甘蓝基因组细菌人工染色体文库的构建

细菌人工染色体（ BAC）文库是克隆甘蓝抗病优质重要基因的基础,以抗枯萎病、富含硫代葡萄糖苷的结球甘蓝自交系R4P1为材料,美国Epicentre公司的CopyControl TM pCC1BACTM为载体,构建了甘蓝细菌人工染色体文库。该文库有73344个克隆,插入片段平均大小约97 kb,空载率＜3％,覆盖甘蓝基因组约10．9倍,筛选到甘蓝任一基因的概率为99．99％,这些结果表明,构建的文库质量较好,可用于后续分析。构建的甘蓝BAC文库不仅可用于枯萎病基因的克隆,而且为克隆其他重要的功能基因及基因组学等的研究奠定了基础。     

甘蓝型油菜基因组文库的构建及与硼高效基因相连锁克隆的筛选

以甘蓝型油菜宁RS-1为材料,构建了含有82944个克隆的甘蓝型油菜的BAC基因组文库.从文库中随机挑取克隆进行DNA长度检测,BAC克隆平均插入片段大小为80 kb,覆盖甘蓝型油菜基因组的5.1倍.随机挑取108克隆进行继代培养100代,分离质粒酶切检测表明不存在插入片段丢失现象,表明该文库的克隆在大肠杆菌中稳定存在;以与硼高效基因相连     

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2300个菌落, 外源插入片段介于9～25 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, 为克隆与红莲型水稻细胞质雄性不育相关的线粒体基     

陆地棉雄性不育恢复系18R的BAC文库构建

 18R雄性不育恢复系农艺性状优良,恢复性状稳定,对不育系能够100%恢复,是研究棉花三系互作的重要材料。本研究以pCC1BAC(BamHI)为载体,构建了18R的细菌人工染色体（BAC）文库。建立的文库包含139,200个BAC克隆。分析结果表明, BAC文库DNA插入片段为50～200 kb,91%的克隆插入片段为80～150 kb,平均102 kb,空载率<2%,覆盖6.3倍基因组。     

中国水仙BAC文库构建的研究

为更深入地挖掘中国水仙的基因组信息,筛选与中国水仙品质相关的功能基因,以中国水仙品种‘金盏银台’的黄化叶片为材料,采用改良Zhang法获得高分子量核DNA,经酶切、连接和转化,首次构建了中国水仙基因组BAC文库。结果表明,采用改良zhang法获取的核DNA分子量大于1 Mb,适合BAC文库的构建;优化了连接、转化体系,发现载体和插入片段的摩尔比为5:1时,连接、转化效率最高;经检测,该文库包括69120个克隆,插入片段的平均大小约87 kb,空载率小于1%,大约50%的克隆大小在80~90 kb之间;Southern blot结果表明该文库未受到细胞器DNA的污染。     

山羊基因组文库的构建研究

构建了山羊基因组文库,为筛选山羊乳蛋白基因,构建山羊乳蛋白基因定点整合的转基因敲入载体奠定基础。全血提取基因组DNA,Sau3AΙ部分酶切,分级分离回收15~23 kb片段,将片段插入Lambda BlueSTAR BamHΙ vector,连接并包装后,NotI酶切鉴定。结果表明,文库滴度为3.4×106;共得到1.7×106个有效克隆,插入片断长度在15~23kb范围,成功构建了西农萨能奶山羊基因组文库。     

棉花细菌人工染色体文库构建的方法优化

以我国抗病、优质、丰产的棉花优良品种中棉所12号为材料,对棉花细菌人工染色体(BAC)文库构建过程中的一些关键技术,如plug的制备、DNA的部分消化、酶切片段的选择、插入片段与载体的摩尔比值、连接产物的浓缩等进行了研究,建立了构建棉花BAC文库的适宜方法体系。依照该方法初步构建了含有38000个克隆的棉花BAC文库。经检测,插入片段平均大小约为120kb,蓝斑率小于0.5%,空载率小于1%。插入片段与载体的最适摩尔比为1∶15,一次转化可以获得约2000个克隆,cfu·μl 1高达4。该方法为进一步构建中棉所12号多倍基因组的BAC文库、从而进行棉花基因组有关研究奠定了基础。     

长片段小麦细菌人工染色体DNA亚克隆文库的构建

为了加快小麦基因组的测序,以Langdon库中的一个BAC克隆(BAC790O10)为材料,利用HydroShear DNA剪切仪将其打断,产生许多大小不同的DNA随机片段,回收其中3～5 kb的片段,并将这些片段随机克隆入TOPO载体,构建了适合于鸟枪法测序的亚克隆文库.     

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。     

大白菜开花相关基因CO的BAC克隆筛选及分析

根据已经公布的大白菜CO基因(Bra008669)序列设计特异引物,采用三维PCR法筛选大白菜BAC文库,获得了3个含有CO的BAC克隆。对筛选到的3个BAC克隆进行PCR扩增,将克隆测序结果与大白菜CO基因进行序列比对,结果表明,相似性达到99.73%,且N端有保守B-BOX结构,C端有保守的CCT结构域,证明所筛选的3个BAC克隆是含有大白菜CO基因的克隆。     

Construction and characterization of a BAC library for sunflower (Helianthus annuus L.)

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.     

