银额果蝇B染色体特异性基因的初步研究

采用代表性差异分析法（RDA）研究了银额果蝇两个单雌系AKM46（含B染色体）和AGZ2（不含B染色体）两基因组间的差异。用AKM46作检测（tester）扩增子,AGZ2作驱赶（driver）扩增子,通过三轮消减杂交后,获得了6个差异片段（100bp～300bp）。亚克隆后,对11个片段测序并与GenBank数据库进行同源性比较分析,获得了9个新的序列。选择clone22 及clone42进行Southern杂交分析,这两个片段仅在检测扩增子及第一、第二、第三轮差异片段中检测到杂交信号,而在驱赶扩增子检测不到杂交信号。证实了这两个片段来自含有B染色体的单雌系AKM46,而且可能是B染色体上的特异基因片段。 Abstract:The genomic difference betweenDrosophila albomicanAKM46 (with B chromosome) and AGZ2 (without B chromosome) was analyzed by RDA (Representational Difference Analysis) technique. In RDA system, the tester amplicon is AKM46 while the driver amplicon from AGZ2. After three rounds of subtractive hybridization, six different products were obtained and identified, and their size was about 100bp～300bp. After subcloning, eleven positive clones were sequenced and blasted with Genbank database. Nine sequences were unmatched with the known sequences. Clone22 and clone42 were selected for Southern hybridization, and positive signals were observed only in tester amplicon, and the first, second, third differente products, not in driver amplicon. The results suggest that the sequence come from AKM46 and might be the specific genes in B chromosome.     

小菜蛾对溴氰菊酯抗性相关的DNA片段的克隆与分析

采用代表性差异分析（representational difference analysis, RDA ）方法,以小菜蛾Plutella xylostella (L.)的敏感品系作为对照组,溴氰菊酯抗性近等基因系（near isogenic line）作为测试组,通过三轮消减杂交得到大小为 150～300 bp的差异扩增带,经亚克隆测序并与GenBank等数据库同源比较,发现差异片段与已知抗性相关基因无同源性;以随机选取的差异片段作探针进行Southern blot分析,在测试组扩增子和三轮差异产物中都获得了阳性结果,而在对照组扩增子中的结果是阴性的。这些结果表明测序片段可能是与抗性相关的新基因序列或调控序列。     

黄鳝单条染色体的显微分离及特异性检测

建立了显微分离黄鳝单条染色体及检测其特异性的方法;在Olympus倒置显微镜下用毛细玻璃微针手工分离黄鳝减数分裂Ⅰ终变期3号染色体,将期DNA作模板进行DOP-PCR扩增后,分别以α^-32P-dCTP和Biotin-11-dUTP标记的单条染色体DNAPCR扩增产物及Biotin-11-dUTP标记的大豆18SrRNA基因作探针进行Southern杂交,FISH和Dot杂交来检测其特异性,结果表明：（1）减数分裂Ⅰ终变期染色体标本是进行染色体显微操作的理想材料;（2）DOP-PCR扩增产物片段在200-1000bp之间,平均600bp左右;（3）杂交结果显示,本研究所获得的单条染色体是黄鳝3号染色体;（4）与显微操作仪和微激光分离相比较。该方法不需要昂贵仪器,在常规实验室即可操作,具有广泛的普及应用意义。     

番木瓜单染色体的显微分离与克隆

用玻璃针显微分离出番木瓜(CaricapapayaL.)单染色体,经过LA-PCR扩增得到80-700bp的DNA片段。Southern杂交表明,扩增片段与番木瓜基因组DNA之间有同源性,从而证明番木瓜单染色体DNA已经被成功扩增。将扩增产物克隆到pGEM-T-Easy载体中,约获得1.18×105个克隆,酶切鉴定插入片段大小为100-400bp。     

结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

用RAPD和染色体原位杂交检测Elymus rectisetus染色体附加到普通小麦中

报道小麦×Elymusrectisetus属间杂种BC2F236个衍生系,通过染色体原位杂交和RAPD分析,检测Elymusrectisetus染色体及其特异染色体DNA标记。原位杂交结果表明,36个衍生系中大多数系含1对异源Erectisetus染色体,少数系含3条以上Ereclisetus染色体;RAPD结果表明,有13个随机引物（OPBA-08、OPB－14OPEA-09、OPF-05、OPF－09、OPJ-05、OPK-03、OPN-12、OPP-20、OPS－12、OPT-20、OPZ-09、OPZ-11）能够在这36个衍生系中分别扩增出普通小麦所没有的Erectisetus特异的染色体DNA片段。其中Erectisetus特异染色体DNA片段OPF－05480bp、OPF－09650bp和OPZ-11350bp只能够在“1048”系统的全部8个株系中扩增出;OPN-12350bp只能够在“1057”系统的全部6个株系中扩增出;OPB-14900bp和OPM-05420bp只能够在“1034”、“1026”2个系统的全部22个株系和“1057”－5－3株系中扩增出。直接获得了3个类型不同的异附加系,省去常规通过测配和附加系两两杂交F1PMCMI细胞学鉴定来确证异附加系之间的异同。由此说明染色体原位杂交和RAPD分析有机结合是鉴定异附加系（异代换系）快速有效的方法,且方法简单、易行．     

