抗药性基因克隆技术研究进展

综述了近几年来抗药性基因克隆技术的研究进展。对RAPD-PCR、单链构象多态性(SSCP)、差异显示PCR(DDRT-PCR)、代表性差异分析技术(representational difference analysis，RDA)、抑制消减杂交法(suppression subtractive hybridization，SSH)、获得全长基因的消减杂交法(full length gene obtainable subtractive hybridization)、基因芯片技术、基因表达系统分析(serial analysis of gene expression，SAGE)的技术和从蛋白质水平克隆抗药性基因的双向电泳技术(two-dimensional gel electrophoresis)、噬菌体表面全套抗体库技术(phage display antibody repertoire library technique)、核糖体展示抗体库技术等方法的特点、优缺点及适用性进行了评述。     

The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes

Whole-genome sequencing (WGS) data present a readily available resource for mitochondrial genome (mitogenome) haplotypes that can be utilized for genetics research including population studies. However, the reconstruction of the mitogenome is complicated by nuclear mitochondrial DNA (mtDNA) segments (NUMTs) that co-align with the mtDNA sequences and mimic authentic heteroplasmy. Two minimum variant detection thresholds, 5% and 10%, were assessed for the ability to produce authentic mitogenome haplotypes from a previously generated WGS dataset. Variants associated with NUMTs were detected in the mtDNA alignments for 91 of 917 (~8%) Swedish samples when the 5% frequency threshold was applied. The 413 observed NUMT variants were predominantly detected in two regions (nps 12,612–13,105 and 16,390–16,527), which were consistent with previously documented NUMTs. The number of NUMT variants was reduced by ~97% (400) using a 10% frequency threshold. Furthermore, the 5% frequency data were inconsistent with a platinum-quality mitogenome dataset with respect to observed heteroplasmy. These analyses illustrate that a 10% variant detection threshold may be necessary to ensure the generation of reliable mitogenome haplotypes from WGS data resources.     

软琼脂克隆法与裸鼠体内接种法检测细胞致瘤性的比较

目的比较体外软琼脂克隆法与裸鼠体内接种法检测细胞致瘤性的敏感性及相关性。方法用软琼脂克隆法检测不同种类、不同来源的细胞的致瘤性,并与裸鼠体内接种法的结果进行比较。结果不同的细胞在体外软琼脂中形成细胞集落的能力及集落形态明显不同。肿瘤细胞在体外可形成肉眼可见的细胞集落,而人二倍体细胞及组织工程用细胞在体外不形成或仅形成极少的细胞集落克隆,其集落形成率与细胞代次没有相关性,在裸鼠体内亦不形成瘤。重组CHO细胞经过遗传改造后,有的在裸鼠体内的致瘤性减弱,但体外软琼脂克隆行为没有发生明显改变。从128至169代次的Vero细胞均可在体外软琼脂中形成不同的细胞集落克隆,高代次细胞的集落形成率明显高于低代次细胞,但所有检测代次的细胞在裸鼠体内均未形成瘤。结论用软琼脂克隆法检测细胞的致瘤性与裸鼠体内接种法不仅具有良好的相关性,而且敏感性高于裸鼠体内接种法。     

Gene Mapping,Cloning and Association Analysis for Salt Tolerance in Rice

Soil salinization caused by the accumulation of sodium can decrease rice yield and quality. Identification of rice salt tolerance genes and their molecular mechanisms could help breeders genetically improve salt tolerance. We studied QTL mapping of populations for rice salt tolerance, period and method of salt tolerance identification, salt tolerance evaluation parameters, identification of salt tolerance QTLs, and fine-mapping and map cloning of salt tolerance QTLs. We discuss our findings as they relate to other genetic studies of salt tolerance association.     

Genome-Wide Association Mapping Identifies New Candidate Genes for Cold Stress and Chilling Acclimation at Seedling Stage in Rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a chilling-sensitive staple food crop, and thus, low temperature significantly affects rice growth and yield. Many studies have focused on the cold shock of rice although chilling acclimation is more likely to happen in the field. In this paper, a genome-wide association study (GWAS) was used to identify the genes that participated in cold stress and chilling accumulation. A total of 235 significantly associated single-nucleotide polymorphisms (SNPs) were identified. Among them, we detected 120 and 88 SNPs for the relative shoot fresh weight under cold stress and chilling acclimation, respectively. Furthermore, 11 and 12 quantitative trait loci (QTLs) were identified for cold stress and chilling acclimation, respectively, by integrating the co-localized SNPs. Interestingly, we identified 10 and 15 candidate genes in 11 and 12 QTLs involved in cold stress and chilling acclimation, respectively, and two new candidate genes (LOC_Os01g62410, LOC_Os12g24490) were obviously up-regulated under chilling acclimation. Furthermore, OsMYB3R-2 (LOC_Os01g62410) that encodes a R1R2R3 MYB gene was associated with cold tolerance, while a new C3HC4-type zinc finger protein-encoding gene LOC_Os12g24490 was found to function as a putative E3 ubiquitin-protein ligase in rice. Moreover, haplotype, distribution, and Wright’s fixation index (FST) of both genes showed that haplotype 3 of LOC_Os12g24490 is more stable in chilling acclimation, and the SNP (A > T) showed a difference in latitudinal distribution. FST analysis of SNPs in OsMYB3R-2 (LOC_Os01g62410) and LOC_Os12g24490 indicated that several SNPs were under selection in rice indica and japonica subspecies. This study provided new candidate genes in genetic improvement of chilling acclimation response in rice.     

