异源表达蒙古沙冬青AmDREB1F基因提高转基因拟南芥的耐逆性

脱水应答元件结合蛋白 (Dehydration-responsive element binding proteins,DREBs) 是一类重要的植物耐逆相关转录因子。蒙古沙冬青Ammopiptanthus mongolicus是中国西北荒漠区特有的强耐逆常绿阔叶灌木。为探明其AmDREB1F基因在耐受非生物逆境中的功能和作用机理,文中对该基因编码蛋白的亚细胞定位、表达模式和转基因拟南芥的耐逆性进行了分析。结果表明：AmDREB1F编码的蛋白质定位于细胞核内;在室内培养幼苗中,该基因在正常条件下不表达,在低温和干旱胁迫下有较明显表达,在高盐和高温胁迫下仅有微弱表达,而在脱落酸 (Abscisic acid,ABA) 处理下不表达;在野外生长植株的叶片中,其表达量在秋末、冬季和早春远高于其他季节,而不同器官相比,其在根和未成熟果荚中的表达量远高于其他器官;将AmDREB1F在拟南芥中组成型表达可提高多个受DREBs调控的胁迫响应基因的转录水平,增强转基因株系对干旱、高盐和低温以及氧化胁迫的耐性,同时导致其生长发育延滞,外施赤霉素3可消除生长延滞现象;将该基因进行胁迫诱导表达也可提高转基因拟南芥对上述非生物胁迫的耐受性,而不影响其生长发育。这些结果说明AmDREB1F可能通过ABA非依赖的信号途径在响应和耐受逆境胁迫中起正调节作用。     

DRE顺式作用元件dsDNA芯片制备

DRE顺式作用元件能与DREB转录因子特异结合,在诱导逆境（干旱、高盐、低温）基因表达过程中起重要作用。dsDNA（double strand DNA）微阵列芯片技术能够有效地检测序列特异性DNA结合蛋白质（转录因子）与大量DNA靶点（顺式作用元件）的特异性结合,可有效分析生物分子结合作用。根据DRE顺式作用元件核心序列设计并化学合成含发夹结构的单链DNA探针,采用Taq DNA聚合酶在片延伸,并对其在片延伸体系的反应温度、Mg^2＋浓度以及单链探针是否变性等条件进行了优化。结果表明,50％的反应温度,2．5mmol／L的Mg^2＋浓度和单链不变性是TaqDNA聚合酶在片延伸的最佳条件。优化方法制备的dsDNA芯片将更有利于DRE顺式作用元件与DREB抗逆转录因子相互作用的研究。     

转录因子DREB1A基因的克隆与植物表达载体的构建

根据GenBank中已发表的转录因子DREB1A基因的cDNA序列设计并合成了一对引物,通过RT-PCR的方法从低温处理的拟南芥总RNA中扩增出DREB1A基因的全长cDNA片段。将其克隆到pMD18 T-vector中。经测序证明该片段与GenBank上报道的序列具有99.8%的同源性。2个碱基的置换导致了一处氨基酸的差异,但这一氨基酸并不在基因的功能结构域上,推测其不会影响基因功能。以植物表达载体pBch为基础,构建了由组成型启动子35S调控的DREB1A基因的植物表达载体pBDR35S,为利用DREB1A基因改良植物抗逆性奠定了物质基础。     

Production of transgenic local rice cultivars (Oryza sativa L.) for improved drought tolerance using Agrobacterium mediated transformation

Rice being the staple food of middle and south India, there is an extensive research undertaken in protecting the species and improving the quality and yield. Several recombinations have been made to the rice genome to impart various qualities which lack in the pure breed. Oryza faces various natural stress, like temperature variance, high salinity, etc., drought is one of the major parameters affecting the growth and yield of the plant. Transgenic rice cultivars can be generated for drought tolerance using the Agrobacterium mediated transformations. The current work aims to impart the gene for drought tolerance in Oryza sativa L. using Agrobacterium mediated transformation. The gene targeted in this context is dehydration response element binding factors (DREB). DREB plays a major role in response to drought mediated stress. Sambha mahsuri (Indica type) and Cotton dora sannalu (Indica type) the two local cultivars have been transformed for the gene AtDREB1A under 35s CaMV promoters (pBIH binary vector) for which the vector used was Agrobacterium. The target plant tissue being used was calli. Optimization of the parameters was performed for a lethal dose of hygromycin, cefotaxime level, and acetosyringone level. PCR amplification was used for the confirmation of the transgenic (T₀) species in which 23% and 18% for Sambha mahsuri and Cotton dora sannalu, respectively. Southern blotting was performed for the genomic DNA. Normal growth was shown by the T₁ transgenic plants whose expression was confirmed by RT-PCR. The T₁ transgenic plants showed good tolerance to drought mediated stress for a total period of one and a half week under greenhouse condition. The study can be concluded by producing a potentially successful drought resistance T₁ species produced using Agrobacterium mediated transformation.     

小叶杨DREB同源基因内SSR的发现及群体结构分析

利用基因特异的POR引物从小叶杨（Populus simonii）经干旱胁迫后构建的cDNA文库中扩增得到长度为671bp的PSDREB cDNA克隆。该基因内部含有完整的且大小为543bp的开放阅读框,可编码181个氨基酸残基。在此基础上,对36株小叶杨基因型个体的DREB基因进行了克隆和序列分析,发现其内部存在以CAC为基本单位的4次（A）、5次（B）、7次（C）、8次（D）、9次（E）、10次（F）和11次（G）7种类型的简单重复序列（SSR）。以该重复序列为扩增对象设计引物,对我国11个省16个群体528株小叶杨进行了群体遗传结构分析。结果表明,在检测群体中各位点等位基因频率大小顺序依次为：D〉C〉F〉E〉A〉G〉B：平均有效等位基因数为3．1670,平均观察和期望杂合度分别为0．4685和0．5315,且在多数群体中存在一定程度的近交效应和显著偏离Hardy-Weinberg平衡：而Ewens-Watterson中立性检验表明,除山西宁武、辽宁朝阳和河南伊川外,其它多数群体均属于中立性选择。研究结果将为利用候选基因内SSR标记来评价林木育种资源的遗传多样性以及保护和利用这些资源提供科学的理论依据。     

Reevaluation of the Subunit Molecular Weights of Soybean 11S Globulin

The acidic and basic subunits are the main constituents of soybean 11S globulin. Each of these two subunits consists of three major polypeptides of similar size. The molecular weights of the acidic and basic subunits have been previously estimated to be 37,000 and 22,000, respectively, by SDS-polyacrylamide gel electrophoresis (Catsimpoolas et al, J. Set Food. Agric., 22, 448 (1971)). Reevaluation of the molecular weights by 6 m guanidine gel chromatography gave the values of 28,000 and 18,000, respectively. These are supported by results of equilibrium sedimentation in the same solvent. The previously reported values seem to have been overestimated, especially for the acidic subunits. The overestimations seem to be related to the high percentage of acidic amino acids, which causes the conformation of the SDS-protein polypeptide complexes of these subunits to deviate from those of proteins usually employed as standards for molecular weight estimations.