Mapping of rhizoctonia root rot resistance genes in sugar beet using pathogen response‐related sequences as molecular markers

Rhizoctonia root and crown rot caused by the fungus Rhizoctonia solani is a serious disease of sugar beet. An F_2:3 population from a cross between a resistant and a susceptible parent has been tested for R. solani resistance and a genetic map has been constructed from the corresponding F₂ parents. The map encompasses 38 expressed sequence tags (ESTs) with high similarity to genes which are involved in resistance reactions of plants (R‐ESTs) and 25 bacterial artificial chromosomes (BACs) containing nucleotide binding site (NBS)‐motifs typical for disease resistance genes. Three quantitative trait loci (QTL) for R. solani resistance were found on chromosomes 4, 5 and 7 collectively explaining 71% of the total phenotypic variation. A number of R‐ESTs were mapped in close distance to the R. solani resistance QTL. In contrast, the NBS‐BACs mapped to chromosomes 1, 3, 7 and 9 with two major clusters of NBS‐BACs on chromosome 3. No linkage between NBS‐BACs and R. solani resistance QTL was found. The data are discussed with regard to using R‐ESTs and NBS markers for mapping quantitative disease resistances.     

小麦BAC shotgun测序文库的构建

为给小麦基因组中某一基因区域的测序与分析奠定基础,以小麦7B和7D染色体上的10个BAC克隆为材料,运用Large-Construct Kit和TOPO TA cloning kit两个商品化的试剂盒及HydroShearDNA剪切仪建立了BAC shotgun文库构建的技术体系,即利用该体系可以获得高纯度的BAC 20-50μg以及DNA 3-5 kb的目标小片段,5-20 min即可完成连接,一次转化可获得2000个重组克隆。利用这一方法,现已构建了10个小麦BAC shotgun文库,这些文库可为小麦基因组特定区段的研究奠定基础。     

关中奶山羊BAC文库构建条件研究

BAC(bacterialartificialchromosome,细菌人工染色体)是一种新发展起来的构建高等生物基因组文库的重要方法。由于关中奶山羊产奶性能好,成为制备乳腺生物反应器的理想动物之一,故对关中奶山羊BAC文库的构建条件进行了研究。用BamH 部分酶切关中奶山羊的基因组,用CHEF(箝位匀强电场)凝胶电泳分离大片段DNA,以电透析法回收DNA后,与酶切脱磷的pBeloBAC11载体连接,然后电激转化感受态细胞DH10β,得到了数千个阳性克隆,插入片段平均为30～40kb。在此过程中摸索了BAC载体制备、高分子量DNA制备、酶切、连接和电激转化等一系列环节对高效转化产生大片段DNA克隆的影响。     

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     

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。     

带BAC质粒的重组伪狂犬病毒的构建及其体外生长特性研究

本研究通过基因克隆技术以伪狂犬病病毒(PRV)弱毒疫苗株Bartha K61株的部分TK基因作为同源重组序列,以绿色荧光蛋白基因(EGFP)作为筛选标记,构建了带LoxP位点的pUCTKAB-EGFP质粒,将pBeloBACII线性化后插入到pUCTKAB-EGFP中构建了转移载体pUCTK-EGFP-BACII.将PRV基因组和转移载体共转染Vero细胞,利用同源重组在Bartha K61株的TK基因中插入了BAC质粒和绿色荧光蛋白筛选标记,经过11轮噬斑纯化和鉴定证实获得了带BAC质粒的重组伪狂犬病毒rv-PRVBAC.通过噬斑试验和一步生长曲线测定等方法对重组伪狂犬病毒rv-PRVBAC的生长特性进行研究,结果发现带有BAC质粒及筛选标记的重组病毒rv-PRVBAC在体外细胞培养中的增殖速度与亲本毒Bartha K61株相比没有明显差别,说明大的基因片段的插入没有影响重组病毒在体外的繁殖速度.重组病毒rv-PRVBAC在Vero细胞上盲传18代,其绿色荧光蛋白仍能够稳定表达,通过PCR鉴定可以检测到插入的外源片段和插入位点两侧的病毒基因序列,表明所获得的重组病毒rv-PRVBAC得到了稳定遗传.本研究成功获得了带BAC质粒的重组伪狂犬病毒rv-PRVBAC,为进一步开展PRV的细菌人工染色体的构建奠定了基础.     

Microstructure of a Brassica rapa genome segment homoeologous to the resistance gene cluster on Arabidopsis chromosome 4

Genome evolution is a continuous process and genomic rearrangement occurs both within and between species. With the sequencing of the Arabidopsis thaliana genome, comparative genetics and genomics offer new insights into plant biology. The genus Brassica offers excellent opportunities with which to compare genomic synteny so as to reveal genome evolution. During a previous genetic analysis of clubroot resistance in Brassica rapa, we identified a genetic region that is highly collinear with Arabidopsis chromosome 4. This region corresponds to a disease resistance gene cluster in the A. thaliana genome. Relying on synteny with Arabidopsis, we fine-mapped the region and found that the location and order of the markers showed good correspondence with those in Arabidopsis. Microsynteny on a physical map indicated an almost parallel correspondence, with a few rearrangements such as inversions and insertions. The results show that this genomic region of Brassica is conserved extensively with that of Arabidopsis and has potential as a disease resistance gene cluster, although the genera diverged 20 million years ago.     

Construction of a universal recombinant expression vector that regulates the expression of human lysozyme in milk

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (hLZ) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb hLZ genomic DNA, and 8.0 kb mWAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L⁻¹, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.