水稻抗白叶枯病基因Xa4位点跨叠BAC克隆群的构建

水稻白叶枯病抗性基因Xa4已被定位于第11染色体长臂末端的分子标记VG181和L1044之间,并与抗性基因同源序列片段RS13共分离。利用这3个标记筛选IRBB56的BAC文库,共得到128个阳性BAC克隆,其中RS13获得18个阳性克隆,这18个克隆中有4个和6个我隆分别同时为G181和L1044的阳性克隆,选其中的12克隆进行分析,构建了一个从G181到L1044区间的BAC跨叠克隆,全长420kb,并且56M22、106P13和104B153个BAC克隆可覆盖整个跨叠克隆群。这一研究结果为进一步分离Xa4基因打下基础。     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Direct Cloning and Sequencing of Bacterial Artificial Chromosome (BAC) Insert Ends Based on Double Digestion

Conventional digestion and ligation was developed into a novel and efficient approach for directly cloning and sequencing the two ends of bacterial artificial chromosome (BAC) clone inserts. Most BAC vectors have two Not I sites. This end isolation method is based on double digestion of the BAC clone DNA with Not I and any blunt-end restriction enzyme for which there is not a restriction site located within the small fragment (containing the cloning site) between the two Not I sites on the BAC vector. Digestion is followed by ligation of the double-digested mixture with a suitable plasmid vector. The pBeloBAC11 and pBlueScriptII SK vectors were used in the present study. The two ends of the BAC insert can be amplified and sequenced with three specific primers, i.e., amplification of the left end with the pBeloBAC11 LF1 and pBlueScriptII KS primers, and the right end with the pBeloBAC11 RR4 and KS primers. They may be directly recovered by transformation if the end fragments are used as probes. More significantly, this simple strategy generally can be applied to any BAC vector with any cloning site.     

Genomic Bacterial Artificial Chromosome Library of the Japanese Flounder Paralichthys olivaceus

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC-lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis. Received March 1, 2000; accepted May 29, 2000.     

关于细菌人工染色体(BAC)文库载体DNA制备的研究

细菌人工染色体（BAC）文库在基因组研究中起着关键作用。构建BAG文库的一个关键步骤就是BAC载体DNA的制备,制备高质量的BAC载体DNA受到包括酶切,脱磷等诸多因素的影响。以BAC载体pECBAC1为材料,分别采用限制性内切酶BamHⅠ和HK脱磷酶对其进行酶切和脱磷,并结合凝胶回收缩化技术,制备了可用于进一步构建BAC文库的线性载体DNA。并在此基础上,确定了制备BAC载体DNA的适宜条件,其中包括确定适宜限制内切酶用量及酶切时间,脱磷酶种类及浓度和凝胶回收纯化线性载体DNA等关系步骤。     

Construction of a Bacterial Artificial Chromosome Library of Phytophthora infestans and Transformation of Clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

Summary A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at     

Serial Analysis of Gene Expression (SAGE) of<Emphasis Type="Italic"> Magnaporthe grisea</Emphasis>: genes involved in appressorium formation

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by E. Cerdá-Olmedo     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

沙鞭不同居群染色体数目及核型分析

该研究采用常规压片法对分布于内蒙古高原的6个沙鞭居群的染色体核型进行研究,探讨沙鞭不同居群的核型特征及其进化关系。结果表明：(1)沙鞭6个居群的染色体数目恒定,均为2n = 2x = 46。(2)染色体有正中部着丝粒(M)、中部着丝粒(m)、亚中部着丝粒(sm)和亚端部着丝粒(st)4种类型,且中部着丝粒类型数量最多。(3)沙鞭不同居群核型公式存在差异。(4)核型类型有1A、2A、1B和2B型4种,染色体平均臂比介于1.29 ~ 1.62,长度比为1.73 ~ 2.68。(5)核型不对称系数处于55.96% ~ 59.95%,核型对称性较高,进化程度较为原始,其中37居群的核型不对称系数最大,进化程度较高,34居群的核型不对称系数最小,进化程度较低。(6)沙鞭6个居群聚为两类,37居群单独聚为一类,其他所有居群聚为一类,表明37居群与其他居群具有相对较远的亲缘关系。该研究首次报道了沙鞭不同居群的染色体核型特征及其进化关系,为以后沙鞭的系统进化和优良种质资源筛选奠定了细胞学证据。     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

