Prophage S2 mutants in Haemophilus influenzae: a technique for their production and isolation

A procedure utilizing nitrosoguanidine has been developed to produce defective and temperature-sensitive mutants of prophage (S2) in lysogenic Haemophilus influenzae. The system should be generally applicable to all temperate phage systems. At saturating concentrations of phage DNA, more than 25 percent of recipient mutant lysogenic bacteria can be transformed to the wild type.     

大肠杆菌vpr基因突变株的蛋白表达及功能研究

 【目的】进一步确定vpr基因及相关蛋白的功能。【方法】在获得vpr同源基因突变株MC1061△vpr的基础上，构建了vpr基因突变株的回复株MC1061-C，并进行了噬菌体裂解试验、细胞壁吸附试验、蛋白表达和印迹试验。【结果】亲本株MC1061和回复株MC1061-C对VT2噬菌体C43b的裂解敏感，突变株MC1061△vpr抵抗噬菌体C43 d的裂解。噬菌体吸附细胞壁试验中，吸附30 min后，亲本株和回复株的上清液中分别存在6.4％和 5.2％的游离噬菌体，吸附到90 min时未有大量的游离噬菌体释放。噬菌体与突变株的细胞壁吸附30 min后，上清液中有85.4％的游离噬菌体存在，表明VT2噬菌体能与亲本MC1061和回复株MC1061-C的细胞壁不可逆吸附，但不能与突变株MC1061△vpr的细胞壁发生不可逆吸附。在蛋白表达中，Western blot显示回复株和亲本株均能转印出特异性90 000蛋白主带，而突变株没有。【结论】vpr基因表达的分子量为90 000的Vpr蛋白与VT2噬菌体的裂解敏感性有关，可能是VT2噬菌体的裂解受体。     

Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy

Electron microscopy of heteroduplex DNA molecules, composed of one strand of Escherichia coli phage lambda(+) DNA annealed to the complementary DNA strand of a lambda deletion or substitution mutant, permits visualization, as well as precise measurements and mapping, of the unpaired single-stranded regions of nonhomology in the otherwise double-stranded molecules. In the lambdab2 mutant, the central segment (13 percent) of the lambda(+) DNA molecule is shown to be deleted. In the hybrid phages lambda(i434) and lambda(i21) a segment of the right arm of the lambda(+) genome (5.5 or 7.6 to 9 percent) is replaced by the corresponding immunity regions of phage 434 (3.3 percent or phage 21 (4 percent) DNA. The b5 region in the lambdab5 mutant appears to be identical to the i(21) segment. From these data it is possible to estimate the size and posiion of those lambda genes which are replaced by the i(434) and i(21) segments. The method permits preparing complete physical maps of viral genomes with a precision heretofore unattainable.     

Measurement of suppressor transfer RNA activity

Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.     

玉米矮杆突变体的筛选与候选基因鉴定

挖掘矮杆基因对玉米耐密植品种选育具有重要意义。利用甲基磺酸乙酯(EMS)诱变郑58自交系获得一系列矮杆突变体,株高降低范围为10%～45%,穗位高降低范围为10%～65%,部分突变体的节间数目也发生了变化。对矮杆突变体细胞大小进行观察发现,突变体的细胞长度均缩短,缩短的范围为25%～75%,突变体株高的降低除了与细胞缩短有关外,还与细胞数目增加有关,为了进一步确定候选基因,对其中的5个突变体构建了F2分离群体,采取BSR高通量测序结合生物信息学分析,获得了多个玉米矮杆突变体的候选基因,为矮杆突变体基因的分离鉴定和开发应用提供了分析方法和新的材料。     

苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8的功能

【目的】前期转录组分析表明苹果树腐烂病菌（Valsa mali）两个多聚半乳糖醛酸酶(polygalacturonase,PG)基因Vmpg7和Vmpg8在病菌侵染定殖过程中显著上调表达。论文旨在分析Vmpg7和Vmpg8在病菌营养生长、致病力、果胶酶活性、对果胶的利用等方面的作用及基因双敲除对同家族内其他基因表达水平的影响,明确Vmpg7和Vmpg8在病菌致病过程中的生物学功能,为进一步解析苹果树腐烂病菌致病分子机理提供基础。【方法】通过qRT-PCR检测基因在病菌侵染过程中的表达水平及基因双敲除后PG家族内其他PG基因的表达量;利用double-joint PCR构建基因敲除载体并通过PEG介导的原生质体转化技术进行基因敲除,经4对引物PCR检测及Southern blot验证获得基因单敲除和双敲除突变体,并利用gap-repair技术对基因进行回复;利用PDA培养基常规培养法观察突变体营养生长状况;通过离体接种富士苹果叶片及枝条的方法检测基因缺失对病菌致病力的影响;通过3,5-二硝基水杨酸（DNS）比色法检测各突变体胞外果胶酶活性;利用Czapek培养基培养法分析突变体对果胶的利用情况。【结果】qRT-PCR分析表明,Vmpg7和Vmpg8在病菌侵染3 d后分别上调表达27.73和8.19倍。通过基因敲除技术,分别获得3个Vmpg7基因单敲除突变体、1个Vmpg8基因单敲除突变体和3个Vmpg7/Vmpg8基因双敲除突变体,并获得了Vmpg7和Vmpg8单敲除突变体的回复突变体。将突变体接种到PDA培养基上,菌落形态和生长速率均没有发生变化;接种到苹果叶片和枝条上,Vmpg7单敲除突变体及Vmpg7/Vmpg8双敲除突变体在叶片和枝条上的致病力显著降低,而Vmpg8单敲除突变体在叶片上的致病力显著降低;进一步对果胶酶活性及对果胶的利用分析,发现Vmpg8单敲除突变体及Vmpg7/Vmpg8双敲除突变体胞外果胶酶活性均显著降低,且所有突变体在果胶培养基上生长均明显减慢。此外,基因回复后突变体的致病力、果胶酶活性及在果胶上的生长速率均回复到野生型菌株03-8水平。重要的是,Vmpg7/Vmpg8双敲除后影响了PG家族中其他PG基因的表达水平,3个基因显著上调表达。【结论】多聚半乳糖醛酸酶基因Vmpg7和Vmpg8可能与同家族内其他基因协同作用,通过调节果胶酶活性参与苹果树腐烂病菌致病过程。     

Target-selected inactivation of the zebrafish rag1 gene

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 F1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.     

The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide

The production of nitric oxide and other reactive nitrogen intermediates (RNI) by macrophages helps to control infection by Mycobacterium tuberculosis (Mtb). However, the protection is imperfect and infection persists. To identify genes that Mtb requires to resist RNI, we screened 10,100 Mtb transposon mutants for hypersusceptibility to acidified nitrite. We found 12 mutants with insertions in seven genes representing six pathways, including the repair of DNA (uvrB) and the synthesis of a flavin cofactor (fbiC). Five mutants had insertions in proteasome-associated genes. An Mtb mutant deficient in a presumptive proteasomal adenosine triphosphatase was attenuated in mice, and exposure to proteasomal protease inhibitors markedly sensitized wild-type Mtb to RNI. Thus, the mycobacterial proteasome serves as a defense against oxidative or nitrosative stress.     

tRNAi(met) functions in directing the scanning ribosome to the start site of translation

The mechanism by which the scanning ribosome recognizes the first AUG codon nearest the 5' end of eukaryotic messenger RNA has not been established. To investigate this an anticodon change (3'-UCC-5') was introduced into one of the four methionine initiator (tRNAi(met) genes of Saccharomyces cerevisiae. The ability of the mutant transfer RNA to restore growth properties to his4 initiator codon mutant yeast strains in the absence of histidine was then assayed. Only the complementary codon, AGG, at the his4 initiator region supported His+ growth. The mutant transfer RNA also directed the ribosome to initiate at an AGG placed in the upstream region of the his4 message. Initiation at this upstream AGG precluded initiation at a downstream AGG in accordance with the "scanning" model. Therefore, an anticodon: codon interaction between tRNAi(met) as part of the scanning ribosome and the first AUG must function in directing the ribosome to the eukaryotic initiator region.     