高抗黑胫病烤烟BAC文库的构建及分析

烟草是重要的模式植物。本研究利用pIndigoBAC536-S载体及Hind III限制性内切酶酶解烟草基因组DNA的方法,构建了烟草新品系14-60的细菌人工染色体(BAC)文库。该文库共包含414,720个克隆,保存在1080块384板中。随机挑选的120个烟草BAC克隆检测结果表明,外源插入片段大小为97.0～145.5 kb,平均约为123 kb,空载率极低(0),覆盖烟草基因组11倍。用烟草hem A基因、eIF4E-1基因、NtFT基因的特异引物进一步验证,该文库质量高、可用性强,为烟草黑胫病抗性基因的克隆以及其他重要农艺性状和品质性状等功能基因克隆研究提供了基础资源。     

棉花黄萎病菌与品种互作的格局分析

 以23个棉花品种(系)接种18个黄萎病菌株的抗感表现(病情指数)为分析对象,在聚类分析的基础上用双标图方法研究了寄主 病原的互作格局。结果表明：23个棉花品种分为6个抗病性不同的品种组,18个菌株聚为5个致病力不同的菌株群。5031和50312组成的品种组G1对所有菌株群具有最高抗性,可作为抗黄育种的高抗质源。来自河北成安的VD25与来自江苏常熟的XS4、XS6和XS7组成的菌株群S4与G5品种组存在互作,其致病力最强,是棉花黄萎病的优势菌株群,在抗黄育种和抗黄鉴定中应予以高度重视。S2和S3菌株群与56%的参试品种构成的3个品种组间存在互作,是潜在的优势菌株群。具平均抗病性的G2品种组是黄萎病菌的优良寄主。     

黄萎病菌诱导海岛棉全长cDNA文库构建及EST分析

采用SMART技术构建了海岛棉黄萎病菌诱导下根全长cDNA文库,并进行了质量鉴定和EST序列分析。结果表明,文库的重组率为92.3%,库容量为1.1×106pfu·mL-1,平均插入片段大于1.5 kb。生物信息学分析表明该文库包含大量抗病调节基因和抗病防御基因。COG功能分类发现了多个参与信号转导、防御反应、细胞骨架以及次生代谢产物合成与分解等不同功能类别的基因成员。文库中出现的高丰度表达基因主要有病程相关蛋白(PR蛋白)、谷胱甘肽硫转移酶基因(GST)、亲环素蛋白、短链乙醇脱氢酶、抗坏血酸过氧化物酶及转录因子等。文库的构建为深入研究棉花抗黄萎病分子机制奠定基础。     

Construction and characterization of a BAC library of soybean

We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.     

Proteomics Analysis of Gossypium barbadense Leaves with Resistance to Verticillium dahliae under Infection Stress

[Objective] The purpose of this study is to analyze the resistance mechanism of island cotton under Verticillium wilt stress and to find possible resistance genes. [Method] The cotton resistance mechanism under the stress of Verticillium dahliae was studied by protein two-dimensional electrophoresis and mass spectrometry at the protein level. [Result] In the leaves, eleven proteins were down-regulated and 15 proteins were up-regulated after 2 hours of inoculation with Verticillium dahliae. The down-regulated proteins were mainly related to photosynthesis and carbon assimilation, and then we deduce that Verticillium dahliae is mainly broken cotton photosynthetic system to cause cotton susceptibility. The up-regulated proteins were mainly related to photosynthesis and benzoquinone reductase, beta-D-galactosidase, 14-3-3f protein and other disease-resistant protein. [Conclusion] It is speculated that the defense mechanism of island cotton on Verticillium wilt may occur at two levels: one is passive defense, it is manifested that the isoproteins of the down-regulated proteins such as chloroplast II AB binding protein and ribulose diphosphate carboxylase are highly expressed to maintain the stability of the island cotton photosynthetic system, the histone and 14-3-3f protein are highly expressed to keep the cell stability; the second is active defense, it is manifested that the high expressed β-D-galactosidase and benzoquinone reductase may be involved in the resistance of island cotton to Verticillium wilt.     

Identification of QTLs Related to Verticillium Wilt Resistance Based on RIL Population

[Objective] The objective of this study was to identify the stable quantitative trait loci (QTLs) related to Verticillium wilt resistance in cotton. [Method] In this study, a population of 111 recombinant inbred lines (RILs) strains was developed by crossing a highly resistant parental line "Changkangmian" to Verticillium wilt and the susceptible parent TM-1. The complete composite interval mapping method was adopted to detect QTLs by Verticillium wilt disease index in multiple environmental conditions and periods in Anyang and Verticillium wilt affected areas of Xinjiang. Simple sequence repeat (SSR) markers of polymorphism were screened for genetic mapping. [Result] The genetic map was constructed by 40 simple sequence repeat (SSR) markers, consisted of 12 linkage groups with total length of 212.5 centimorgan (cM). A total of six QTLs related to the resistance to Verticillium wilt were obtained. The likelihood of odd (LOD) values ranged from 2.51 to 5.55. The maximum phenotypic variation explained (PVE) 20.34%, and the minimum PVE 6.93% were recorded. Among detected QTLs, qVR-D05-1 was detected in both Verticillium wilt affected fields in Anyang and Xinjiang in July 2015 and July 2016 with PVE of 12.96% and 20.34%, respectively. [Conclusion] This study can provide a potential reference for mapping stable QTLs related to resistance to Verticillium wilt.