矮化早熟高粱突变体的RAPD分析

以随机扩增多态DNA(RandomAmplifiedPolymorphicDNA,RAPD)标记方法对航天搭载得到的矮化早熟高粱突变体进行遗传特性分析,利用适合玉米的引物对高粱进行引物筛选,从31种随机引物中共扩增出4条多态性片段,反映出突变体的遗传差异性。     

蒲公英耐盐突变体‘滨蒲1号'的耐盐性研究

土壤盐渍化是当今农业生产上的重要危害之一,它严重影响当地农作物的生长、发育及产量的提高。为了提高盐碱地的利用效率,该文以蒲公英耐盐突变体‘滨蒲1号’及其亲本叶片为材料进行丙二醛(MDA)含量等8种生理指标的测定,同时利用cDNA-RAPD技术对蒲公英耐盐突变体及其亲本根胁迫0、12、24 h的差异表达基因进行分析。结果表明:(1)突变体‘滨蒲1号’叶片中脯氨酸含量、叶绿素含量、可溶性蛋白含量、CAT活性、POD活性、SOD活性在不同胁迫时间点均大体高于亲本; MDA含量、相对电导率低于亲本。(2)以筛选出的10条RAPD引物进行cDNA-RAPD分析,共扩增出22条清晰的条带,差异条带10条,多态性为45.4%。(3)扩增产物片段大小在150～1 000 bp之间,主要为盐抑制基因片段,推测蒲公英耐盐突变体‘滨蒲1号’的耐盐性既与渗透调节物质脯氨酸及多种抗氧化酶上升引起的保护作用有关,也与根中一些与耐盐相关的基因表达变化有关。该研究为进一步克隆蒲公英耐盐基因并利用基因工程手段培育耐盐优质的蒲公英新品系奠定一定的理论基础。     

利用SSR分析红菜苔的遗传多样性

红菜苔是我国独有的蔬菜资源,深受消费者喜爱。利用63对SSR引物检测来自全国的45份红菜苔种质资源的遗传多样性,63对引物扩增出124条带,平均每条引物扩增出近2条带,扩增产物片段大小都在150～300bp之间,相似系数在0.56～0.89之间。结果表明,红菜苔具有丰富的遗传多样性,本研究为菜苔资源的利用和育种提供了分子生物学依据。     

茄子耐盐种质资源遗传多样性的SRAP和ISSR分析

利用SRAP和ISSR分子标记,研究了14份耐盐茄子种质资源的遗传多样性,结果表明,2种标记均能揭示材料间较高的遗传多样性,其中ISSR标记多态性略高于SRAP标记。在SRAP分析中,每对引物组合可扩增出8-15条DNA片段,平均为12．12条：26对SRAP引物组合共扩增出315条DNA片段,其中263条具有多态性,多态性比率为83．49％;材料间遗传相似系数变化范围为0．212～0．923,平均值为0．755。在ISSR分析中,每个引物可获得5～16条DNA片段,平均为10．87条;15个ISSR引物共扩增出163条DNA片段,其中141条具有多态性,多态性比率为86．50％;材料间遗传相似系数变幅为0．333-0．957,平均值为0．736。聚类分析表明,2种标记都能将供试材料完全区分开来,聚类结果具有一定的相似性,但也存在明显差异。Mantel相关分析表明,SRAP分析与ISSR分析的相关性达到极显著性水平（r=0．904,P〈0．01）。     

巨型引物PCR法对抗CD3改型单链抗体

亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.     

利用RAPD和ISSR标记分析青麻种质遗传多样性

利用RAPD和ISSR分子标记检测来自全国11个省(市)的48份青麻种质资源的遗传多样性,为青麻资源利用和育种提供分子生物学依据.在48份青麻种质资源中,17条RAPD引物扩增出191条带,多态条带比率为87.43%;9条ISSR引物扩增出82条带,多态条带比率为88.89%,扩增产物片段大小都在0.1～3.0kb之间.两种分子标记的结果呈显著正相关(r=0.80).基于UPGMA聚类,野生种和栽培种各自聚为相应的类别.     

