residues peculiar to S-RNase in Japanese pear and one histidine residue essential for RNase activity. We compared nucleotide sequence similarity of the exon regions of ten pear S-RNase alleles. The nucleotide sequence of S₁ showed a high similarity to S₄ (97.4%) and the new S₁₀ shows 77.8% (S₅) to 84.4% (S₄) similarity with the other pear S-RNase alleles. S₁₀ had a unique restriction endonuclease site for ‘HhaI’, with digests yielding fragments of 1235 and 491 bp. The S-genotype of pear cultivar (‘Chengsilri’) was determined to be S₅S₁₀ by PCR–RFLP (restriction fragment length polymorphism). Cluster analysis of 49 known S-RNase alleles of the Rosaceae separated into two divergent groups are as follows: group I: pear and apple, group II: almond, sweet cherry and mume.     

Mapping a new source of resistance to powdery mildew in mungbean

Both restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) analyses were employed to map a new source of resistance to powdery mildew in mungbean. Disease scores of an F₂ population derived from the cross between a moderately resistant breeding line VC1210A and a susceptible wild relative (Vigna radiata var. sublobata, accession TC1966) showed a continuous distribution and was treated as a quantitative trait. Although no significant quantitative trait loci (QTL) that can explain the variation was detected by QTL analysis based on the reconstructed RFLP linkage map, new marker loci associated with resistance were discovered by AFLP analysis. The RFLP loci detected by two of the cloned AFLP bands are associated with resistance and constitute a new linkage group. A major resistance quantitative trait locus was found on this linkage group that accounted for 64.9% of the variation in resistance to powdery mildew. One of the probes developed in this study has the potential to assist in breeding for powdery mildew resistance in mungbean.     

Relationships among ancestral species of sugarcane revealed with RFLP using single copy maize nuclear probes

Summary DNA restriction fragment length polymorphism (RFLP) analysis was performed on 50 wild and old cultivated sugarcane accessions. Ninety-four maize low copy nuclear DNA sequences of known chromosomal position were screened for hybridization to digested sugarcane genomic DNA blots. Seventy-five (80%) gave very strong hybridization signals and usually yielded many bands and detected profuse polymorphism. Twenty-nine probes and 36 probe/enzyme combinations were selected on the basis of the scorability of the banding profiles. A total of 1110 fragments were separately identified among the 50 genotypes. Multivariate analyses of the data allowed the separation of the three basic species, Saccharum spontaneum, S. robustum and S. officinarum, showed that S. spontaneum had structure which could be related to the geographic origin of the clones and supported current hypotheses on the origin of secondary species S. barberi and S. sinense. The use of more probes did not improve the resolution between the various species examined but identified a few key polymorphisms which were not accounted for by current phylogenetic hypotheses and can guide future analyses. RFLPs in sugarcane will be useful essentially for depicting the genomic constitution of modern varieties of interspecific origin.     

The conversion of a RFLP assay into PCR for the determination of purity in a hybrid pepper cultivar

Summary A RFLP assay which had been developed for distinguishing between a hybrid pepper and its parents, has been converted into a simple applicable PCR assay, suitable for large-scale quality-control assessment of the commercial hybrid. For this conversion the sequences of the ends of the probe used in the previous RFLP assay were determined. From these sequences suitable primers were devised for inverse PCR of heterogeneous DNA fragments derived from the male parent. The inverse-PCR product was cloned and partially sequenced. These sequences, in turn, made it possible to determine primers on both sides of the locus of mutation, and to develop the reported conventional PCR assay.     

Assessment of the degree of genetic variation in beet based on RFLP analysis and the taxonomy of Beta

Summary Clones derived from Beta vulgaris and Beta maritima were assayed for their ability to detect restriction fragment length polymorphisms (RFLPs) in different beet accessions. The clones able to detect polymorphism were used as genetic markers to assess the degree of genetic variation existing between and within different species of the genus Beta. The data support the current taxonomy of the Beta vulgaris section, while the great genetic similarity found between Beta webbiana and Beta procumbens indicates that they could belong to the same species.Enough variation was found between Beta vulgaris cultivars, allowing the isolation of a sufficient number of genetic markers for the construction of detailed genetic maps.     

The Use of RFLPs (Restriction Fragment Length Polymorphisms) Detects Germplasm Introgressions from Wild Species into Potato (Solanum tuberosum ssp. tuberosum) Breeding Lines

The germplasm of the potato (S. tuberosum ssp. tuberosum) has been modified since the beginning of this century by breeders who introgressed important agronomic traits, for example disease resistance genes, from several wild and cultivated Solanum species of the Americas. In this paper we show that the Rflp analysis of potato-breeding material can be used to detect chromosomal regions descended from more-distantly related Solanum species. The Rflp patterns of individuals of ten Solanum species, ten breeding lines of S. tuberosum ssp. tuberosum and the cultivated variety ‘Bintje’ were analyzed. Rflp data for each of eight single loci of known genomic position were used for the computation of locus-specific phenograms by distance matrix methods. Several of the potato-breeding lines deviated clearly from the clustered species S. tuberosum, S. stenotomum and S. canasense for one or more of the loci considered. These deviations indicated the presence of “exotic” germplasm at a particular locus. The possibility of detecting such germplasm has implications for mapping the agronomic traits for which the wild species were introgressed and which may still be linked to “foreign” chromosome fragments.