抗马铃薯晚疫病质粒的分子克隆及其体外PCR扩增

从一株假单胞菌中分离其质粒DNA,并将其转化到大肠杆菌DH5α中。通过晚疫病抑菌实验证明此菌对马铃薯晚疫病有很强的抑制作用,对该质粒进行纯化和限制性酶切位点分析,将此质粒重组到含有绿色发光蛋白基因的质粒载体中。获得的重组质粒在体外经PCR扩增证明抗晚疫病的重组质粒已克隆到含有绿色光蛋白基因的质粒载体中。     

一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

玉米线立体类质粒的克隆和鉴定

S型细胞质雄性不育玉米的线粒体类质粒S-1为一个长6.4kb的双链线状DNA分子。本文对基本上全长的S-1（只用限制内切酶PstI在两端各切掉128bp）在pUC18和pBR322两种质粒中进行了克隆得到4种杂合的质粒。限制性酶切图谱证明它们分别是S-1以不同方向克隆在两种质粒中的结果,Southern印迹杂交也证明S-1克隆在pBR322中.     

一步法快速质粒鉴定方法

对《分子克隆实验指南》(第三版)中的牙签法小量制备质粒DNA的方法做了改进,减少了 加样次数,免去冰浴、离心等步骤,改进后的方法在琼脂糖凝胶电泳前仅需一步加样和加热过程, 提高了实验效率。同时结合使用多孔道移液器和96孔板,更适合于在高通量筛选中鉴定阳性克 隆。实验对改进后的方法与《分子克隆实验指南》上的方法进行了比较,表明“一步法”的检测效 果、准确率与重复性均有到较好的结果。     

用于扩增较大片段DNA的PCR方法

用于扩增较大片段ＤＮＡ的ＰＣＲ方法方向东,陈淳（中科院上海生物化学研究所国家分子生物学实验室,上海２０００３１）诸江（上海第二医科大学上海血液学研究所,上海２０００２５）关键词ＤＮＡ扩增,ＰＣＲ自从Ｔａｑ（Ｔｈｅｒｍｕｓａｑｕａｔｉｃｕｓ）ＤＮＡ聚合...     

萘质粒ND1.860HindⅢ片段的分子克隆和限制酶图谱

萘质粒ND1.860经限制性核酸内切酶HindⅢ完全消化和部分消化所产生的限制片段,分别在大肠杆菌质粒pBR322中克隆。通过对含有ND1.860HindⅢ片段的17个重组质粒进行限制酶分析,建立了ND1.860质粒的HindⅢ、EcoRⅠ和XbaⅠ种内切酶26个切点的酶切图谱。     

基于PCR的质粒快速定点诱变方法建立与评价

基因突变对生命的进化具有重要意义,针对质粒的DNA定点诱变技术也是基因工程、蛋白质工程研究中的重要手段之一。为了提高质粒定点诱变的效率,本研究利用引物部分重叠的设计方案,使用Tm值相对固定的引物设计模式,将同一引物分为重叠区（Tm=50±2℃）和非重叠区（Tm=60±2℃）,并在严格控制模板用量（2 pg/kb）的基础上,通过20个PCR循环对目标质粒进行定点诱变扩增,随后取0.5 μL产物直接用于转化。在FastPfu Fly酶系中,利用此法构建了6个含碱基替换、缺失和插入的质粒,均获得成功,突变效率可达96%以上,阳性克隆获得数达70个以上。此外,利用4种不同PCR酶系对该法的适用性进行了评价,结果表明突变效率均可达93%以上,阳性克隆获得数均在10个以上。通过适当增加PCR模板用量（10 pg/kb）并使用纯化后的PCR产物进行转化,该法可适用于转化效率大于106（cfu/μg）的任意感受态细胞,对应的突变效率可大于91%,阳性克隆获得数大于20。根据本法的作用原理,该方案适合质粒中10～20 bp（因重叠区GC含量及碱基序列的不同而改变）以内的任意碱基替换和插入,以及任意长度的DNA片段缺失。且具有通用性强、耗时少、诱变成功率高、成本低、对感受态及转化效率无特殊要求等优点,适合各实验室的日常研究使用。     

质粒拯救法克隆细菌基因组大片段

[目的]细菌基因组大片段尤其是基因簇的克隆与操作,是细菌基因功能分析的一个难点.基因组测序工作的不断完成和序列信息的大量积累,为细菌基因组:DNA的操作提供了方便.本文报道了利用细菌的全基因组信息和质粒拯救法的原理建立的一种克隆细菌基因组大片段的方法.[方法]首先,根据基因组序列信息,在待克隆片段的一侧扩增一段DNA,并将其克隆到自杀载体上构建打靶质粒,然后,将打靶质粒整合到细菌的基因组中构建重组菌,提取重组菌的基因组DNA,酶切,自连,转化,将自杀质粒与待克隆的目的片段一起拯救出来.最后,根据需要将拯救的DNA片段亚克隆到新的载体中.[结果]我们利用该方法克隆了布鲁氏菌中长度为11kb的virB操纵子,并构建了互补质粒.将该质粒导入到virB的突变株中后使virB操纵子的转录活性得到了恢复,表明该策略切实可行.[结论]这种重组克隆策略给我们提供了一种新的对细菌基因组大片段进行操作的方法.     