RAPD分析在绢丝昆虫亲缘关系研究中的应用：Ⅱ．柞蚕品种间的遗传差异

采用RAPD技术,对5个柞蚕品种的遗传差异进行比较研究,结果表明,所采用40个随机引物中,有27个引物扩增谱清晰且重复性较好,扩增总片段数253条,单个引物的扩增片段数在4－16之间,片段大小在0．33－3．0kb之间。不同柞蚕品种间的遗传差异较小,遗传距离（D）在0．066－0．1659之间,根据D值,由UPGMA聚类分析软件绘制了它们的分子进化树。     

辐射诱发唐菖蒲复色花突变体的AFLP分析

以唐菖蒲品种'新秀'的种球为材料,经不同剂量50Co γ射线诱变后,进行生物学性状观察,并以'新秀'为对照.用AFLP分子标记对突变体进行多态性分析.结果显示,(1)诱变后代出现了广泛变异,并从75 GY剂量组M:代中选出了花瓣呈粉白相间的复色花突变体.(2)突变体的大多数引物扩增产物带型与对照带型差异显著;64种引物中50对引物检测出DNA分子的多态性,产生1 600条清晰谱带,多态性位点112个,多态性13.08%,相似性系数为93%.差异片段主要集中于100～700 bp之间.研究表明,复色花突变体和对照之间的差异与遗传物质的改变相关,其中一些可能与花色形成基因有关.     

应用RAPD技术分析奥利亚罗非鱼和尼罗罗非鱼的基因多态性

以随机扩增多态DNA技术(RAPD）分析了奥利亚罗非鱼和尼罗罗非鱼两个养殖群体的群体内及群体间遗传关系,并探讨了该技术在种群鉴定中的应用。RAPD引物筛选结果表明,所测试的20个随机引物中(Table 1),除一个引物未扩增出任何片段外,其余19个引物均扩增出1～11个大小不等的片段,长度大部分在500—3000bp之间,共扩增出220个片段,平均每个引物产生55个片段。两群体间共有片段70条,大部分引物的扩增产物具有种间多态性,种群间相似系数为0.727。以筛选的引物对两种群不同个体(Fig．1,Table 2)及种群混合样品(Fig.2,Table 3)进行RAPD分析。结果表明,不同引物在扩增图谱上表现很大差异：奥利亚罗非鱼不同个体间表现为一致的扩增图谱,种内相似系数达1000,显示了其种群内遗传变异的缺乏;尼罗罗非鱼种内相似系数为0．827,个体间存在不同程度的多态性;两个种群间的相似系数分别为0．767和0.742,表明种间有较高的同源性,遗传距离为0．235,略低于国外的报道．此外,两个养殖群体间的扩增图谱比较也暗示了遗传渐渗现象的存在。实验表明,RAPD标记可以作为一种可靠的遗传标记,用于不同鱼类种群的鉴定,RAPD分析方法是一种快速,简便且行之有鼓的鉴定鱼类种群的方法。     

应用RAPD技术分析奥利亚罗非鱼和尼罗罗非鱼的基因多态性(英文)

以随机扩增多态ＤＮＡ技术（ＲＡＰＤ）分析了奥利亚罗非鱼和尼罗罗非鱼两个养殖群体的群体内及群体间遗传关系,并探讨了该技术在种群鉴定中的应用。ＲＡＰＤ引物筛选结果表明,所测试的２０个随机引物中（Ｔａｂｌｅ　１）,除一个引物未扩增出任何片段外,其余１９个引物均扩增出１～１１个大小不等的片段,长度大部分在５００～３０００ｂｐ之间,共扩增出２２０个片段,平均每个引物产生５．５个片段。两群体间共有片段７０条,大部分引物的扩增产物具有种间多态性,种群间相似系数为０．７２７。以筛选的引物对两种群不同个体（Ｆｉｇ．１,Ｔａｂｌｅ２）及种群混合样品（Ｆｉｇ．２,Ｔａｂｌｅ３）进行ＲＡＰＤ分析。结果表明,不同引物在扩增图谱上表现很大差异。奥利亚罗非鱼不同个体间表现为一致的扩增图谱,种内相似系数达１０００,显示了其种群内遗传变异的缺乏;尼罗罗非鱼种内相似系数为０．８２７,个体间存在不同程度的多态性;两个种群间的相似系数分别为０．７６７和０．７４２,表明种间有较高的同源性,遗传距离为０．２３５,略低于国外的报道、此外,两个养殖群体间的扩增图谱比较也暗示了遗传渐渗现象的存在。实验表明,ＲＡＰＤ标记可以作为一种可靠的遗传标记,用于不同鱼     

Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes

We isolated a collection of chemotaxis mutants and characterized them for chemotactic phenotype and genotype. The mutations of most of these mutants mapped in the region between pyrD and thyA. However, the mutation in the gene specifying the chemotactic methyltransferase mapped very close to aroF. From a bank of phages containing Bacillus subtilis DNA we identified two lambda charon4 phages that contained genes specifying chemotactic functions. The inserted DNAs were removed by digestion with restriction endonuclease EcoRI and were found to share a 4.0-kilobase (kb) fragment. One of these DNAs also contained a 7.7-kb fragment, and the other also contained a 10.9-kb fragment. We identified mutants that were complemented by each fragment. The fragments were each ligated into plasmid pFH7 and were incorporated into lysogenic SP beta c2 or a deletion mutant of SP beta c2 in order to form transducing phages. The mutants in the collection containing mutations that mapped in the region between pyrD and thyA were tested for complementation by transducing phages containing the 4.0-kb fragment, the 7.7-kb fragment, the 4.0-kb fragment plus the 7.7-kb fragment, and the 10.9-kb fragment. A total of 24 mutants were complemented by the 4.0-kb fragment, 7 were complemented by the 7.7-kb fragment, 9 were complemented by the 4.0-kb fragment plus the 7.7-kb fragment, 15 were complemented by the 10.9-kb fragment, and 25 were complemented by none of the fragments.     

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides .     

Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts

Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture. The mutants were classified into three groups. Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome. The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment. Attempts to complement mutants of this second group with cloned DNA were unsuccessful. The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants. The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants. The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants. Mutants in each of these groups were mated with R. leguminosarum strains that nodulated peas (R. leguminosarum biovar viciae) or clovers (R. leguminosarum biovar trifolii). Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R. leguminosarum biovar viciae or R. leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases. These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.     

The molecular nature of spontaneous and chemically induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene in rat liver epithelial cells

The molecular nature of mutations in 6-thioguanine-resistant hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient clones of an adult rat liver (ARL) epithelial cell line mutated by benzo[a]pyrene or aflatoxin B1 was studied. DNA from these clones or spontaneous HGPRT-deficient mutants was subjected to Southern blotting using an HGPRT probe following DNA digestion with the restriction enzymes BamH1, EcoR1, HindIII or XbaI. With either the chemically induced or spontaneous mutants, no difference in restriction fragment pattern was observed between any of the mutants and their wild-type parent. However, differences were found between two lines ARL 6 and ARL 14 and the lines ARL 18, ARL 19 and DNA from Fischer rat hepatocytes. Although the variants did not display loss of HGPRT activity. It is suggested that deletion or loss of a pseudogene sequence could be the basis for the alterations in restriction fragment patterns.     

Pleiotropic effects of mutants in gene A of bacteriophage phi chi 174.