橄榄CaICE1基因的克隆和表达分析

为了解橄榄(Canarium album)抗寒相关转录因子ICE1的调控功能,采用RT-PCR技术克隆了‘福榄1号’的ICE1,命名为CaICE1,并进行生物信息学、qRT-PCR表达模式和相关miRNA预测分析。结果表明,CaICE1 cDNA序列的开放阅读框长度为1 650 bp,可编码549个氨基酸(GenBank登录号MG459422)。Ca ICE1为不稳定亲水性蛋白质,含有跨膜结构、磷酸化位点以及HLH保守结构域,定位于细胞核,与枳的ICE1亲缘关系较近。CaICE1密码子偏好性较弱,AGA、AGG、TGG和CCA可能为其最优密码子群。CaICE1主要在橄榄花、种子和叶中大量表达,-3℃低温胁迫下CaICE1表达水平比常温显著上升。psRNAtarget预测结果表明,CaICE1可能是miR825、miR477、miR5658、miR1436和miR394等多个逆境响应miRNA的靶基因。因此,CaICE1可能在橄榄低温胁迫过程中发挥重要调控作用,且可能受miRNA的调控。     

不同产地木耳菜的染色体核型分析

为了研究木耳菜核型特征及不同产地间的进化关系,以来自7个产地的8个木耳菜品种为材料,采用常规压片法进行核型分析,并进行核型进化趋势分析和主成分分析。结果表明:(1)所有木耳菜的染色体数目均为2n=2x=44,未见异常染色体,染色体类型均为中部着丝粒染色体(m)或近中部着丝粒染色体(sm),且m数量多于sm。(2)不同产地的木耳菜在染色体核型公式、核型类型、随体位置、染色体长度比、臂比及核型不对称系数等指标均存在明显差异;随体均为1对,但随体位置不同。(3)核型类型为1A、1B和2A型,其中1A型5种,数量最多。(4)染色体长度比范围为1.51~2.06,平均臂比值范围为1.30~1.48,仅有吉林‘利丰’和江西‘航城’存在臂比大于2的染色体。(5)核型不对称系数范围为56.25%~59.17%,核型的对称程度较高,推测木耳菜的进化程度较为原始,其中河北‘金发’是最原始,江西‘航城’最进化。研究结果为木耳菜的细胞遗传学研究提供了参考依据。     

茶树CsLEA5基因的克隆及表达分析

该研究以茶树‘龙井长叶’为材料,克隆获得了茶树胚胎发育晚期丰富蛋白基因CsLEA5的cDNA序列,该序列全长515 bp,包含一个375 bp的开放阅读框,编码124个氨基酸,预测蛋白分子量为13.5 kD,理论等电点为5.92。蛋白序列分析结果显示,CsLEA5为高亲水性和稳定性蛋白,且含有一个典型的LEA_3保守结构域,属于LEA蛋白中LEA_3亚家族成员。CsLEA5基因启动子区域包含多种与逆境响应相关的顺式作用元件,如乙烯响应元件（ERE）、胁迫响应元件（STRE）、创伤响应元件（WUN motif）及MYB、MYC转录因子识别位点等。qRT PCR分析显示,CsLEA5基因表达具有明显的组织特异性,在叶片中的表达量最高,其次是嫩茎,而在其他组织器官中的表达量较低,且CsLEA5基因表达受低温和干旱胁迫的诱导。研究表明,CsLEA5基因可能在茶树响应低温和干旱胁迫过程中发挥重要作用。该研究对了解茶树抗逆分子机制,筛选抗性候选基因资源提供了重要理论依据。     