志贺菌毒力大质粒大片段敲除方法的建立

目的:构建志贺菌毒力大质粒大片段缺失突变体库。方法:首先利用λ-Red重组系统构建弗氏2a志贺菌301株毒力大质粒特定位点缺失株,再在距离此位点20 kb处缺失另一突变位点,最后根据重组酶识别远端FRT位点的特性,将两个远端FRT位点之间的DNA序列全部缺失。结果:敲除了毒力大质粒24 kb的DNA序列。结论:利用λ-Red重组系统及FLP-FRT位点特异性识别重组系统可以对志贺菌毒力大质粒逐步进行大片段的敲除,构建大质粒大片段缺失突变体库。     

一种高效克隆PCR产物的方法

利用TaqDNA聚合酶3′末端非模板依赖性加碱基的特性,制备了3′末端带T载体,与18种PCR产物进行连接反应。结果,阳性克隆率达40％—70％,而对照组小于10％。证明该方法是一种高效节省适用范围广的好方法。     

长距离反向PCR技术高效扩增已知DNA片断的侧翼序列

为解决传统反向PCR技术扩增片段短、假阳性多的不足,建立了长距离反向PCR(LD I-PCR)扩增技术:0.5μg DNA/mL的反应体系使DNA酶解片段充分自身环化连接,其产物用25 nt~30 nt的序列特异引物进行长距离PCR。结果表明该方法能特异地扩增出长达16 kb的序列,在已知DNA片段的侧翼序列克隆方面具有高效、简便、特异的优点。     

Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice

We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community.     

Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library

We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes.     

转基因抗虫棉Bt基因插入区碱基组成分析

利用TAIL－PCR的方法克隆不同来源的转基因抗虫棉中外源基因插入区的侧翼序列并对其进行序列和结构分析,结果表明,同一个较基因的单构自交得到的不同株系中外源基因插入区的两侧DNA序列完全相同,不同的转基因抗虫棉虫的外源基因插入位置各不相同,不同来源的转基因品种外源基因插入的上游侧翼片段含有一段残留质粒片段,外源基因插入的下游侧翼片段为富含AT碱基结构,其中泗棉3号转基因抗虫品系中下游侧翼片段的AT碱基高达92％,Southern杂交结果显示这些侧翼序列为高AT含量的多拷贝序列,序列中没有发现拓扑异构酶的结合位点。     

Ds因子在烟草染色体插入位点旁邻顺序的克隆

用ＮｅｓｔｅｄＰＣＲ 方法从含Ｄｓ 因子的转基因烟草ＤＮＡ 中克隆了Ｄｓ 因子在烟草染色体插入位点的９ 个旁邻ＤＮＡ 片段,以这些片段作探针,和野生型烟草的ＤＮＡ 进行Ｓｏｕｔｈｅｒｎ 杂交,以检测这些片段在烟草基因组中的拷贝数,结果表明它们都属于烟草染色体上的低拷贝ＤＮＡ。另外,对这些ＤＮＡ 片段进行核苷酸序列测定,并将它们的顺序与Ｇｅｎｂａｎｋ 数据库中已有的核苷酸序列相比较,其中长度为１２８ 核苷酸的片段１ 和荷兰芹的４ＣＬ２ 基因的一个区段有５７ ．８ ％ 同源性,而另一长度为１６９ 核苷酸的片段３ 和百合中的反转座子的一个区段有６０ ．９ ％ 的同源性。这都表明Ｄｓ 因子在异源植物烟草中插入染色体单拷贝基因的机率很高,这对转座子标签法克隆基因是非常有利的。     

Structural Characterization of the virB Operon on the Hairy-root-inducing Plasmid A4

The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferringtumorigenic symptoms on plants upon infection by its host bacterium,Agrobacterium rhizogenes. The virB operon on pRiA4 has beensequenced and found to be composed of 11 genes, virB1 to virB11,whose products mostly appear to be associated with the cellmembrane. A novel structural characteristic is frequent overlappingsbetween the translation termination and initiation codons ofadjacent genes. This is indicative of fine tuning of relativetranslation frequencies for each VirB protein. These resultssupport the view that VirB multisubunit complexes provide facilitiesfor T-DNA transfer at the bacterial cell membrane. The structuralorganization of the pRiA4 virB operon was essentially identicalto that of the previously reported virB operons of tumorinducingplasmids, pTiC58 and pTiA6, and the corresponding VirB proteinsof the three plasmids were extremely homologous to one another.On the basis of the structural similarity of each VirB protein,the phylogenetic relationship among pRiA4, pTiC58, and pTiA6is discussed.     

具有原核基因启动子活性的蓖麻蚕染色体DNA片段

利用大肠杆菌启动子克隆载体pHE5,研究了蓖麻蚕染色体的Hind Ⅲ酶解片段在大肠杆菌中作为四环素抗性基因启动子的功能作用。蓖麻蚕染色体的Hind Ⅲ片段与pHE5重组,在大约10~6个重组子中获得953株启动子重组体。测定了这些含有重组质粒的菌株抗四环素能力,其中有4株在平板上抗四环素水平超过225微克/毫升。对其中pARP201的插入片段进行了限制性图谱分析和启动子活性区域的缺失定位,证明了pARP-DB(由pARP201衍生而来)中的约0.5kb的外源插入片段具有完整的原核启动子功能。我们分析了这段DNA的部分核苷酸顺序,发现它与原核基因启动子极其相似。     

Transgene structures in T-DNA-inserted rice plants

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3 ends or two 5 ends of the transferred DNAs. The 3 ends were mostly linked through 3–6 bp of the complementing sequence, whereas the 5 ends were linked through either precise junctions or imprecise junctions with filler DNA.     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。