尼罗罗非鱼六个性别相关标记的FISH分析

从尼罗罗非鱼（Oreochromis niloticus）BAC基因文库5个克隆中提取和纯化含有6个性别连锁或相关标记（CLC5，GM204，GM271，GM354，UNH995和UNH104）的重组质粒DNA作为模板，以简并核苷酸为引物，通过PCR制备原位杂交探针。探针用荧光素进行标记，并与尼罗罗非鱼中期相染色体进行荧光原位杂交以确定这些标记在尼罗罗非鱼染色体上的位置和分布。结果显示，这些性别连锁或相关标记都位于尼罗罗非鱼第一对染色体长臂近末端，从分子细胞学角度验证了第一对染色体是尼罗罗非鱼的性染色体。另外由于这些标记的荧光信号在XY个体的2条性染色体上都有，一方面说明这些标记在罗非鱼上还不是性别特异的；另一方面也验证了尼罗罗非鱼的性染色体还处于分化的早期阶段。【中国水产科学，2006，13（4）：525—529】     

Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

ABSTRACT:   The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae.     

小麦-鹅观草易位系T7A/1Rk#1的选育与鉴定

将小麦-鹅观草del1Rk#1L二体添加系的花粉用10 Gy 60Co γ射线照射处理,给小麦中国春授粉,获得杂种。综合利用C-分带、GISH、顺次C带/45SrDNA-FISH和顺次GISH/45SrDNA-FISH等分子细胞遗传学技术在M2代筛选和鉴定出1个涉及小麦7A和鹅观草1Rk#1染色体的相互易位染色体系,并获得1Rk#1染色体的1个45SrDNA位标,该位标和其对应的红色GISH-带纹能够特异地识别1Rk#1染色体短臂。对M2代群体染色体组成分析和测交分析表明,两条易位染色体在后代中以共分离方式成对出现,且易位通过雌配子的传递率高于雄配子。综合2004、2005和2006 3年的赤霉病抗性鉴定结果表明：该易位系对赤霉病表现部分抗病,但抗性表现在不同年份、不同地点有差异。试验还证明花粉辐射是诱导小麦-外缘物种染色体易位的有效方法。     

小伞山羊草染色体的FISH核型分析

采用荧光原位杂交(Fluorescence in Situ Hybridization,FISH)技术分析小伞山羊草染色体的核型特点。结果表明,小伞山羊草共包含7对染色体,其中1 U和5 U染色体各含有1对随体。分别以Oligo-pSc 119.2-1(绿色)与(GAA)7(红色)重复序列为探针对小伞山羊草根尖细胞染色体进行FISH分析,发现Oligo-pSc-119.2-1信号主要分布在染色体的端部及近端部,不同染色体之间的信号强度有所差别。(GAA)7信号主要集中于染色体着丝点附近,不但比Oligo-pSc-119.2-1信号的亮度高,分布丰富,而且在染色体上的分布可与Oligo-pSc-119.2-1信号互补。因此,同时采用Oligo-pSc-119.2-1与(GAA)72种探针能准确地辨别小伞山羊草的每对染色体,建立了小伞山羊草的FISH核型。     

大白菜延伸DNA纤维的制备和检测(英文)

[目的]制备和检测大白菜延伸DNA纤维。[方法]以大白菜幼叶为材料,采用刀切分离细胞核、SDS释放DNA、盖玻片滑动牵拉DNA的方法,首次获得大白菜DNA延伸纤维。[结果]以基因组DNA和25SrDNA为探针进行原位杂交检测,该DNA纤维长度大约为1.93Mb,25SrDNA在大白菜基因组中的拷贝数在258-687之间。该方法获得的DNA延伸纤维平行、清晰、适合FISH分析。[结论]该研究将会促进大白菜基因图谱的构建和组织分析。     