批量筛选水稻T-DNA插入突变体库获得生殖发育相关突变体

针对花器官形态和种子发育突变表型对大型水稻T-DNA插入突变体库进行筛选,获得了大量突变体信息及材料,在9 760个突变体家系中筛选得到177个花器官形态和数量异常的突变家系,突变频率为1.81%;对9 760个家系中的3 432个家系筛选得到179个种子发育缺陷的突变家系,突变频率为5.22%。对所获得的270个突变家系进行了T-DNA插入的阳性检测,阳性率为64.8%。利用公共数据库RMD(Rice MutantDatabase,RMD)给定的侧翼序列,鉴定了其中1个结实率较低的突变体家系,表明其突变表型和T-DNA插入共分离,为深入研究该基因的功能提供了重要的遗传材料。     

水稻披垂叶突变基因研究进展

水稻是重要的粮食作物,全球有超过50%的人口以稻米作为主食。同时水稻又作为一种模式生物被各大研究机构所研究。水稻披垂叶突变体是由于控制水稻中脉形成的基因发生突变,导致基因发生功能缺失,使水稻叶片中脉的形成不能正常进行,从而出现披垂叶的性状。目前已经发现2个基因位点与水稻中脉形成有关,DL和DL2基因正是这2个不同的位点。研究水稻披垂叶突变基因,对研究水稻叶中脉的形成以及水稻理想株型的选育有重要的意义。对水稻披垂叶突变基因的研究进展进行综述,以对水稻这一类特殊株型得到更好地了解。     

3个水稻叶色突变体的遗传分析及基因定位

利用甲基磺酸乙酯对常规粳稻‘秀水09’进行诱变处理,获得了3个黄叶突变体Y1-1,Yl-2和Yl-3,多代自交稳定遗传.突变体与野生型‘秀水09’相比,分蘖减少、植株变矮、苗期叶片表现明显的黄色,随着生长发育的进行,叶片逐渐返绿.遗传分析表明这3个突变体的黄叶性状均受1对隐性核基因控制.利用SSR分子标记,将突变基因Yl-1定位在第3号染色体RM282与RM6080之间的7.5 cM范围内,将突变基因Yl-2和Yl-3定位在第5号染色体分子标记5-43w与RM1237之间,遗传距离分别为2.0 cM和6.8 cM.     

水稻果胶甲基转移酶OsTSD2基因突变体的分析

探讨水稻果胶甲基转移酶OsTSD2基因的功能,尤其是对生长发育的影响,筛选并鉴定了OsTSD2基因突变的3个突变体tsd2a、tsd2b和tsd2c。表型观察显示:突变体水稻结实率下降、种子粒长变短、粒宽变窄、千粒重下降;突变体的颖壳颜色变暗,颖壳的破裂程度加重并出现新的背部破裂;荧光免疫标记试验研究发现,突变体种子萌发率下降可能是由于突变体种子盾片中的甲基化果胶含量低所致。结果表明,OsTSD2基因的下调导致水稻的种子在发育和萌发过程中存在明显的缺陷。     

两个新的水稻叶色突变体形态结构与遗传定位研究

【目的】对2个新的水稻叶色突变体进行形态结构与遗传分析,并且初步定位这2个突变基因。【方法】在水稻育种材料中分别发现了一株白色条纹叶突变体和一株黄叶突变体,经多代自交已形成稳定的突变系。对突变体的主要形态特征与叶绿素组分等进行分析,观察叶绿体的超微结构,并以这2个突变系杂交产生的F2群体作为定位群体,应用SSR标记对突变基因进行初定位。【结果】与其野生型相比,白色条纹叶突变体的单株穗数减少12.86%,生育期延长11.27%,黄色叶突变体的株高降低31.08%,千粒重减少14.55%,生育期延长17.86%,并且2种突变体的叶绿素含量都显著低于其野生型。电镜观察结果表明：2种突变体的类囊体结构异常,与野生型水稻相比,黄色叶突变体的类囊体片层数变少,白色条纹叶中条纹部分的类囊体片层结构几乎消失,正常绿色部分的类囊体结构没有变化。遗传分析表明：这2种突变性状均受1对隐性核基因控制,位于不同染色体上,将突变基因暂时命名为st9(t)(stripe)、chl12(t) (Chlorophyll-deficit)。将st9(t)定位到第一染色体短臂最末端,与分子标记RM1331相距9.6 cM,且与标记RM3252等共分离;将chl12(t)定位到第三染色体短臂,与分子标记RM411、RM8208之间的遗传距离分别是1.2、5.1 cM。【结论】发现了2个叶色突变新基因,为下一步的基因克隆与功能分析奠定了基础。     

