糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

低浓度苯并[a]芘诱导赤子爱胜蚓HSP70和HSP90转录上调研究

为寻找土壤低浓度多环芳烃污染分子生物标记物,采用了抑制消减双杂交的方法构建了赤子爱胜蚓在苯并[a]芘（BaP）人工土壤污染胁迫下的差异表达cDNA文库,经测序和基因比对分析后,在上调文库中分别发现2个与热休克蛋白HSP70和1个与HSP90显著匹配的cDNA克隆.经定量PCR验证了0。1 mg·kg^-1和1。0 mg·kg^-1 BaP对赤子爱胜蚓HSP70和HSP90的诱导作用,表明这两个新克隆到的赤子爱胜蚓热休克蛋白基因可作为土壤污染监测的备选分子生物标记物.     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素.在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化.为研究水稻(Oryza sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库.通过文库筛选和测序获得18个已知基因和47个功能未知基因.这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应.Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应.     

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin (α2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.     

激活蛋白处理水稻引发基因差异表达的研究

利用抑制性消减杂交技术(Suppression Subtractive Hybridization,SSH)成功构建了植物激活蛋白处理水稻与非处理组水稻中差异表达的消减cDNA文库。从文库中一共筛选到1756个克隆,通过反向Northern杂交,从中得到264个有效克隆并测序,利用BLAST在GenBank数据库进行序列相似性比对分析,获得28个上升表达差异基因。通过分析这些基因与植物的光合作用、营养物质的运输代谢等多种植物的生理生化功能相关。本研究为揭示激活蛋白的作用机理奠定基础。     

凡纳滨对虾ANT2 基因的克隆及低温表达谱分析

为研究凡纳滨对虾适应低温的分子机理, 实验对从低温处理凡纳滨对虾抑制性消减文库中筛选出的一个360 bp EST 序列进行了研究。首先, 同源比对显示该EST 片段与其他物种的ANT2 基因高度同源, 命名为凡纳滨对虾ANT2 基因(LVANT2); 其次, 通过构建凡纳滨对虾肝胰腺全长cDNA 文库, PCR 扩增获得LVANT2 基因的全长cDNA1540 bp, 其中包括1011 bp 的完整开放阅读框, 编码336 个氨基酸残基。然后, 对基因进行了不同组织和低温处理的表达谱分析: (1)组织表达谱的结果显示, 该基因在凡纳滨对虾肌肉组织中表达量最高; (2)在不同低温处理下的表达结果显示, 该基因在15℃处理下基因表达量发生显著变化, 13℃开始呈下调表达, 11℃时表达量又升高; 13℃低温处理不同时间发现该基因在12h 内表达量发生显著变化,48h 后表达量最高。LVANT2 基因的低温诱导表达模式说明其可能在凡纳滨对虾低温适应中发挥作用