CAJ | 학술논문

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。
위극복조성형계동자계동외원기인과량표체인기적제다문제，동원극륭(Mo-molybdopterin cofactor sulfurase)기인（ABA3）적계동자(ABA3s)서렬，병용PlantCARE연건분석기비생물역경응답원건,실시정량PCR검측 ABA3기인재비생물역경유도하적차이표체후。연후，용해계동자구건계동 GUS（β-glucuronidase）기인적표체재체,기인창법전화옥미유상조직。경조직화학염색법검측기표체후,재고삼、고염、저온협박처리급ABA유도하검측 GUS매형광치여형광소매(내삼)발광치적비치(GUS/LUC),이차평개 ABA3s계동자재비생물역경협박하적계동활성。결과표명, ABA3기인재모의간한、저온、고온、고염협박급ABA、을희유도하차이표체,설명해기인적계동자(ABA3s)구유비생물역경유도활성。서렬분석표명, ABA3s계동자전장777 bp,함유ARE、HSE、MBS、TGA、Circadian등다충비생물역경협박응답원건。용ABA3s계동GUS기인구건적표체재체전화적옥미유상조직,향응간한、저온、고온、고염협박등다충비생물역경협박,급ABA화을희유도, GUS검측정양성。재8%감로순고삼조건하, GUS/LUC비치비공백대조고6배。상술결과표명, ABA3s계동자구유비생물역경유도특성,경진일보험증기공능후,가용우옥미항역전기인연구。
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.