黄鳝二价染色体上地高辛配基标记随机引物原位DNA合成的研究

在黄鳝二价染色体上, 运用地高辛配基标记的随机引物原位DNA合成技术诱导出了明暗相间、基本稳定且类似于R带的带状结构。本文对该结果进行了较详细的分析和讨论。 Abstract:The dark bands alternating with light bands,relatively stable and R-band-like structure was showed on the pachytene bivalents of Rics-field eel(Monopterus albus Zuiew)by using the random-primed in situ digoxigenin-labelled DNA synthesis technique,and the results were analysed and discussed in detail.     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

蓖麻蚕DNA导入家蚕引起遗传变异的研究-基因组DNA的RAPD检测

借助于精子介导,在家蚕受精的过程中将蓖麻蚕DNA转入家蚕卵内,从它们后代获得了新的变异品系。本文采用RAPD技术对这些品系基因组DNA进行了分析。结果表明,所用50种10mer随机引物中有49个检测出DNA的多态性,统计分析图谱中各类扩增带,其中变异品系与其相应受体的差异带占其总带数的26-37%,提示外源DNA导入受体后引起后代基因组的显著变异,并对这些变异的意义了讨论。 Abstract:With the aid of domesticated silkworm sperms,eri silkworm DNA was transferred into domesticated silkworm eggs during insemination,and variant strains were obtained from the progenies.Genomes of three new strains were analyzed using RAPD assay.Polymorphic fingerprints were obtained from 49 out of 50 primers.Different kinds of amplified bands in RAPD patterns were calculated and analyzed,the variant bands between variants and their recipients counted for 26~37% of the total bands of each variant.The results indicated that exogenous DNA introduced into recipients induced remarkable variation in progeny genomes.The significance of the variation was discussed.     

有和无甘油的聚丙烯酰胺胶在检测突变时的差别

有文献报道在非变性的聚丙烯酰胺中加入甘油可提高SSCP检测的灵敏度。我们的实验结果建议研究者在进行SSCP筛查未知突变时最好采用不加甘油的非变性的聚丙烯酰胺胶,这既省力省钱,又灵敏。在判读SSCP胶时,千万不要看到在双链带位置有一条比正常迁移率慢的带就判定为插入突变。此时要判定突变的性质,最好测序。 Abstract：It was reported that glycerol in the non-denatured SSCP polyacrylamide gel could increase the sensibility of detecting mutation. We detected the mutation of PKD 1 gene in the patients with autosomal dominant polycystic kidney disease.PCR com bined with SSCP(single-strained conformation polymorphism),the non-denatured 10% polyacrylamide gel without glycerol or 10% polyacrylamide gel with 5% glycerol and DNA sequencing method were used.Our results showed that four single strand b ands were found in the non-denatured polyacrylamide gel without glycerol while t wo single strand bands were found in the polyacrylamide gel with glycerol in the same patient.Sequence showed there is a deletion of G in one DNA molecular and a G→A substitution in another DNA molecular in the patient with abnormal shift SSCP bands.Therefore, our experiment suggested that non-denat ured polyacrylamide gel was better than the polyacrylamide gel with glycerol in detection mutation,and it will save labor and money.It also suggeste d that one basedeletion can cause a slow double-strand DNA following the normal double strand band,which was caused by the heterogeneous DNA molecule formed bet ween the normal DNA strand and the one base deletion DNA strand with the protrud ing base.Our results suggest that when judging mutation in SSCP gel,it is not re liable to decide that mutation is inversion according to slow mobility in the ge l,and when the characteristic of mutation need to be judged,it must be sequenced .     

Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t-1 DNA was extracted from its genomic DNA, labeled with biotin-1 1-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization （FISH） signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both Cot-1 DNA FISH banding patterns and chromosome morphology.     