GTPase RABE1b参与拟南芥胚胎发育的研究

[目的]研究影响拟南芥早期胚胎发育分裂模式的基因。[方法]从拟南芥T-DNA插入的突变体库中分离到2个T-DNA插入突变体,通过表型观察胚胎发育情况。[结果]PCR-WALKING表明T-DNA插入位点分别位于基因At4g20360的5＇非编码区和启动子区,基因编码的GTPase RABE1b蛋白为Rab蛋白家族的成员,将这2个突变体分别命名为Atrabe1 b-1和Atrabe1b-2。透明分析发现此突变体在胚胎发育早期球形胚时期分裂模式出现异常,RT-PCR分析发现此基因在拟南芥中以组成型表达。[结论]基因At4g20360影响拟南芥早期胚胎发育的分裂模式,推测其编码的蛋白GTPaseRABE1b可能对控制拟南芥胚胎发育过程中的细胞分裂起重要作用。     

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

水稻短宽粒基因SWG1的图位克隆

【目的】水稻产量由单位面积有效穗数、每穗粒数和粒重3个因素构成,其中,粒重主要由水稻的籽粒形态决定。筛选和鉴定新的粒型突变材料和基因,可为产量性状的分子设计育种奠定基础。【方法】在籼稻保持系西大1B(XD1B)的甲基磺酸乙酯（EMS）诱变群体中鉴定到一个短宽粒突变体short and widen grain 1(swg1);分析籽粒形态和其他农艺性状,并对颖壳进行组织细胞学观察分析;运用BSA法进行基因定位;通过遗传互补试验确定候选基因;采用qRT-PCR分析该基因的表达模式及其他粒型相关基因和细胞发育基因的表达水平。【结果】农艺性状分析发现,与野生型相比,swg1突变体粒长显著降低,粒宽显著增加,表现出短宽粒的表型;进一步组织和细胞学分析,发现突变体颖壳纵向细胞变短是粒长变短的主要原因,而粒宽增加是由于颖壳横向细胞数目和细胞大小同时增加。遗传分析结果表明,该突变性状受隐性单基因控制,通过图位克隆与遗传互补验证,确定候选基因为LOC＿Os07g42410,编码一个植物特异转录因子。qRT-PCR分析发现该基因表达无明显的组织特异性,在茎、叶、幼穗中表达强烈。通过对已知粒型相关基因、细胞...     

烟草叶形突变体的形态特征与关键基因表达差异研究

为分析烟草叶片大小差异的原因,以‘红花大金元’野生型和两个叶片突变体（突变体1、突变体2）为材料,对叶片形态、解剖结构以及叶发育关键基因的表达差异进行研究。结果表明：突变体1的叶长、叶宽、叶面积分别为野生型的61.88%、25.87%、10.12%,比叶重是野生型的1.91倍;突变体2的叶长、叶宽、叶面积分别为野生型的78.82%、106.63%、83.92%,比叶重是野生型的1.35倍。突变体1同一层海绵组织细胞总数为野生型的10.93%,海绵组织细胞体积较野生型减小,叶片厚度显著增加,增幅为23.15%;突变体2同一层海绵组织细胞总数为野生型的48.12%,海绵组织细胞体积与野生型相比增加,叶片厚度显著增加,增幅为18.03%。基因NtARF2-1、NtDA1、NtTOR1、NtARF10-1、NtEBP1、NtGRF8、NtGRF16在突变体1中的表达量与野生型相比差异显著,其中NtEBP1上调表达最显著,上调2.22倍;NtGRF8、NtGRF16下调最显著,表达量分别为野生型的16.06%、13.07%。与野生型相比,NtTOR1、NtTOR2、NtARF10-1、NtEBP1的表达量在突变体2中显著上调,其中NtTOR1表达量上调最显著,上调3.17倍;NtARF2-1、NtARF2-2、NtDA1、NtGRF8、NtGRF16的表达量在突变体2中显著下调, NtGRF8、NtGRF16下调最显著,表达量分别为野生型的13.43%、21.68%。以上结果表明,突变体1与突变体2叶面积的减小与细胞总数的减少密切相关,突变体1、突变体2叶片细胞大小与数目的改变可能与基因NtTOR1、NtEBP1、NtGRF8、NtGRF16表达有关。     

蛋白质定向进化的高通量筛选方法

定向进化是改造蛋白质分子的有效新策略。创建突变库的方法已经有很多而且比较通用,而建立有效的高通量的筛选方法是蛋白质定向进化成功的关键。本文综述了近几年发展起来的用于定向进化的高通量的文库筛选方法,介绍了各种展示技术及流式细胞分选技术的原理及其在文库筛选中的应用,分析了存在的问题及发展趋势。