玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

Cloning and functional validation of promoter of mo-molybdopterin cofactor sulfurase gene in maize

为克服组成型启动子启动外源基因过量表达引起的诸多问题,同源克隆(Mo-molybdopterin cofactor sulfurase)基因(ABA3)的启动子(ABA3s)序列,并用PlantCARE软件分析其非生物逆境应答元件,实时定量PCR检测ABA3基因在非生物逆境诱导下的差异表达后。然后,用该启动子构建启动GUS(β-glucuronidase)基因的表达载体,基因枪法转化玉米愈伤组织。经组织化学染色法检测其表达后,在高渗、高盐、低温胁迫处理及ABA诱导下检测GUS酶荧光值与荧光素酶(内参)发光值的比值(GUS/LUC),以此评价ABA3s启动子在非生物逆境胁迫下的启动活性。结果表明,ABA3基因在模拟干旱、低温、高温、高盐胁迫及ABA、乙稀诱导下差异表达,说明该基因的启动子(ABA3s)具有非生物逆境诱导活性。序列分析表明,ABA3s启动子全长777 bp,含有ARE、HSE、MBS、TGA、Circadian等多种非生物逆境胁迫应答元件。用ABA3s启动GUS基因构建的表达载体转化的玉米愈伤组织,响应干旱、低温、高温、高盐胁迫等多种非生物逆境胁迫,及ABA和乙稀诱导,GUS检测呈阳性。在8%甘露醇高渗条件下,GUS/LUC比值比空白对照高6倍。上述结果表明,ABA3s启动子具有非生物逆境诱导特性,经进一步验证其功能后,可用于玉米抗逆转基因研究。

To overcome the problems caused by the over-expression of exogenous genes under the control of constitutive promoters, the promoter （ABA3s） sequence of maize （Zea mays） mo-molybdopterin cofactor sulfurase gene （ABA3） was cloned homologously, analyzed for its abiotic stress-responsive elements by the PlantCARE software, and detected for dif-ferential expression of the ABA3 gene under the abiotic stresses by real-time quantitative PCR. Then, this promoter was used to construct expression vector to start GUS （β-glucuronidase） gene, and transform maize calli by biolistics. After iden-tification by histochemcal staining, the ratio of the GUS activity relative to the luciferase activity （internal control） （GUS/LUC） was measured under the stresses of hypertonic, high salt, low temperature, and the induction of ABA, and used to evaluate the activity of the ABA3s promoter in response to abiotic stresses. The results showed that the ABA3 gene was differentially expressed under the stress of simulative drought, low temperature, high temperature, high salt, and the induc-tion of ABA and ethylene, indicating that the promoter （ABA3s） of this gene is induced by abtiotic stress. The sequence analysis showed that the ABA3s promoter is 777 bp long, and contains abiotic stress-responsive elements ARE, HSE, MBS, TGA and circadian. The transformed calli by the expression vector of the GUS gene under the control of the ABA3s promo-ter showed positive in GUS detection in response to the abiotic stresses of drought, low temperature, high temperature, high salt, and the induction of ABA and ethylene. The GUS/LUC ratio was six folds higher than the blank control under the hypertonic stress of 8%mannitol. It is concluded that the promoter ABA3s is inducible in response to abiotic stresses, and might be applied to transgenic research of maize for abiotic tolerance after further functional evaluation.