It has previously been established that the functional gene A product of phi chi X 174 is required for double-stranded DNA replication and that mutants in gene A affect the lysis of the host cell. We report here other alterations of normal phenotype for a subset of gene A mutants suggesting additional functions of gene A. Mutants in the subset failed to terminate cellular DNA synthesis and were unable to efficiently inactivate the colony-forming ability of the host. Two mutants in a second group retained the ability to kill the infected cell, although only one of these mutants efficiently terminated cellular DNA synthesis. Normal termination of cellular DNA synthesis did not occur by the production of random multiple breaks in the DNA, although it may have occurred by the selective production of breaks in newly synthesized DNA. It has previously been shown that two protein products are produced from the gene A region, the smaller of which is a C-terminal fragment of the larger. The separate phenotypes reported here for the two groups of mutants in gene A are consistent with separate functions for the two gene products previously reported.     

Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.

To facilitate the determination of the genomic location of the vaccinia virus gene(s) encoding alpha-amanitin resistance (alpha r) (Villarreal et al., J. Virol. 51:359-366, 1984), a collection of alpha r, temperature-sensitive (ts) mutants were isolated. The premise of these experiments was that mutants might be found whose dual phenotypes were the result of a single or two closely linked mutations. Genetic analyses of the alpha rts mutant library revealed two mutants, alpha rts7 and alpha rts12, that apparently fit this criterion; in alpha rts7 the two lesions were indistinguishable, whereas in alpha rts12 the two mutations were closely linked but separable. Cloned vaccinia virus HindIII DNA fragments were used to marker rescue the temperature-sensitive phenotype of these two dual mutants. The temperature-sensitive lesion of alpha rts7 was rescued by the HindIII N fragment (1.5 kilobases), whereas alpha rts12 was rescued by the neighboring HindIII M fragment (2.0 kilobases). The progeny virions of the alpha rts7 HindIII-N rescue reverted to an alpha-amanitin-sensitive phenotype, whereas the alpha rts12 HindIII-M progeny were still resistant to the drug. Taken together, these data indicate that the gene encoding alpha-amanitin resistance maps to the HindIII N fragment and provides evidence for the existence of essential vaccinia virus genes in a region of the genome previously believed to be nonessential for replication in tissue culture. Biochemical analyses revealed that both mutants were capable of synthesizing DNA as well as early and late viral proteins at the permissive and nonpermissive temperatures. At the nonpermissive temperature alpha rts12 and alpha rts7 were unable to process the major core precursors P94 and P65 into VP62 and VP60.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.

We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .     

Studies of the Physiological and Genetic Basis of Acid Tolerance in Rhizobium leguminosarum biovar trifolii

Acid-tolerant Rhizobium leguminosarum biovar trifolii ANU1173 was able to grow on laboratory media at a pH as low as 4.5. Transposon Tn5 mutagenesis was used to isolate mutants of strain ANU1173, which were unable to grow on media at a pH of less than 4.8. The acid-tolerant strain ANU1173 maintained a near-neutral intracellular pH when the external pH was as low as 4.5. In contrast, the acid-sensitive mutants AS25 and AS28 derived from ANU1173 had an acidic intracellular pH when the external pH was less than 5.5. The acid-sensitive R. leguminosarum biovar trifolii ANU794, which was comparatively more sensitive to low pH than mutants AS25 and AS28, showed a more acidic internal pH than the two mutants when the three strains were exposed to medium buffered at a pH of less than 5.5. The two acid-sensitive mutants had an increased membrane permeability to protons but did not change their proton extrusion activities. However, the acid-sensitive strain ANU794 exhibited both a higher membrane permeability to protons and a lower proton extrusion activity compared with the acid-tolerant strain ANU1173. DNA hybridization analysis showed that mutants AS25 and AS28 carried a single copy of Tn5 located in 13.7-kb (AS25) and 10.0-kb (AS28) EcoRI DNA fragments. The wild-type DNA sequences spanning the mutation sites of mutants AS25 and AS28 were cloned from genomic DNA of strain ANU1173. Transfer of these wild-type DNA sequences into corresponding Tn5-induced acid-sensitive mutants, respectively, restored the mutants to their acid tolerance phenotypes. Mapping studies showed that the AS25 locus was mapped to a 5.6-kb EcoRI-BamHI megaplasmid DNA fragment, whilst the AS28 locus was located in an 8.7-kb BglII chromosomal DNA fragment.