Construction and Characterization of Large-Insert Genomic Libraries (BAC and Fosmid) from the Ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis> and Initial Physical Mapping of a Histocompatibility Locus

The colonial protochordate Botryllus schlosseri is genetically manipulable and represents a potential model organism for a variety of biological disciplines, including immunology, stem cell biology and development. This article presents the construction and characterization of both BAC and fosmid genomic libraries of the 725-Mbp B. schlosseri genome. The BAC library currently consists of 2× genome coverage with an average insert size of 80 kb. The fosmid library is at 11× genome coverage with an average insert of 40 kb. B. schlosseri is a small organism containing a large number of compounds that hinder DNA purification. Thus a number of protocols had to be modified in order to make purified, high molecular weight inserts for cloning, including both gel purification and insert concentration techniques. Both libraries were characterized by using them in initial physical mapping of a single histocompatibility locus, and were found to be representative and functional. These libraries are important tools for physical mapping and positional cloning in the B. schlosseri genome, and the techniques adapted to make them are suitable for use on other organisms in which high molecular weight DNA is difficult to purify.     

玉米生物钟基因ZmPRR1-2的克隆及表达分析

为探究玉米生物钟基因ZmPRR1-2的功能及表达特性,解析玉米光周期途径调控开花的机理,该研究以玉米骨干自交系‘黄早4’为材料,克隆ZmPRR1-2基因的cDNA序列并进行生物信息学分析;利用qRT-PCR技术对该基因进行组织特异性表达分析和48 h的昼夜节律表达分析。结果表明：(1)成功克隆获得ZmPRR1-2基因的编码区全长1 554 bp,编码517个氨基酸,编码的蛋白属于PRR基因家族,含有1个REC结构域和1个CCT结构域,多序列比对和系统进化分析显示ZmPRR1-2基因在禾本科植物中高度保守;ZmPRR1-2蛋白属亲水性蛋白,不包含跨膜结构域和信号肽。(2)ZmPRR1-2基因在玉米叶片中的表达量最高,显著高于其他7个组织,表明该基因主要在叶片中发挥功能,而在果穗和花丝中表达量相对较低,且显著低于雄穗中的表达量。(3)昼夜节律表达分析显示,在短日照条件下,ZmPRR1-2基因的表达量于光照3 h后开始逐渐上升,在光照结束后3 h时达到表达高峰;在长日照条件下,于光照6 h后ZmPRR1-2基因的表达量才开始逐渐上升,且在光照结束时达到表达高峰。研究认为,ZmPRR1-2基因...     

甜荞柠檬酸转运蛋白基因FeFRD3的克隆及表达分析

该研究采用同源克隆策略,从甜荞中克隆到1个柠檬酸转运蛋白基因FeFRD3(GenBank登录号为MG462907)。FeFRD3基因含一个1 554bp开放阅读框,编码517个氨基酸,预测蛋白分子量为55.83kD,等电点为8.48。生物信息学分析显示,FeFRD3蛋白含有8个跨膜区,定位于质膜和液泡膜上。蛋白序列分析结果表明,FeFRD3与拟南芥、大豆和水稻的FRD3同源蛋白有较高的序列一致性。系统进化树分析表明,FeFRD3属于具有将铁由根向地上部位长距离转运功能的柠檬酸转运蛋白,且与拟南芥AtFRD3亲缘关系最近。qRT-PCR分析结果表明,FeFRD3基因在甜荞根、茎、叶和种子中均有表达,但在根中的表达量最高,在种子中的表达量最低;缺铁胁迫没有影响FeFRD3基因在根中的表达,但高铁胁迫明显诱导了该基因在根中的表达。研究结果为进一步深入研究FeFRD3基因在甜荞铁长距离转运中的功能奠定了基础。