Inhibition of shoot induction by 5-azacytidine and 5-aza-2′-deoxycytidine in Petunia involves DNA hypomethylation

Shoot bud regeneration from Petunia leaf disks was inhibited when they were cultured with the demethylating agents, 5-azacytidine (AzaC) and 5-aza-2′-deoxycytidine (AzadC), in shoot induction (SI) medium. Explants induced shoot primordia if they were transferred after 1 week from the medium containing the drugs to medium without drugs. The fresh weight of leaf disks cultured on SI medium for 2 weeks in the presence of the drugs was 60–80% lower when compared to control shoot-forming cultures. Internode length was reduced when shoots were transferred to phytohormone-free Murashige and Skoog medium containing the drugs. However, no other morphological abnormalities were seen in these shoots, even at 20 μm AzaC or 5 μm AzadC. Coupled restriction enzyme digestion (with HpaII and MspI) and random amplification of genomic DNA was performed to detect the level of methylation of CCGG sites in the DNA of the explants exposed to AzaC and AzadC. Over 15 amplified bands were detectable in the control. Five of these bands were absent in the amplified products when digested DNA from the drug-treated explants was used as the template, showing that hypomethylation of DNA had occurred. This suggests that inhibition of shoot bud formation in the presence of the drugs AzaC and AzadC may be due to the altered methylation status. Received: 7 January 1997 / Revision received: 17 February 1997 / Accepted: 1 March 1997     

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.     

Bachelor groups in primate multilevel society facilitate gene flow across fragmented habitats

In the face of ongoing habitat fragmentation,many primate species have experienced reduced gene flow resulting in a reduction of genetic diversity,population bottlenecks,and inbreeding depression,including golden snub-nosed monkeys Rhinopithecus roxellana.Golden snub-nosed monkeys live in a multilevel society composed of several 1 male harem units that aggregate to form a cohesive breeding band,which is followed by one or more bachelor groups composed of juvenile,subadult,and adult male members.In this research,we examine the continuous landscape resistance surface,the genetic diversity and patterns of gene flow among 4 isolated breeding bands and 1 all-male band in the Qinling Mountains,China.Landscape surface modeling suggested that human activities and ecological factors severely limit the movement of individuals among breeding bands.Although these conditions are expected to result in reduced gene flow,reduced genetic diversity,and an increased opportunity for a genetic bottleneck,based on population genetic analyses of 13 microsatellite loci from 188 individuals inhabiting 4 isolated breeding bands and 1 all-male band,we found high levels of genetic diversity but low levels of genetic divergence,as well as high rates of gene flow between males residing in the all-male band and each of the 4 breeding bands.Our results indicate that the movement of bachelor males across the landscape,along with their association with several different breeding bands,appears to provide a mechanism for promoting gene flows and maintaining genetic diversity that may counteract the otherwise isolating effects of habitat fragmentation.     

Loss of Genetic Diversity of Domesticated Panax notoginseng F H Chen as Evidenced by ITS Sequence and AFLP Polymorphism： A Comparative Study with P. stipuleanatus H T Tsai et K M Feng

In the present study, we evaluated the genetic diversity of Panax notoginseng F H Chen, a domesticated species, and P. stipuleanatus H T Tsai et K M Feng, an endangered wild species in southeastern Yunnan and adjacent areas in Vietnam, using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and amplified fragment length polymorphism (AFLP) markers. Twenty-four accessions from three plantations of P. notoginseng and 51 samples from eight populations of P. stipuleanatus were assayed. A total of 694 bp of partial sequences of 18S, ITS 1, 5.8S, ITS2, and partial sequences of 26S were obtained. No sequence variation was detected within P. notoginseng and nine sites (1.30%) were variable in P. stipuleanatus. Two-thirds of the variable sites were found between Langqiao and other populations. In P. notoginseng, four pairs of AFLP primer combinations generated 312 bands, of which 240 (76.9%) were polymorphic and 60.15% of the polymorphisms were harbored within plantations. Approximately 41.0% and 66.9% of bands were polymorphic in population D7 and 5589, respectively. In P.stipuleanatus, the same four primer combinations produced 346 bands, of which 334 (96.5%) were polymorphic and approximately 62.14% of polymorphisms were maintained within populations. Considerable variations were observed. The percentage of polymorphic bands ranged from 50.2% to 84.9% and the average over populations was 70.9%. Cluster analysis did not show correlation of genetic differentiation with the distinctive leaf morphology of P. stipuleanatus (i.e. one form with bipinnatifid leaflets and the other with undivided leaflets). Because over 40% of genetic variations were maintained among populations and because of the very restricted distribution of P. stipuleanatus, all natural populations of this species should be conserved in situ. Considering that there are variations in P. notoginseng within and among plantations, we suggest establishing a genetic resource conservation garden or reintroducing P. notoginseng into its native habitats in southwestern China. Such reintroduction should be carefully executed after large-scale screening of genetic variation within the species.