A Dual-Color Tyr-FISH Method for Visualizing Genes/Markers on Plant Chromosomes to Create Integrated Genetic and Cytogenetic Maps

In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refining genetic maps and validation genome assembly at the chromosomal level. Despite the tremendous progress in genome sequencing, the plant genome assembly at the chromosome level remains a challenge. Recently developed optical and Hi-C mapping are aimed at assistance in genome assembly. For high confidence in the genome assembly at chromosome level, more independent approaches are required. The present study is aimed at refining an ultrasensitive Tyr-FISH technique and developing a reliable and simple method of in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH to find out the reasons behind the flaws of this technique. The accurate visualization of markers/genes appeared to be significantly dependent on the means of chromosome slide preparation, probe design and labeling, and high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.6 Kb with a frequency of 51.6%. Based on our results, we developed a more reliable and simple protocol for dual-color Tyr-FISH visualization of unique short DNA sequences on plant chromosomes. This new protocol can allow for more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.     

The Use of DArTseq Technology to Identify Markers Linked to Genes Responsible for Seed Germination and Seed Vigor in Maize

Seed vigor and seed germination are very important traits, determined by several factors including genetic and physical purity, mechanical damage, and physiological condition, characterized by maintaining a high seed vigor and stable content after storage. The search for molecular markers related to improvement in seed vigor under adverse condition is an important issue in maize breeding currently. Higher sowing quality of seeds is necessary for the development of the agriculture production and better ability to resist all kinds of adversity in the seeds’ storage. Condition is a very important factor affecting the yield of plants, thanks to the construction of their vitality. Identification of molecular markers associated with seed germination and seed vigor may prove to be very important in the selection of high-yielding maize varieties. The aim of this study was to identify and select new markers for maize (SNP and SilicoDArT) linked to genes influencing the seed germination and seed vigor in inbred lines of maize (Zea mays L.). The plant material used for the research was 152 inbred maize lines. The seed germination and seed vigor were analyzed. For identification of SNP and SilicoDArT markers related to the seed germination and seed vigor, the SilicoDarT technique developed by Diversity Arrays Technology was used. The analysis of variance indicated a statistically significant differentiation between genotypes for both observed traits. Positive (r = 0.41) correlation (p < 0.001) between seed germination and seed vigor was observed. As a result of next-generation sequencing, the molecular markers SilicoDArT (53,031) and SNP (28,571) were obtained. Out of 81,602 identified SilicoDArT and SNP markers, 15,409 (1559 SilicoDArT and 13,850 SNP) were selected as a result of association mapping, which showed them to be significantly related to the analyzed traits. The 890 molecular markers were associated with seed vigor, and 1323 with seed germination. Fifty-six markers (47 SilicoDArT and nine SNP) were significant for both traits. Of these 56 markers, the 20 most significant were selected (five of these markers were significant at the level of 0.001 for seed vigor and at the level of 0.05 for seed germination, another five markers were significant at the level of 0.001 for seed germination and at the level of 0.05 for seed vigor, five markers significant at the level of 0.001 only for seed vigor and five significant at the level of 0.001 only for seed germination also selected). These markers were used for physical mapping to determine their location on the genetic map. Finally, it was found that six of these markers (five silicoDArT—2,435,784, 4,772,587, 4,776,334, 2,507,310, 25,981,291, and one SNP—2,386,217) are located inside genes, the action of which may affect both seed germination and seed vigor. These markers can be used to select genotypes with high vigor and good seed germination.     

选煤厂三相交流电动机缺相保护的方法及一种简单保护方法的应用

文章针对选煤厂三相异步电动机缺相运行产生的原因、现象进行阐述,列举了缺相故障的几种常见保护措施。同时针对运行较早矿区或厂房等,提出了一种简单的保护方法,该方法设计简单、方便易行,在生产中取得了较好的效果。