Azure mutants: a type of host-dependent mutant of the bacteriophage f2

A new type of host-dependent mutant, azure mutant, of bacteriophage f2 has been isolated. Growth of these mutants was restricted specifically by amber suppressor genes in the host bacteria. Restriction of the formation of infective centers by different bacterial suppressor genes was 98 percent, 90 percent, and 70 percent with Su-3, Su-1, and Su-2 genes, respectively. Restriction, like suppression, was the dominant phenotype. The block in growth of the mutants occurred in an early stage of the infection cycle. Once infection was established, however, an infected cell produced approximately the same number of progeny phage as a cell without the suppressor genes did. It is proposed that the azure codon is the same as the amber codon (uracil, adenine, guanine) and that restriction results from improper termination of protein chains of the phage RNA polymerase. Similar mutants may exist in other systems.     

Molecular Genetics of the Bithorax Complex in Drosophila melanogaster

The bithorax complex in Drosophila melanogaster is a cluster of homeotic genes that specify developmental pathways for many of the body segments of the fly. The DNA of the bithorax complex has been isolated, and a region of 195,000 base pairs that covers the left half of the complex is described here. The lesions associated with many of the bithorax complex mutants have been identified, and most are due to DNA rearrangements. Most of the spontaneous mutants have insertions of a particular mobile element named "gypsy." This element affects the functions of sequences removed from the site of insertion. Mutant lesions for a given phenotypic class are distributed over large DNA distances of up to 73,000 base pairs.     

Immune control of tuberculosis by IFN-gamma-inducible LRG-47

Interferon-gamma (IFN-gamma) provides an essential component of immunity to tuberculosis by activating infected host macrophages to directly inhibit the replication of Mycobacterium tuberculosis (Mtb). IFN-gamma-inducible nitric oxide synthase 2 (NOS2) is considered a principal effector mechanism, although other pathways may also exist. Here, we identify one member of a newly emerging 47-kilodalton (p47) guanosine triphosphatase family, LRG-47, that acts independently of NOS2 to protect against disease. Mice lacking LRG-47 failed to control Mtb replication, unlike those missing the related p47 guanosine triphosphatases IRG-47 or IGTP. Defective bacterial killing in IFN-gamma-activated LRG-47-/- macrophages was associated with impaired maturation of Mtb-containing phagosomes, vesicles that otherwise recruited LRG-47 in wild-type cells. Thus, LRG-47 may serve as a critical vacuolar trafficking component used to dispose of intracellular pathogens like Mtb.     

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。     

Landscape of somatic retrotransposition in human cancers

Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.     

禾谷镰孢碳源代谢调控因子FgCreA的功能

【目的】敲除禾谷镰孢（Fusarium graminearum）中碳源代谢调控因子FgCreA,并对其营养生长、有性生殖和致病力等方面进行研究,为研究禾谷镰孢中碳源代谢机制提供依据。【方法】根据酵母数据库SGD和NCBI数据库中的序列,利用酿酒酵母中碳源代谢调控因子Mig1确定禾谷镰孢中的碳源调控因子FgCreA;在NCBI数据库中检索其他物种中碳源代谢调控蛋白,使用ClustalW2软件进行多重序列比对,并用MEGA5软件构建系统进化树,同时在InterProScan网站上预测其蛋白结构域;在NCBI数据库中检索与禾谷镰孢碳源吸收相关的结构基因和真菌毒素DON生物合成的相关基因,调取起始密码子上游1 000 bp的片段,预测FgCREA基因结合位点位置;用Primer5软件设计引物,利用Split-PCR和PEG介导原生质体转化的方法进行基因敲除,并通过PCR和Southern blot验证获得FgCREA基因敲除突变体Fgcrea。根据突变体Fgcrea营养生长、有性生殖和致病力等方面的变化分析FgCREA的功能。【结果】通过生物信息学方法,明确禾谷镰孢中只有一个碳源代谢调控因子基因FgCREA(FGSG_09715),氨基酸序列416 aa,共包含两个保守的C2H2锌指结构区域。FgCreA同源蛋白在各类真菌中均存在一定程度的同源性,具有较高的保守性。禾谷镰孢碳源吸收相关结构基因（XYL2、ARA1、ICL1、PG1、SUC2）和真菌毒素DON生物合成相关基因（TRI1、TRI3、TRI4、TRI5、TRI6、TRI7、TRI8、TRI10、TRI12、TRI101）启动子区均含有FgCREA DNA结合位点。采用Split-PCR技术和PEG介导的原生质体转化,通过PCR和Southern blot获得并验证了两个FgCREA基因敲除突变体。表型观察发现,突变体Fgcrea的生长速度与野生型相比下降90%;分生孢子形态正常,但产孢量与野生型相比降低88%;有性生殖阶段,突变体Fgcrea可以产生正常的子囊壳、子囊和子囊孢子,但需要比野生型长20-28 d;突变体Fgcrea对钠盐较敏感,侵染小麦穗不发病。【结论】获得禾谷镰孢碳代谢调控因子FgCreA敲除突变体,明确FgCreA参与营养生长和有性生殖,并能够影响致病过程。但是否影响相关结构基因的转录水平和DON生物合成仍需进一步验证。     