刺鳅X染色体DNA文库的构建

刺鳅（Mastacembelus aculeatus）是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割（Microdissection）和兼并引物PCR（DOP-PCR）方法,从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA,利用T载体和电转化方法,建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp,理论上包含X染色体98％以上的序列。当用荧光原位杂交（FISH）来验证文库的专一性时发现,在无竞争性DNA杂交条件下,整个X和Y染色体上都表现出强烈的杂交信号,并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时,常染色体上的信号消失,仅性染色体上异染色质区域保留有较强信号。就此,本文对刺鳅性染色体上的序列类型进行了探讨。     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd∶YAG激光微束将处于有丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收。将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增。Southern杂交证明4个特定区域的DNA确实来自于小麦基因组。用一系列(42对引物)位于6B染色体和其他染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证。结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体。将6B染色体4个片段的第二轮PCR产物克隆到pGEMT-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×10~5、2.74×10~5、2.45×10~5和2.93×10~5个重组子克隆。每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证。结果显示:插入片段大小在300～1800 bp之间,平均大小为820～870 bp,其中43％～48％的克隆为低/单拷贝序列,42％～47％为中/高拷贝序列。本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础。     

糖多孢红霉菌同源片段长度与染色体重组率关系的研究

为了探索同源片段长度与糖多孢红霉菌染色体同源重组率的关系,化学合成或用重叠PCR合成带有突变位点、在突变位点两侧长度为（26bp+27bp）、（500bp+576bp）和（1908bp+1749bp）的同源序列,克隆于糖多孢红霉菌同源重组载体pWHM3后,分别构建了pWHM1113、 pWHM1116和 pWHM1119质粒。以PEG介导转化糖多孢红霉菌A226原生质体,3个质粒分别获得每皿30个、69个和170个转化子,但pWHM1113质粒不能与染色体有效整合,pWHM1116质粒与染色体整合率为转化子的2%,而pWHM1119质粒与染色体整合率达到转化子的19%。 pWHM1116和 pWHM1119质粒均可进行有效的染色体二次重组,将突变位位点引入染色体。因此,同源片段长度为（500bp+576bp）或更长时,可与糖多孢红霉菌染色体进行有效的单重组和双重组。     

大豆单染色体的显微分离及体外扩增

采用玻璃针分离法,通过显微操作器成功地分离到大豆（ＧｌｙｃｉｎｅｍａｘＬ．）单染色体。将分离到的两条大豆染色体分别放入两个０．５ｍＬＥｐｐｅｎｄｏｒｆ管中,经Ｓａｕ３Ａ酶切,并在染色体ＤＮＡ片段两端加上Ｓａｕ３Ａ人工接头后,进行两轮ＰＣＲ扩增,得到０．３～３ｋｂ之间的ＤＮＡ片段。Ｓｏｕｔｈｅｒｎ杂交表明,这些大豆单染色体扩增片段与大豆基因组ＤＮＡ之间有同源性,从而证明两条单染色体ＤＮＡ确实已被成功地扩增了,同时表明两条不同的大豆单染色体扩增产物存在一定的差异。在常规的倒置显微镜下对小型染色体进行了显微分离,为小型单染色体ＤＮＡ的体外扩增及微克隆奠定了基础。     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Representational difference analysis using minute quantities of DNA.

Representational difference analysis (RDA) is a differential hybridization method which can effectively isolate unique DNA sequences from complex and highly related genomes or cDNA libraries. A major drawback of the RDA analysis is the requirement for pure driver and relatively pure tester samples, ruling out the analysis of whole tissue biopsies. To circumvent this problem, we have modified the technique for the analysis of very small quantities of DNA so that pure cell populations isolated by micromanipulation from tissue sections can be analyzed. Using this modified technique, as few as 50 diploid cells ( approximately 500 pg of DNA) can be analyzed.     