动物性别决定的分子机理及性别鉴定与控制新技术

哺乳动物中位于Ｙ染色体短臂临界区域的ＳＲＹ基因启动雄性性状的发育。该基因在生殖腺脊中的表达可激发下游基因ＭＬＳ的转录,引起缪氏体抑制物和睾酮分泌,促使睾丸组织器官的发育。针对该基因制备特异探针或产该区域片段得到引物对就可用ＦＩＳＨ或ＰＣＲ准确,快速鉴定出植入母体前的胚胎性别,以及对精子筛选分离的结果作出准确评价。与ＰＣＲ相比,ＦＩＳＨ技术更具检测优势。     

Ability of chromosome 4H to compensate for 4D in response to drought stress in a newly developed and identified wheat–barley 4H(4D) disomic substitution line

A spontaneously developed wheat–barley 4H(4D) disomic substitution line was identified cytogenetically using genomic in situ hybridization (GISH), multicolour fluorescent in situ hybridization (FISH) and microsatellite markers. The ability of the barley 4H chromosome to compensate for wheat 4D in response to mild drought stress was also investigated. In the barley cv. 'Betzes' and the 4H(4D) substitution line, mild osmotic stress induced intensive stomatal closure, resulting in reduced water loss through transpiration and unchanged relative water content in the leaves. As the CO₂ assimilation rate remained relatively high, the water use efficiency, which is an important factor associated with drought tolerance, increased extensively under mild osmotic stress in these lines. In the case of the parental wheat genotypes, however, mild drought stress induced less intense stomatal closure and a greater decrease in the CO₂ assimilation rate than in barley or in the substitution line, resulting in unaugmented or reduced water use efficiency. The results demonstrate that genes localised on the 4H chromosome of barley were able to increase the water use efficiency of the wheat substitution line, which is suitable for improving wheat drought tolerance through intergeneric crossing.     

内蒙古绒山羊Hoxa7基因在毛囊中的表达

利用原位杂交技术检测Hoxa7基因在毛囊不同生长时期的表达模式,结果表明,Hoxa7基因分别在胚胎期的95 d初级毛囊的内根鞘表达、在115 d次级毛囊的内根鞘和绒干表达、在125 d初级毛囊内根鞘和毛干皮质表达、在兴盛期10月的初级毛囊毛干皮质和次级毛囊的绒干中表达。揭示在毛囊发育中,Hoxa7基因对于毛囊细胞的增殖可能起一定的作用。     

Fluorescent in situ hybridization as an aid to introducing alien genetic variation into wheat

Summary Fluorescent in situ hybridization (FISH) has been used to assess the occurrence and frequency of wheat-alien chromosome pairing in a wheat/Thinopyrum bessarabicum hybrid and in wheat/rye hybrids with different levels of chromosome pairing by examining pollen mother cells at metaphase I of meiosis. The use of FISH to identify the presence and size of alien chromatin in a wheat background is also demonstrated.The value of FISH as an aid to the introgression of alien genetic variation into wheat is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization     

基因组原位杂交技术在植物研究中的应用

基因组原位杂交是以亲本之一的总基因组DNA做探针,另一亲本的基因组DNA做封阻,在荧光原位杂交技术的基础上发展起来的一种染色体/染色质检测技术。在其发展的十几年里,已在植物的基因组研究中发挥了重要的作用。应用这一技术可对多倍体中基因组之间的亲缘关系、基因组组成及起源进行研究;对杂交种中染色体组的组成进行分析;对代换系、附加系和易位系进行有效的鉴定,并对其中的外源染色体或染色体片段的来源、大小、数目及发生位点进行检测和定位。此外,利用基因组原位杂交技术还有助于确定物种间的同源性;研究杂交种中来源不同的染色质在核中的分布;探索B染色体的起源、染色体间的配对、重组、交换等现象。随着基因组荧光原位杂交技术体系的不断发展、完善和改进,其应用范围不断拓展,在植物基因组研究领域中发挥了越来越重要的作用。