Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.     

Coupling mechanism of multi-force interactions in the myosin molecular motor

The dynamics of the myosin molecular motor as it binds to actin filaments during muscle contraction are still not clearly understood. In this paper, we focus on the coupling mechanism of multi-force interactions in the myosin molecule during its interaction with actin. These forces include the electrostatic force, the van der Waals force and the Casimir force in molecular dynamic simulations of the molecules in solvent with thermal fluctuations. Based on the Hamaker approach, van der Waals and Casimir potentials and forces are calculated between myosin and actin. We have developed a Monte Carlo method to simulate the dynamic activity of the molecular motor. We have shown that because of the retardation effect, the van der Waals force falls into the Casimir force when the distance between the surfaces is larger than 3 nm. When the distance is smaller than 3 nm, the electrostatic force and the van der Waals force increase until the myosin becomes attached to the actin. Over the distances studied in the present work, the electrostatic force dominates the attractive interactions. Our calculations are in good agreement with recently reported experimental results.     

Isolation of tomato proteinase inhibitor II gene and the function of its intron

The genomic DNA sequence of tomato proteinase inhibitor II gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucleotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.     

基因转录的活细胞成像研究技术

转录是基因表达过程中的一个关键调节步骤,也是人类发育和疾病的基础。最近,用活细胞成像方法研究基因转录引起了人们广泛的兴趣．综述了此领域当前的技术进展,并讨论了将来的发展趋势．     

三叶青两个肌动蛋白基因片段的克隆及其分析

根据GenBank中登录的植物肌动蛋白保守序列设计1对引物,对三叶青的块根进行RT‐PCR ,在1次扩增中得到2个不同的肌动蛋白基因片段,分别命名为 ThAct1和 ThAct2．测序结果显示：ThAct1基因片段长度为867 bp ,编码252个氨基酸;ThAct2基因片段长度为1079 bp ,编码152个氨基酸．经Blast分析,ThAct1和ThAct2基因均属于NBD＿sugar‐kinase＿HSP70＿actin superfamily家族,与其它物种Actin基因序列和编码的蛋白质均具有同源性．RT‐PCR结果初步表明：在茎、普通根和块根中 ThAct1和 ThAct2基因的表达未见差异;但在叶中,ThAct1的表达稍强,而 ThAct2的表达稍弱．另外,在茎、普通根和块根中,ThAct1表达比ThAct2强;但在叶中 ThAct1表达比ThAct2弱．结果为分析肌动蛋白基因在三叶青块根发育中的功能奠定了基础;获得的肌动蛋白也可以作为内参基因,在三叶青功能基因组的研究中用于其它基因的定量表达分析．     

衣藻的核基因组转化及外源基因转录分析

通过珠磨法将衣藻表达载体pSP108转入到莱茵衣藻细胞壁缺陷型CC-400藻株中,经抗性筛选及PCR鉴定,获得了24个转化藻株.在抗性基因ble编码序列的中间部位和末端部位分别设计两对不同的探针引物,运用实时荧光定量PCR对转基因藻株进行外源基因转录水平的分析.结果发现:不同转化藻株外源基因的转录水平有明显差异;同一转化藻株两对探针引物,外源基因的转录水平也存在差异.说明莱茵衣藻在转化过程中,外源基因整合到基因组上的片段数量及片段长度和完整性具有不确定性.     

Transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.