稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

东乡野生稻双元细菌人工染色体(BIBAC)文库的构建

双元细菌人工染色体(binarybacterialartificialchromosome,BIBAC)是能直接将大片段DNA转入植物的载体,是植物基因图位克隆和构建植物基因嵌入突变体库的重要工具。该研究以东乡野生稻为材料,构建其BIBAC文库。该文库由14592个克隆组成,平均插入片段大小为65kb,覆盖率为2倍基因组。稳定性检测结果表明,东乡野生稻基因组DNA能够在BIBAC载体中稳定存在。     

米曲霉(Aspergillus oryzae) RIB40全长cDNA文库的构建

【目的】构建米曲霉RIB40的全长cDNA表达文库,为米曲霉功能基因的开发以及次生代谢产物合成途径相关基因的筛选与克隆奠定基础。【方法】采用RNAiso法从米曲霉RIB40菌体中提取总RNA。选用PolyATract mRNA Isolation System Ⅲ试剂盒分离纯化mRNA。以5μg mRNA为模板,按照ZAP-cDNA Synthesis Kit试剂盒说明书要求合成单、双链cDNA,使用CHROMA SPIN-400柱离心层析纯化后连接于Uni-ZAP XR表达载体上,体外包装后转染Escherichia coli XL1-Blue宿主菌。【结果】构建了米曲霉RIB40的全长cDNA文库,初级文库滴度约为2.96×106 CFU/mL,重组率约为97.8%,插入片段平均长度大于1.5 kb,达到一个高质量cDNA文库的要求。文库扩增后,滴度达到3.4×1010 CFU/mL。【结论】米曲霉RIB40全长cDNA表达文库的成功构建,将会对米曲霉基础生物学研究及相关基因的筛选与克隆奠定基础。     

利用高效的大片段DNA回收方法改良Fosmid文库的构建

外源DNA插入片段为40 kb左右的Fosmid文库在基因组学研究中有广泛的应用,但长期以来,40 kb外源片段的分离与纯化依赖于传统的切胶并电洗脱至透析袋的方法,难以得到足够量的DNA片段,极大降低Fosmid文库构建的成功率。通过改进全自动核酸/蛋白质回收系统SageELF的操作流程,建立一种简单、便捷、高效地回收40 kb左右DNA片段的方法,并用其成功地构建高质量的Fosmid文库。从文库中随机挑选的25个单克隆,经过测序及酶切分析,发现该文库中插入的DNA片段大小为37.9±5.2 kb。以上结果表明,利用改良的操作方法回收40 kb基因组DNA,操作简捷、高效,片段大小精准;另外,用该DNA片段构建的Fosmid文库,插入片段比较集中,有利于后续的基因组学分析。     

适用于链霉菌大片段基因组DNA 克隆和异源表达的细菌人工染色体(BAC)载体的构建及应用

【目的】很多链霉菌来源的天然产物的生物合成基因簇往往很大,用传统的cosmid载体很难完整的克隆和异源表达。本研究通过载体改造,成功构建出一个新的细菌人工染色体(BAC)载体,用于链霉菌来源的天然产物生物合成基因簇的克隆及异源表达实验。【方法】从复合型载体pCUGIBAC1出发,通过λRED介导的PCR-targeting方法,用链霉素抗性基因替换掉原有的氯霉素抗性基因标记,同时插入链霉菌中常用的安普拉霉素抗性标记、转移起始位点oriT、φC31整合酶基因int、整合位点attP等元件。【结果】成功构建出可装载链霉菌大片段DNA的BAC载体pMSBBACs。使用pMSBBACs构建出链霉菌U27的基因组BAC文库,平均插入片段大小为100 kb。选取其中一个大小为140 kb的BAC质粒进行功能验证,实验证明通过接合转移和原生质体转化的方法都能够将这个大型BAC质粒导入链霉菌模式菌株,并通过位点特异性重组整合到染色体中进行异源表达。【结论】BAC载体pMSBBACs可成功用于放线菌大片段基因组DNA的克隆和异源表达实验。     

温室黄瓜根结线虫发生地土壤微生物宏基因组文库的构建及其一个杀线虫蛋白酶基因的筛选

【目的】本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。【方法】采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库,同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛。【结果】该文库库容31008个克隆,平均插入片段36.5kb,包含1.13Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选,通过功能初筛,筛选到1个含杀线虫蛋白酶基因的Fosmid克隆(pro12)。进一步构建和筛选出亚克隆(espro124a5),通过对基因结构进行了初步分析发现:espro124a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10(accession no.YP_756822at NCBI)的丝氨酸蛋白酶S15仅有45%的同源性,是一种新型的丝氨酸蛋白酶,有其保守的催化三元组:Asp469、His541和Ser348。【结论】密度梯度离心法提取到的DNA纯度高、片段长,完全能满足构建宏基因组Fosmid文库的要求;同时,构建的宏基因组Fosmid文库库容大,有利于我们从中筛选其他的微生物基因资源。     

新疆军垦型细毛羊基因组BAC文库的构建

选用新疆军垦型细毛羊为材料,.构建了含有190 464个克隆的BAC文库,文库平均插入片段大小为133 kb,同时文库92.5%的克隆插入片段大于100 kb,而且有部分克隆甚至大于300 kb,这将满足大多数基因筛选的要求.假定绵羊的基因组含有3x106 kb,根据文库的平均插入片段大小,计算的文库基因组覆盖率为8倍.因此,从文库筛选到目的片段的概率为98.2%.由于该文库中插入的外源片段来自新疆军垦型细毛羊的基因组,这对于研究新疆军垦型细毛羊的特殊性状的基因与其他绵羊品种和物种之间的差异,及构建其全基因组物理图谱和基因图谱地完善是非常有利的.     