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。     

安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。     

水稻组织培养中Tos17诱发体细胞无性系变异纯合体筛选技术初探

利用PCR技术对从日本引进的Tos17插入水稻极限糊精酶基因变异体后代进行了插入位点鉴定和纯合体筛选分析。结果表明,利用Tos17末端2 6bpDNA序列和插入位点上下游各2 0 0bp和30 0bp处的目标基因序列片段设计引物,可以有效地进行Tos17插入突变纯合体筛选,为突变体的进一步利用提供了分子依据。     

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。     

高效、新T-DNA侧翼序列分离技术——Actail-PCR

 【目的】建立一种有效分离T-DNA插入突变体侧翼基因组序列的方法。【方法】分离侧翼的目标基因组序列是利用T-DNA标签法研究植物功能基因组学的一个关键步骤,笔者发展稳定高效的Actail-PCR分离技术。该技术一侧引物来自于T-DNA载体,另一侧引物为一个复性控制引物。复性控制引物在3'端为一个14 bp的随机简并引物,在5'端非目标尾部序列接上一个通用引物,中间由5个多聚脱氧次黄嘌呤核苷（poly（dI））连接。【结果】Actail-PCR技术将随机简并引物重新编辑成复性控制引物,在40℃的低严谨条件下,3'端的随机简并引物可以与T-DNA插入突变体的侧翼目标区段随机的结合,扩增得到目标片段,随后利用5'端的通用引物与T-DNA载体上的巢式引物依次组合,在65℃（10个循环后逐步降温至58℃）的高严谨条件下逐步扩增特异片段,同时抑制非特异性扩增,可以显著提高目标片段的扩增效率,减少假阳性。【结论】相对传统的TAIL-PCR,Actail-PCR技术可以更高效地分离T-DNA插入突变体的侧翼基因组序列。     

Transfer of Agrobacterium DNA to Plants Requires a T-DNA Border But Not the virE Locus

Agrobacterium tumefaciens induces tumors in plants by transferring and integrating oncogenes (T-DNA) into the chromosomes of host plant cells. Agrobacterium strains were used to transfer complementary DNA copies of a potato spindle tuber viroid (PSTV) to plant cells at a wound site on tomato plant stems. Subsequently, infectious viroid RNA was found in the leaves of these plants, indicating systemic PSTV infection. This process utilized the T-DNA transfer mechanisms of Agrobacterium since PSTV infection required most virulence genes (vir) as well as one of the DNA sequences that flank either side of the Agrobacterium T-DNA. However, transfer still occurred from virE mutants of Agrobacterium, strains that fail to induce tumors even though a completely functional T-DNA is present. The virE gene seems to be directly involved in the integration of foreign DNA into plant chromosomes.     

优质水稻黄华占雄性不育突变体的筛选及初步分析

利用EMS(甲磺酸乙酯)处理优质水稻品种黄华占种子,经过田间筛选和对M1、M2代连续的遗传分析得到317份独立的、能够稳定遗传的、由单隐性核基因控制的雄性不育突变材料.通过对花器官形态观察及花粉染色分析,初步将这些突变体材料分为6类,其中花粉发育突变体在不同的花粉发育时期出现异常.这些突变材料为深入研究水稻雄性不育分子机制及开展分子设计育种提供充足的材料.98个无粉型雄性不育突变体用于雄性不育基因TDR的测序分析,其中3个突变体在3个不同的位置上出现单碱基突变.     

