Research on the Construction of Bacterial Artificial Chromosome Vector DNA and the Potentials in Application

[ Objective] The aim of this study was to provide a method for solving the problems in preparing BAC vector with High-copy plasmid pUC119- Bluelox BAC. [ Method ] With selecting a proper single restriction site, sequences of a single copy BAC vector plasmid were inserted into proper site of High-copy plasmid pUC119 vector. [ Result] The gene sequence of BAC vector lost control function of single copy number in new plasmid pUC119-BAC and was copied through High-copy form. The gene sequence of BAC vector basic function was completely cutted off through single enzyme digestion and the control function of single copy could be recovered by auto-connection. [ Conclusion] The High-copy pUC119-BAC plasmid was used to copy and amplify high copy of basic function gene sequence in BAC vector, besides that it could be used to construct transfer vector of molecular cloned recombinant virus or BAC library.

Construction of a full bacterial artificial chromosome(BAC) library of Oryza sativa genome

We have constructed a full BAC library for the superior eaxly indica variety of OryZa sativa, Guang Lu Ai4. The MAX Efficiency DHlOB with increased stabilltyof inserts was used as BAC host cells. The potent pBelo BACII with double selection markers was used as cloning vector. The cloning efficiency we have reached was as high as 98%, and the transformation efficiency was raised up to 1Oo transformants / pg of large fragment DNA. The BAC recombinant transformants were picked at random and analyzed for the size of inserts, which turned out to be of 120 kb in length on average. We have obtained more than 20,000 such BAC clones. According to conventional probabillty equation, they covered the entire rice genome of 420,000 kb in length. The entire length of inserts of the library obtained has the 5- to 6- fold coverage of the genome. To our knowledge, this is the first reported full BAC library for a complex genome.

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

Transgenic Rice Plants Harboring Genomic DNA from Zizania latifolia Confer Bacterial Blight Resistance

Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

核型分析联合其他遗传学检测技术在高龄孕妇产前诊断中的应用

目的探讨核型分析联合单核苷酸多态性微阵列技术(single nucleotide polymorphism array,SNP array)或荧光原位杂交技术(fluorescence in situ hybridization,FISH)或细菌人工染色体微珠标记技术(BACs on Beads,BoBs)在高龄孕妇产前诊断中的应用价值。方法回顾性研究因高龄行产前诊断的930例孕妇,全部行核型分析,联合BoBs检测420例,联合SNP array检测343例,联合FISH检测167例,分析其染色体结果及妊娠结局。结果930例胎儿中,核型异常192例(20.7%),三体综合征中以21-三体居多,性染色体异常中以克氏征居多。在核型联合其他检测中,联合SNP array检测可显著提高胎儿染色体异常检出率,差异有统计学意义(χ^(2)=6.191,P=0.013)。对于染色体嵌合体的诊断,核型联合FISH检测可更精准地确定嵌合类型及比例。孤立性高龄胎儿染色体异常风险相对较低,高龄合并无创高危胎儿染色体异常率最高,在高龄合并超声软指标中,随超声软指标数目增多,染色体异常率随之升高(P=0.001)。高龄孕妇年龄与核型异常、非整倍体尤其21三体的发生率呈正相关(P<0.05)。结论孤立性高龄孕妇胎儿染色体异常风险相对较低,高龄合并无创高危胎儿染色体异常的风险高,随超声软指标数目增多、孕妇年龄增长胎儿染色体异常的风险增加,核型联合FISH检测可更精准地确定嵌合类型及比例,核型分析联合其他检测尤其与SNP array技术可显著提高染色体异常检出率,有利于遗传咨询及再生育指导,是高龄孕妇首选的产前诊断方案。

Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and named tms1. Several AFLP markers which tightly linked to the tms1 gene have been identified recently. In order to develop a detailed physical map of the tms1 gene-encompassing region and finally clone the tms1 gene, a bacterial artificial chromosome (BAC)

Construction of a Bacterial Artificial Chromosome Library of TM-1,a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondria! and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tms 1 gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.