云南药用野生稻核基因组细菌人工染色体(BAC)文库的构建与分析

通过云南药用野生稻核基因组BAC文库的构建,保存处于濒危状态的云南药用野生稻遗传资源,该文库包含27 500个克隆,随机挑取80个克隆检测,插入片段平均大小为80kb,文库容量相当于水稻基因大小的5.1倍。     

新疆细毛羊2倍基因组BAC文库PCR筛选系统的构建与使用

本研究所构建的BAC文库覆盖了8倍新疆细毛羊的基因组,平均插入片段的大小为133kb,同时文库92．5%的克隆插入片段大于100kb,而且有部分克隆甚至大于300kb,假定绵羊的基因组含有3×10~6kb,根据文库的平均插入片段大小为133kb,从文库筛选到目的片段的概率为98．208%。为了验证文库有较好的覆盖率,构建了2倍基因组文库PCR筛选系统,并对位于新疆细毛羊20号染色体MHC基因邻近区段的DMB_EX2、MCMA36、CP73和BM1258 4个分子标记进行了筛选,得到的平均阳性克隆数为1．5个,从筛选结果来看,这与文库插入片段估计的8倍基因组覆盖率相当接近并且没有偏向,这使得本文库成为研究绵羊的功能基因、位置克隆和完善基因组物理图谱的极为有用的资源。     

对虾养殖水环境宏基因组Fosmid文库的构建

采用包埋法提取大片段基因组DNA,通过低熔点琼脂糖酶法回收40 kb左右的DNA,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EP1300-T1R,构建对虾养殖水环境Fosmid文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存8 000个克隆,包括了大概8×108个微生物细胞.用几丁质酶筛选平板对文库进行初步筛选,筛选到4个阳性克隆子.本试验构建Fosmid文库,初步获取了对虾养殖生态系统的微生物遗传信息,对于从虾池原位环境鉴定并筛选具有潜在益生作用的功能微生物意义重大.     

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis.

A cosmid clone containing the entire hCG beta gene cluster has been isolated. The restriction map of this clone has been determined by an indirect-end-label FIGE (field inversion gel electrophoresis) method. Analysis of this cosmid clone shows that there are 6 hCG beta genes in human genomic DNA. A previously uncloned portion of the hCG beta cluster, termed the "gap" region, has been shown not to contain any sequences homologous to the hCG beta cDNA. The restriction mapping method employed in this study takes advantage of the superior resolution of FIGE for high molecular weight DNA fragments in the size range 15-50 kb. This method is broadly applicable and permits rapid and accurate restriction mapping for extended regions of genomic DNA that have been cloned into cosmid or lambda vectors.     

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.     

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.     

Simplified cosmid vectors for gene transfer to cultured mammalian cells: isolation of the gene for elongation factor 2 from the mouse

We constructed a series of cosmid vectors that carry two tandemly arranged lambda cos and mammalian selective markers. We achieved cloning efficiencies of 1-3 x 10(7) and greater than 10(6) colony-forming units per microgram of insert, using a cloned 42-kb BamHI fragment and Sau3AI fragments of 40-50 kb from mouse genomic DNA, respectively. The modified Ca.phosphate coprecipitation method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607-616] considerably improved the efficiency of gene transfer of cosmids into cultured mammalian cells: when genes encoding thymidine kinase from herpes simplex virus type 1 and aminoglycoside 3'-phosphoribosyltransferase from Tn5 were selected, the efficiencies of gene transfer into mouse L cells were about 10(-6). The mouse genome contains one copy of the functional gene for elongation factor 2 (EF2) per haploid genome and multiple copies of the EF2-related gene. We isolated a cosmid that carried functional full-length mouse EF2 from a cosmid library of L-cell genomic DNA, by colony hybridization and subsequent gene transfer of candidate cosmids into human 143B cells.     

A new high-capacity cosmid vector and its use

The construction of a cosmid, MUA-3, designed for the convenient cloning of eukaryotic DNA segments up to 48 kb in length is described. The cosmid contains all of the plasmid pBR322 with approx. 400 bases of lambda DNA, including the cohesive end site, inserted at the pBR322 PstI endonuclease recognition site. Methods for using this vector to construct several types of Drosophila melanogaster genomic DNA libraries are given, and libraries made by these methods are characterized. A sheared Drosophila DNA-EcoRI linker library is shown to stably maintain average Drosophila DNA inserts of over 40 kb and up to 48 kb, and the efficiency of producing clones by a partial restriction and ligation method is shown to be over 3 X 10(5) clones/microgram of Drosophila DNA.