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.     

分子生物学技术在产甲烷古菌研究中的应用

鉴于产甲烷古菌在环境和能源两大方面的特殊作用,对它的研究一直是微生物学研究的焦点之一。现代分子生物学技术的发展与应用,进一步推动了对产甲烷古菌的研究。本文将综述产甲烷古菌基因克隆研究进展以及几项有代表性的分子生物学技术在产甲烷古菌研究中的应用。     

半滑舌鳎含sox9基因BAC克隆的筛选及BAC-FISH定位

以本实验室构建的半滑舌鳎BAC文库为基础,通过对其进行有序混合,构建了两步PCR筛选体系;对性别决定相关的sox9基因的序列设计特异性引物,筛选获得阳性单一BAC克隆。利用BAC-FISH技术将包含sox9基因的BAC克隆定位在半滑舌鳎染色体上。结果显示,sox9基因在雌、雄半滑舌鳎的一对常染色体的长臂上分别存在两个杂交信号位点,信号稳定且特异。研究证实了该文库筛选体系的有效性;首次实现了对性别相关的sox9基因在半滑舌鳎染色体上的定位,其结果为揭示sox9基因参与鱼类性别控制的机制提供了重要基础。     

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。     

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。     

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.     

小麦-簇毛麦6VS/6AL易位染色体在不同小麦背景中的遗传稳定性及其在配子中的传递

为研究小麦-簇毛麦6VS/6AL易位染色体在不同小麦背景中的遗传稳定性及其在配子中的传递,利用高抗白粉病的普通小麦-簇毛麦6VS/ 6AL易位系92R137与不抗白粉病的栽培品种百农64、百农9310、邯5310、小偃54、淮麦20、徐麦856进行杂交,并用这些品种分别与杂种F1进行正反回交.对杂种F1花粉母细胞减数分裂中期Ⅰ的细胞学观察表明,在减数分裂中期Ⅰ易位染色体6VS/ 6AL通常以6AL与6A 染色体配对形成棒状二价体.各杂种F1高抗白粉病,位于6VS上的抗白粉病基因Pm21呈显性,在F2中有69.0%～74.0%的植株抗白粉病,接近1对显性基因遗传的理论值.由于6VS与6AS在通常情况下不发生配对交换,因此可通过白粉病抗性鉴定并结合利用6VS上的分子标记来跟踪6VS/ 6AL易位染色体的传递.测交结果表明,以F1作母本6VS/ 6AL易位染色体通过雌配子的传递率为49.2%(45.8%～54.9%);以F1作父本6VS/ 6AL易位染色体通过雄配子的传递率为44.7% (43.1%～46.8%),均接近50%的理论值.但在各组合中,6VS/ 6AL易位染色体通过雄配子的传递率均低于通过雌配子的传递率.     

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