In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by i...In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapid-ness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.展开更多
海藻糖是昆虫的血糖,在昆虫体内主要通过海藻糖合成酶(trehalose-6-phosphate synthase,TPS)催化合成。本研究通过同源克隆和cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术,从异色瓢虫Harmoniaaxyridis中克隆得到了TPS...海藻糖是昆虫的血糖,在昆虫体内主要通过海藻糖合成酶(trehalose-6-phosphate synthase,TPS)催化合成。本研究通过同源克隆和cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术,从异色瓢虫Harmoniaaxyridis中克隆得到了TPS基因的cDNA全长序列,命名为HaTPS(GenBank登录号:FJ501960),全长2949bp,包含3'非翻译区为505bp,5'非翻译区为26bp,开放阅读框长2418bp,共编码805个氨基酸。软件分析显示该基因编码蛋白的分子量为90.58kD,等电点为7.01,包含两个糖基化位点,无信号肽和跨膜结构。同源比对分析发现,昆虫中TPS基因高度保守,包含两个保守的结构域。同时,采用实时荧光定量PCR技术对异色瓢虫HaTPS在不同发育阶段、低温诱导条件下的表达量进行了研究。结果表明:HaTPS在预蛹期的表达量最高;在短时低温诱导条件下,HaTPS的表达量随着温度的降低而显著升高,在降温和升温处理条件下,HaTPS的表达量呈现先升高后下降的表达趋势。结果表明,TPS基因在昆虫抗逆中起到了重要的调节作用。昆虫经过低温诱导,其TPS基因的调控能力得到提升。展开更多
基金This work was supported by the National High Science and Technology Program "863" (Grant Nos. 2001AA212041 and 2001AA222251)the National Natural Science Foundation of China (Grant No. 39989001)the National Special Program for Research and Industr
文摘In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapid-ness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.
文摘海藻糖是昆虫的血糖,在昆虫体内主要通过海藻糖合成酶(trehalose-6-phosphate synthase,TPS)催化合成。本研究通过同源克隆和cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术,从异色瓢虫Harmoniaaxyridis中克隆得到了TPS基因的cDNA全长序列,命名为HaTPS(GenBank登录号:FJ501960),全长2949bp,包含3'非翻译区为505bp,5'非翻译区为26bp,开放阅读框长2418bp,共编码805个氨基酸。软件分析显示该基因编码蛋白的分子量为90.58kD,等电点为7.01,包含两个糖基化位点,无信号肽和跨膜结构。同源比对分析发现,昆虫中TPS基因高度保守,包含两个保守的结构域。同时,采用实时荧光定量PCR技术对异色瓢虫HaTPS在不同发育阶段、低温诱导条件下的表达量进行了研究。结果表明:HaTPS在预蛹期的表达量最高;在短时低温诱导条件下,HaTPS的表达量随着温度的降低而显著升高,在降温和升温处理条件下,HaTPS的表达量呈现先升高后下降的表达趋势。结果表明,TPS基因在昆虫抗逆中起到了重要的调节作用。昆虫经过低温诱导,其TPS基因的调控能力得到提升。
