棉铃虫膨胀线粒体基因组的鉴定与分析

以已公布的棉铃虫线粒体DNA序列对来自4头棉铃虫雄蛹的DNA的三代测序数据进行筛选,获得了11条与线粒体DNA有同源性的三代read序列,并根据其中的read 66003鉴定出了一种膨胀的线粒体基因组。该线粒体基因组大小为27 113 bp,其保守区域包含13个蛋白编码基因、2个rRNA基因、22个tRNA基因以及1个AT富集区,与已公布的棉铃虫线粒体基因组的结构相似。膨胀区域位于cox1基因编码区内部,大小为11 467 bp,经预测含有一个完整的真核基因(依赖ATP的RNA解旋酶)以及多种转座元件的片段,但与线粒体DNA无同源性,也无I类或Ⅱ类内含子存在的证据。对田间和室内棉铃虫DNA样品的PCR扩增未能检测到膨胀线粒体基因组的存在。以上结果表明膨胀片段可能是细胞核DNA序列通过偶然的水平转移事件而整合到线粒体基因组中的,且该种膨胀方式的发生概率极低。本文报道的膨胀线粒体基因组为日后动物线粒体基因组学的研究提示了一种独特的变异方式。     

甘蓝黑腐病菌rPf基因簇对胞外蛋白酶Ⅰ基因表达的调控

甘蓝黑腐病黄单胞菌（ＸａｎｔｈｏｍｏｎａｓｃａｍｐｅｓｔｒｉｓＰｖ．ｃａｍｐｅｓｔｒｉｓ）产生的胞外蛋白酶Ⅰ在致病的早期阶段起重要作用，该酶以及其它胞外酶和胞外多糖的合成受一致病因子调控基因簇（ｒＰｆ基因簇）的正向调控。本研究利用带有β－半乳糖苷酶报道基因（ｌａｃＺ）的转座子Ｔｎ５－Ｂ２０诱变蛋白酶Ⅰ基因克隆，获得了ｌａｃＺ在蛋白酶Ⅰ基因启动子控制下表达的Ｔｎ５－Ｂ２０插入突变质粒。通过将这种突变质粒导入野生型和各ｒｐｆ基因突变体菌株后，测定ｌａｃＺ基因在细胞生长周期中的表达水平，不仅进一步证实了这些ｒｐｆ基因对蛋白酶Ⅰ基因的正向调控作用，而且明确了它们的调控水平．发现ｒｐｆＡ、ｒｐｆＣ、ｒｐｆＥ、ｒｐｆＧ或ｒｐｆＨ突变后，蛋白酶Ⅰ基因的转录会降低９０％左右，而ｒｐｆＢ突变后，蛋白酶Ⅰ基因的转录只降低４８％。     

转座子mini-Tn10介导的运动突变对溶藻弧菌毒力的影响

采用转座子mini-Tn10/Km构建了致病性溶藻弧菌的突变库,采用半固体双层平板初筛到73株运动缺陷突变株,初筛的突变株纯化及重复筛选后获得运动性缺陷表型稳定的突变株ND-01MM,Southern鉴定确定其为单位点插入。野生型菌株ND-01和运动缺陷突变株ND-01MM对致病性溶藻弧菌的天然宿主大黄鱼粘液的趋化、粘附以及在大黄鱼吞噬细胞内存活能力等生理功能的比较研究发现,溶藻弧菌运动缺陷后趋化及粘附能力均极显著下降(P<0.01),但在吞噬细胞内的存活能力则与野生型菌株差异不显著(P>0.05)。采用腹腔注射和浸泡两种方式感染大黄鱼,结果发现运动缺陷对浸泡感染的影响较为明显,但对腹腔注射的感染方式影响不大。     

转基因植物中删除选择标记基因的研究进展

选择标记基因广泛应用于植物转基因工程中,然而这些基于除草剂和抗生素类抗性选择标记基因的利用,在转基因筛选和鉴定完成后功能多余。当前转基因植物的标记基因及相关产物对环境污染和食用安全的潜在影响,已经引起了人们的广泛关注。随着转基因技术的不断发展和人们对转基因植物和食品安全性的关注,删除转基因植物中的选择标记基因非常重要。文章着重讨论了植物转基因过程中,选择标记基因的利用,删除选择标记基因的几种方法,包括共转化法、位点特异性重组、染色体内重组、转座子介导的重组、删除叶绿体转化中选择标记基因,以及无选择标记基因植株的直接再生等。同时,还对这些技术的发展作了展望。     

