粤油7号花生AhNCED1基因启动子克隆及其活性分析

MOLECULAR CLONING AND GUS-AIDED ACTIVITY ASSAYING OF PROMOTER SEQUENCE OF AhNCED1 GENE FROM Arachis hypogaea L.cv.Yueyou 7

AhNCED1是干旱胁迫下花生中调控脱落酸(abscisic acid,ABA)生物合成的关键基因。本文采用基于PCR的基因组DNA步移法,从抗旱性强的粤油7号花生中克隆AhNCED1基因起始密码子ATG上游2392bp启动子序列,构建该启动子驱动报告基因GUS的植物双元表达载体pAhNCED1p∷GUS,转化野生型拟南芥获得AhNCED1p∷GUS转基因植株。通过GUS染色及其酶活性测定分析AhNCED1p∷GUS转基因拟南芥中AhNCED1p启动子的活性。结果表明,AhNCED1p∷GUS转基因拟南芥叶片中AhNCED1p启动子活性最强,脱水胁迫显著增强7 d龄转基因拟南芥幼苗叶片中AhNCED1p启动子活性。300mmol·L-1山梨醇胁迫或100μmol L-1外源ABA处理3 h明显增强25d龄AhNCED1p∷GUS转基因拟南芥中AhNCED1p启动子活性。结果说明,粤油7号花生AhNCED1基因启动子受渗透胁迫和ABA诱导,与生物信息学预测AhNCED1基因启动子序列中存在逆境胁迫和ABA响应元件的结果相一致。

The nine-cis-epoxycarotenoid dioxygenase（NCED） is considered to be a rate-limiting enzyme involved in abscisic acid（ABA） biosynthesis in plants.The AhNCED1 gene was previously characterized from drought-resistant peanut variety,and was confirmed to be the key gene regulating ABA biosynthesis in peanut plants under drought stress.The levels of drought stress-induced AhNCED1 transcript and endogenous ABA in leaves of drought-resistant peanut were significantly higher than those in drought-sensitive peanut variety.The molecular mechanism of the difference in the levels of AhNCED1 gene expression and ABA accumulation between drought-resistant and drought-sensitive peanut varieties in response to drought stress still needs to be elucidated.In present study,a 2392-bp promoter sequence of 5＇ upstream of initial codon of AhNCED1 gene from drought-resistant Arachis hypogaea L.cv.Yueyou 7 was cloned through a PCR-based genomic DNA walking method.The promoter sequence was analyzed bioinformatically in PlantCARE cis-acting element database,and fused with GUS gene originated from pCAMBIA1301 to construct a binary vector pAhNCED1p∷GUS,which was introduced into wild type Arabidopsis through Agrobacterium-mediated transformation,generating AhNCED1p∷GUS transgenic plants after hygromycin screening and PCR detection.The AhNCED1 promoter activity in AhNCED1p∷GUS transgenic Arabidopsis was determined through GUS histochemical staining and fluorometric assay.The GUS staining showed that the fusion gene AhNCED1p∷GUS was dominantly expressed in leaves of seven-day-old transgenic seedlings,and that the AhNCED1 gene promoter activity in leaves of seven-day-old transgenic seedlings was significantly enhanced by an 8 hours dehydration treatment.The fluorometric GUS assay revealed that the AhNCED1 gene promoter activity in 25-day-old AhNCED1p∷GUS transgenic Arabidopsis seedlings was significantly increased by a 3 hours hydroponical treatment of 300mmol·L-1 sorbitol or 100μmol L-1 exogenous ABA,implying that AhNCED1 gene promoter is osmotic stress-and ABA-induced which might be attributed to the predicted important cis-acting elements,including stress-responsive elements and ABA responsive element（ABRE）,in the promoter sequence.