水稻白叶枯病菌hrp调节基因hrpXoo的克隆与序列分析

 用化学方法诱变水稻白叶枯病菌PXO99A 菌株 ,获得 6株hrp-突变体 ,此突变体除丧失在非寄主烟草上激发过敏反应和在感病寄主水稻上的致病能力外 ,有些缺乏激发烟草产生HR的信号物质 ,有些在胞内存在此信号物质 ,而不能泌至胞外。来自Xanthomonasoryzaepv .oryzaeJXOIII粘粒基因文库的hrp基因克隆pUHRX2 4 5 ,所携hrp基因片段大小为 36 .8kb。系列亚克隆 36 .8kbhrp基因片段及各亚克隆对hrp-突变体功能互补作用的结果显示 ,3.3kbSacI片段为最小酶切功能片段。序列测定和分析结果表明 ,3.3kbhrp片段含hrpX oo基因和含与热激蛋白 90家族有关的 2个开放阅读框hspORF1和hspORF2。HspORF1在蛋白质数据库中未发现序列蛋白。HspORF2与Hsp90 Xo的同源性达 99%。hrpXoo与黄单胞菌中已报道的hrpX基因有很高的同源性(90 %以上 )。在黄单胞菌中高度保守的编码α 螺旋 转 α 螺旋结构的 6 0 bp核苷酸序列 ,在交叉功能互补时是必需的。不含此结构的hrpXoo (1.1kb)片段 ,可使JXOIII的hrp-突变体在水稻上具致病性和在非寄主烟草上激发产生HR ,但不能使来自PXO99A 和RS10 5的hrp-突变体恢复在烟草上激发产生HR的功能。黄单胞菌中已知HrpX的同列比较显示 ,X .oryzae和X .campestris种间在 88、196和 2 4 7位点的氨基酸上有     

根癌农杆菌介导的稻瘟病菌近等菌系的构建与初步应用*

 利用根癌农杆菌C58C1介导转化1株致病力极弱的野生型稻瘟病菌菌株CY2,成功地获得3000多个转化子,混合接种于携带25个不同抗病单基因的水稻近等基因系上,获得4株单胞致病突变体;继代培养和PCR检测表明,这些突变体均为T-DNA插入CY2的后代,而非自发突变或外源菌株污染。将4株突变体与实验室已保存的20株致病突变体分别回接水稻近等基因系,推断其携带的致病基因类型,结果表明突变体分别针对13个抗性单基因的18个单基因系发生了突变,其中针对Pi-a,Pik-s,Pi-sh单基因系表现出较高的侵染频率。由于这些突变体均来自同一个菌株,仅致病性存在差异,属于近等菌系,利用其分析出102个水稻品种中的34个品种的抗病基因组成,其中,抗病基因Pik-s,Pi-a,Pi-sh,Pi-19(t)分布频率较高,分别达70.59%,61.76%,52.94%和44.12%,且带有这类基因的糯稻品种多于杂交水稻。     

A gene expression map for Caenorhabditis elegans

We have assembled data from Caenorhabditis elegans DNA microarray experiments involving many growth conditions, developmental stages, and varieties of mutants. Co-regulated genes were grouped together and visualized in a three-dimensional expression map that displays correlations of gene expression profiles as distances in two dimensions and gene density in the third dimension. The gene expression map can be used as a gene discovery tool to identify genes that are co-regulated with known sets of genes (such as heat shock, growth control genes, germ line genes, and so forth) or to uncover previously unknown genetic functions (such as genomic instability in males and sperm caused by specific transposons).     

Genetic engineering of novel genomes of large DNA viruses

Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.     

耐辐射异常球菌高温胁迫下双组份系统 dtrAB基因的功能分析

分析预测了耐辐射异常球菌(Deinococcus radiodurans)基因组中的一对双组份蛋白DtrA（DR_A0009）和DtrB（DR_A0010）。生物学软件分析结果显示,DtrA含有组氨酸激酶（HisKA_3）结构域和ATP酶（HATPase_c）结构域,且有4个跨膜区;DtrB含有接收结构域和HTH结构域,且有Lux家族的调控蛋白。两者共同构成一个转录单元。分别构建了dtrA和dtrB基因缺失突变株（ΔdtrA和ΔdtrB）,发现42℃高温胁迫条件下,2个突变株的生长和生存率明显低于野生型菌株,而正常培养温度（30°C）条件下,突变株和野生型菌株的生长无差异。QRT\|PCR分析显示,dtrA或dtrB基因突变导致了热激相关基因发生下调。推测双组份蛋白DtrAB参与了耐辐射异常球菌在高温胁迫下的调控作用。