小麦类CTR1基因的克隆和特性分析

Cloning and Characterization of a Putative CTR1 Gene from Wheat

【目的】克隆小麦类CTR1基因(TaCTR1),研究其在各种胁迫条件下的表达、亚细胞定位和过量表达TaCTR1对转基因烟草耐盐性的影响。【方法】利用RACE结合RT-PCR技术克隆TaCTR1,利用半定量RT-PCR研究TaCTR1在各种非胁迫及ABA处理条件下的表达,通过烟草转化研究过量表达TaCTR1对转基因植株耐盐性的影响。【结果】TaCTR1全长2635bp,编码759个氨基酸,在其羧基端有一个非常保守的丝氨酸/苏氨酸蛋白激酶结构域,该结构域含有ATP结合位点和一个钙调素结合位点;TaCTR1与其它植物中CTR1的氨基酸序列同源性较高。TaCTR1的表达受NaCl和干旱胁迫的诱导,当用30μmol·L-1ABA对小麦幼苗进行处理时,TaCTR1的表达受到抑制,当将小麦幼苗转移至无ABA的Hoagland培养液时,TaCTR1的表达又逐渐升高。利用洋葱表皮细胞进行瞬时表达显示TaCTR1可能定位于细胞质膜。过量表达TaCTR1的转基因烟草植株耐盐性下降。【结论】TaCTR1是小麦中克隆的第一个CTR1基因,TaCTR1:GFP可能定位于细胞质膜,过量表达TaCTR1的烟草植株耐盐性下降说明TaCTR1是植物耐盐信号传导途径的负调控因子。

[Objective] CTR1 is a key negative regulator in ethylene signal transduction. A salt-induced CTR1 like gene （TaCTR1）was cloned from wheat, the expression of TaCTR1 under abiotic stresses and ABA treatment, the subcellular localization and the effect of overexpression of TaCTR1 on salt tolerance in tobacco was studied. [ Method ] A putative CTR 1 gene was cloned and characterized from wheat via rapid amplification of eDNA ends （RACE） and RT-PCR, TaCTR1 expression under stresses was analyzed using semi-quantitative RT-PCR, and the effect of overexpression of TaCTR1 on salt tolerance was conducted in tobacco. [Result] The full-length eDNA of TaCTR1 is 2 635 bp which codes for a polypeptide of 759 amino acids. There is a conserved serine/threonine protein kinase domain at the carboxyl terminus containing an ATP-binding site. Southern analysis revealed that TaCTR1 is consisted of a gene family in wheat. The amino acid homologies of CTR1 among different organisms share higher similarities. Expression analysis revealed that TaCTR1 was induced by NaCI and drought stress but inhibited by ABA treatment. Transient expression of TaCTRI：GFP in the onion epidermal cells revealed that TaCTR1 was probably localized to the plasma membrane. Over expressing TaCTR1 decreased salt tolerance of transgenic tobacco （Nicotiana tabacum L.） plants compared with control. [Conclusion] TaCTR1 is the first CTR1 gene cloned in wheat and may involve in various abiotic stresses. Overexpression of TaCTR1 decreased the salt-tolerance in tobacco suggested that TaCTR1 may act as a negative regulator of salt stress in plants.