玉米Mutator转座子结构特征和基因克隆的研究进展

Mutator（Mu）转座子是已发现的植物中转座活性最强的转座子,其较高的转座频率以及趋向于插入单拷贝功能基因转座的特性,使其成为构建玉米突变体库的一个主要方法。当前玉米B73品系基因组测序已经基本完成,利用反向遗传学方法,研究玉米基因的功能更为方便。文中简要介绍Mu转座子的结构特征以及Mu的分类,介绍3种克隆Mu转座子标签插入位点侧翼序列的技术,对Mu研究所存在的问题进行分析,展望Mu的应用。     

Characterization of nonautonomous Tc1-like transposable elements of channel catfish (Ictalurus punctatus)

Putative nonautonomous transposable elements from channel catfish (Ictalurus punctatus) were identified. They were named Tipnon elements for Tc1-like transposable elements from channel catfish that are nonautonomous. These elements were defined by their terminal repeats that share identity to members of the known Tc1/mariner transposon superfamily. They show structural similarities to Tc1-like elements, but share little sequence identity beyond the terminal inverted repeats. They do not harbor any amino acid blocks that show similarities to the Tc1-like or other transposases and thus may represent truly nonautonomous transposons in channel catfish. They are abundant in the channel catfish genome with a copy number of 32000, having 500 base pair per copy, this family of nonautonomous transposon-like elements account for 1.6% of the channel catfish genomic DNA. Their high abundance and transposon-like terminal repeats indicate that they may play important roles in gene evolution and in genomic architecture of catfish. Similarity search for potential coding capacity of the Tipnon elements revealed that they contain sequence blocks that can potentially encode amino acid blocks similar to the para-type sodium channel proteins in cockroaches or house flies, proteins that function in the central nervous system as voltage-gated sodium transporters. Sequences surrounding the terminal inverted repeats are divergent from those used by the reconstructed Sleeping Beauty fish transposase.     

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins. Resistance to the pathogen and insensitivity to the toxins are conferred by the Asc locus on chromosome 3L. Sensitivity to AAL-toxins is a relative character; the toxins inhibit development of all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars. In addition to tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action of AAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity. To molecularly isolate Asc, transposon tagging and positional cloning strategies are applied. As a first step, transposon insertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asc locus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers are used to identify yeast artificial chromosomes (YACs) with tomato DNA inserts. Once an Asc-insertion mutant and/or a YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable. Elucidation of the molecular role of Asc will illuminate the specificity of host recognition by Alternaria alternata f. sp. lycopersici.Abbreviations AAL-toxin Alternaria alternata lycopersici-toxin - A. a. lycopersici Alternaria alternata f. sp. lycopersici - Asc Alternaria stem canker - HST host-selective toxin     

拟南芥抗病基因克隆的策略及利用

生物技术的快速发展为植物抗病基因的克隆奠定了坚实的基础。克隆抗病基因不但有利于深入研究植物与病原物的分子互作机理,而且为植物重要病害的防治提供了有效措施。迄今为止,在拟南芥中已经克隆了十几个抗病基因。对拟南芥抗病基因的种类和特性、克隆策略以及克隆抗病基因的应用前景进行了综述。详细介绍了定位克隆技术和转座子标签技术的原理及其在拟南芥抗病基因克隆中的应用。     

GFP标记的短小芽胞杆菌（Bacillus pumilus）转座突变株的构建初探

以水稻叶面附生菌短小芽孢杆菌(B.pumilus)为研究对象,通过构建pMOD-egfp-Tetr转座复合体,采用电击转化法对短小芽孢杆菌DX01菌株进行了Tn5转座插入诱变,获得了大量的转化子。对转化子进行了遗传稳定性分析和分子鉴定,并且在荧光显微镜下观察到了强烈的绿色荧光,研究结果表明外源DNA已经成功整合进细菌的基因组中,gfp基因在短小芽孢杆菌中实现了组成型表达。     

超氧化物歧化酶与苏云金芽胞杆菌高温诱导产黑色素相关

为探讨苏云金芽胞杆菌高温诱导产黑色素机制,利用含mini-Tn10转座子载体pIC333构建苏云金芽胞杆菌油松亚种4CA1,随机插入突变体库,以寻找相关调控基因。通过高温诱导筛选得到1株在42℃产黑色素减弱的菌株;克隆得到mini-Tn10中断基因的部分序列,通过测序及同源性比对分析,确定中断基因为超氧化物歧化酶基因sodA2。