Highly Efficient Base Editing in Viral Genome Based on Bacterial Artificial Chromosome Using a Cas9-Cytidine Deaminase Fused Protein

Viruses evolve rapidly and continuously threaten animal health and economy,posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine.We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase.We cloned pseudorabies virus genome into bacterial artificial chromosome,and used CRISPR-guided cytidine deaminase to directly convert cytidine(C)to uridine(U)to induce premature stop mutagenesis in viral genes.The editing efficiencies were 100%.Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus(PRV)genomes.Notably,in our study viral genome exists as a plasmid in E.coli,suggesting that this method is virus species-independent.This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.

Construction of a bacterial artificial chromosome library for Gossypium herbaceum var. africanum

Gossypium herbaceum var. africanum is the only wild cotton species within the cultivated diploid G. herbaceum. As the A sub-genome donor of tetraploid cotton, it is characterized by its resistance to insects, diseases, and other adversities. We have constructed the first bacterial artificial chromosome library (BAC) for G. herbaceum var. africanum. With high quality and broad coverage, this library includes 75000 clones, with an average insert size of about 115 kb and fewer than 4% empty clones. Our library is approximately five-fold the size of the A-genome (1667 Mb) and it provides 99.3% probability for isolating genes of interest or their sequences. Using nine SSR markers that are located on five different chromosomes and linked with resistance to Verticillium wilt, seven of nine could amplify the 40 superpools and got 1-14 hits. Because of its moderate wide coverage and relative large insert size, this library will be an important genomic resource for classifying and analyzing the evolution of cotton species, as well as for isolating disease-resistance genes and control elements.

高覆盖率水稻BAC库的构建及抗病基因相关克隆的筛选

利用含Xa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系IRBB56构建了一个水稻细菌人工染色体文库。该文库包含55 296个克隆，平均插入片段为132kb。按水稻基因组为450Mb计，该文库覆盖14倍基因组，筛选出任一水稻基因或序列的概率为99.99%。用均匀分布的3个叶绿体基因和4个线粒体基因克隆作探针筛选文库，结果显示该文库中含细胞器基因组DNA同源序列的克隆数小于1%。用分布于水稻3条不同染色体、分别与Xa4、xa5和xa13连锁的DNA标记筛选文库，分别检测出11～106个阳性克隆，为克隆这些基因打下了基础。 该文库对水稻基因组的高度覆盖率和较大的插入片段，非常适合于物理作图和基因的分离和克隆。

荷斯坦奶牛瘤胃微生物元基因组BAC文库的构建与分析

采用未培养技术和脉冲场电泳技术直接从瘤胃微生物提取到大小在2Mb左右混合微生物DNA,经HindⅢ不完全酶切获得50～100kbDNA片段,将其连接在pCC1BAC载体上,转化E.coliEPI300,得到瘤胃微生物BAC文库,经对文库的鉴定分析,该文库的平均插入片段54.5kb,空载体率小于2%,库容837Mb,共保存15360个克隆。通过对该文库进行部分酶活性筛选,获得具有淀粉酶活性的克隆16个;纤维素酶活性的克隆26个,而且能降解纤维素的克隆中25个呈现多酶活性。这些结果表明该文库具有重要研究价值。

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BAC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 ,并结合凝胶回收纯化技术 ,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等关键步骤。

奶牛瘤胃微生物BAC文库中脲酶克隆的筛选与分析

利用脲酶选择性培养基,从奶牛瘤胃微生物BAC文库的15 360个克隆中筛选得到了12个脲酶克隆。利用酚-次氯酸法对脲酶克隆子的酶学性质分析,表明均具有不同的尿素分解能力,酶的活力范围是30～2 466 U/mg。脲酶阳性克隆插入片段大约是60 kb,限制性内切酶HindⅢ酶切图谱表明,各个克隆具有不同DNA片段,其中脲酶Urease 1、Urease 3、Urease 4和Urease 9的最适pH8.0,最适温度为60℃。本研究从奶牛瘤胃微生物BAC文库中筛选到了具有脲酶酶活特性的克隆。

