赤桉抗寒转录因子ICE1基因的分子克隆与表达分析

ICE1属于一种类似MYC的bHLH转录因子,可特异地结合到CBF3启动子的MYC作用元件并诱导CBF/DREB1下游基因的转录表达。本文以拟南芥ICE1蛋白序列为信息探针,搜索桉树基因组和EST数据库的同源序列并进行拼接、设计引物,通过RT-PCR从赤桉克隆了桉树的第一个ICE1基因。其cDNA长1792bp,含有完整的开放阅读框,可编码523个氨基酸。BLAST分析表明,cDNA序列及其推导的氨基酸序列均与拟南芥、芥菜、小麦和甜杨ICE1存在着较高的同源性,预示所获得的cDNA可能是赤桉ICE1基因（EcaICE1）。EcaICE1基因表达分析结果显示,EcaICE1在赤桉根、茎、叶中均表达,而且表达水平不受低温胁迫处理时间的影响,这表明EcaICE1是组成型表达。此外,EcaICE1的超表达可以提高转基因烟草的耐低温能力。上述结果为进一步研究EcaICE1在赤桉耐低温胁迫过程中基因表达的调控机制打下基础。     

Pancreatic beta-cell damage mediated by beta-cell production of interleukin-1. A novel mechanism for virus-induced diabetes

Viral infection is one environmental factor that may initiate beta-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta-converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma-induced iNOS expression by primary beta-cells. In addition, poly(I-C) + IFN-gamma-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a nitric oxide-dependent fashion.     

萝卜低温胁迫转录因子的克隆及遗传转化

采用RT-PCR和电子克隆技术从抗寒植物萝卜(Raphanus sativusL.)中分离了一个低温胁迫转录因子的cDNA全长序列,命名为RsICE1(GenBank登录号为HQ891287).序列分析显示,该基因全长1 375 bp,编码区为1266 bp,编码421个氨基酸.序列比对表明,该蛋白C端含有一个典型的bHLH结构域,与其他植物的ICE1蛋白具有较高的同源性.进化树分析表明,RsICE1编码的蛋白与油菜的ICE1编码蛋白亲缘关系最近,处在同一进化分枝.半定量RT-PCR分析表明,RsICE1是冷诱导条件下差异表达的基因.构建该基因的植物表达载体,利用农杆菌介导法转化粳稻品种龙粳24,经PCR和RT-PCR分子检测,证明RsICE1基因已经整合到水稻基因组中,并正常表达.与对照相比,低温处理后转基因株系存活率和脯氨酸含量明显增加,相对电导率积累速率明显下降,提高了抗低温胁迫能力.