Labile DNA sequences in flax identified by combined sample representational difference analysis (csRDA)

Flax (Linum usitatissimum) has a genome in which changes have been associated with environmental factors. The inbred flax variety, Stormont Cirrus (Pl), served as the parent, and several lines (termed genotrophs) were derived from this parent. The phenotypes of the genotrophs were stable in a number of different growth environments, unlike the original Pl line in which changes associated with environmental factors continued to occur. These genotrophs differed from the original line in a number of characteristics, but the only known phenotypic characteristic that is shared by all the genotrophs and different from the parental, Pl, line is the lack of changes associated with the original environmental factors. However, some of these genotrophs have changed in both phenotype and nuclear DNA subsequent to their original growth and differentiation from Pl. Representational difference analysis (RDA) has been used to identify differences between Pl and all the genotrophs in an attempt to identify the loci controlling these aspects of plasticity. Subtractions between Pl DNA as a tester (target) and one of the genotrophs (individual RDA) or a mixture of different types of genotroph (L6, S6, C2, and LH) DNAs as a driver were done (combined sample RDA; csRDA). In addition, contrary RDA, where of the genotroph DNA was used as a tester and Pl DNA as a driver, was also executed. Three difference clones (163-4-2, 123-5-2, and 163-13), from 74 primary clones obtained after three rounds of subtractions with Pl DNA as tester were further characterized. In addition, 2 difference products (213-r1 and 213-r9) were characterized from contrary RDA. The clones 163-4-2 and 163-13 from the csRDA showed polymorphisms between Pl and all the genotrophs when PCR was done with primers derived from sequences of the clones, but only the clone 163-13 polymorphism was confirmed by Southern blot analysis. Four of 5 clones (163-4-2, 123-5-2, 163-13 and 213-r9) that have been characterized appear to be associated with structural changes in the DNA. From the contrary csRDA, it was observed that no clones could be recovered from subtractions between a mixture of genotrophs as a tester and Pl as a driver, and several possible explanations have been proposed.     

Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

Leptospirosis is a globally important zoonotic diseasecaused by the pathogenic species of the spirochete genus,Leptospira including L. interrogans, L. kirschneri, L.noguchii, L. borgpetersenii, L. santarosai, L. weilii andetc. [1]. Pathogenic leptospires …     

The Isolation of differentially expressed mRNA sequences by selective amplification via biotin and restriction-mediated enrichment

Molecular analysis of development frequently implies the isolation and characterization of genes with specific spatial and temporal expression patterns. Several methods have been developed to identify such DNA sequences. The most comprehensive technique involves the genomewide probing of DNA sequence microarrays with mRNA sequences. However, at present this technology is limited to the few organisms for which the entire genome has been sequenced. Here, we describe a subtractive hybridization technique, called selective amplification via biotin and restriction-mediated enrichment (SABRE), which allows the selective amplification of cDNA fragments representing differentially expressed mRNA species. The method involves the competitive hybridization of an excess of driver cDNA fragments (D) to a trace of tester cDNA fragments (T), and the subsequent purification of tester homohybrids (in which both strands are contributed by the tester cDNA). After competitive hybridization, cDNA fragments that are more abundant in the tester than in the driver are enriched in the tester homohybrids. However, as the fraction of tester homohybrids is very small [T(2)/(D + T)(2)], their purification requires highly efficient procedures. In SABRE, the isolation of tester homohybrids is afforded by a combination of three successive steps: removal of biotinylated terminal sequences from most of the heterohybrids by S1 nuclease digestion, capture of biotinylated hybrids with streptavidin-coated paramagnetic beads, and specific release of homohybrids from the beads by restriction nuclease digestion. If several rounds of SABRE selection are performed in series, even relatively rare differentially expressed mRNA sequences may result in the production of predominant cDNA fragments in the final tester homohybrid population.     

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.     

Comparison of mealybug (Planococcus lilacinus) and fruit fly genomes: isolation and analysis of conserved sequences and their utility in studying synteny in the mealybug

By using ligation-mediated PCR products from mealybug DNA as tester and biotinylated fly DNA as driver, we recovered a fraction of the tester that remains hybridized to driver following high-stringency washing conditions. This fraction is expected to contain mealybug sequences conserved in the fly (MCF). Reciprocal experiments enabled the isolation of fly sequences conserved in the mealybug (FCM). Coding sequences among MCF show amino acid identities >40% with fly proteins, allowing a reliable identification of orthologs. Three sequences from the fly cytogenetic positions 98-99 were hybridized onto mealybug chromosomes and the results identified differences in synteny between the two species. Taken together, our results present a method for direct isolation of sequences conserved between an 'orphan' (mealybug) genome and a 'reference' (fly) genome and showed that these sequences can be used to study chromosome synteny in the mealybug.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。