高纤维强力棉花种质系苏远7235 BAC文库的构建

苏远7235是我国利用异常棉等多个野生种创造的高纤维强力棉花种质系,是开展棉花纤维品质研究的重要材料。本研究以pIndigoBAC-5(HindIII-cloning ready)为载体,构建了苏远7235的细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库,该文库包含30336个BAC克隆。分析结果表明,重组克隆苏远7235 DNA插入片段为50-140 kb,平均120 kb,空载率2.1%,89.6%的克隆插入片段大于100 kb。

稻属不同染色体组着丝粒BAC克隆的分离和鉴定

着丝粒在真核生物有丝分裂和减数分裂染色体正常的分离和传递中起着重要的作用。通过构建5个稻属二倍体野生种的基因组BAC文库,采用菌落杂交和FISH技术,筛选和鉴定了各染色体组着丝粒克隆,并且分析了这些克隆在不同基因组间的共杂交情况,结果表明:(1)C染色体组的野生种O.officinalis和F染色体组的野生种O.brachyantha具有各自着丝粒特异的卫星DNA序列,并且O.brachyantha着丝粒还具有特异的逆转座子序列;(2)A、B和E染色体组的野生稻O.glaberrima、O.punctata和O.australiensis着丝粒区域都含有与栽培稻着丝粒重复序列CentO和CRR同源的序列;(3)C染色体组野生稻O.officinalis的2条体细胞染色体着丝粒具有CentO的同源序列,同时也发现其所有着丝粒区域都包含栽培稻CRR的同源序列。这些结果对克隆稻属不同染色体组的着丝粒序列、研究不同染色体组间着丝粒的进化关系和稻属不同着丝粒DNA序列与功能之间的关系均具有重要意义。

红莲型水稻不育系和保持系线粒体基因组BAC文库的构建

以红莲型 (HL )细胞质雄性不育系和保持系为材料 ,构建了水稻线粒体基因组的 BAC文库。每个文库保存约2 30 0个菌落 ,外源插入片段介于 9～ 2 5 kb之间。以线粒体基因为探针对文库进行菌落原位杂交验证 ,均筛选到了阳性克隆。构建的两个文库为进一步研究水稻线粒体基因组的结构特点 。

棉花细菌人工染色体的荧光原位杂交(BAC-FISH)技术

细菌人工染色体荧光原位杂交(BAC-FISH)技术是植物染色体识别、物理作图等分子细胞遗传学研究的重要工具,但对于某些物种尤其是多倍体植物,由于大量重复序列的存在等问题,使得该技术应用受到很大的限制.通过选择棉花分子遗传图中高重组区的微卫星位点(simple sequence repeats,SSR)标记的策略,筛选到不含或含有少量重复序列的细菌人工染色体(BAC)克隆,同时,在通用FISH技术程序基础上,通过改进发根、变性、洗脱条件等步骤,构建出适合于棉花的BAC-FISH技术,简化了操作流程的同时,获得稳定的杂交结果及较高的检出率;并通过将一随机获得的BAC进行染色体的物理定位,进一步引入双探针、双色及重复杂交技术,显示了该技术的成熟与良好的应用前景和价值.

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Ｇ１８１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ＩＳ个克隆中有４个和６个克隆分别同时为Ｇ１８１和Ｌ１０４４的阳性克隆．选其中的１２个克隆进行分析，构建了一个从Ｇ１８１到Ｌ１０４４区间的ＢＡＣ跨叠克隆群，全长４２０ｋｂ，并且５６Ｍ２２、１０６Ｐ１３和１０４Ｂ１５ ３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基因打下了基础。