小麦抗逆相关基因TaSAP1的5′非翻译区内含子功能分析

Function analysis of 5′ untranslated region introns in drought-resistance gene TaSAP1

逆境相关蛋白(stress associated protein,SAP)是植物中一类具有A20/AN1锌指结构域的蛋白,而A20/AN1锌指蛋白主要参与植物逆境响应。小麦中TaSAP1基因参与植株对各种逆境的响应,其5′非翻译区含有2个内含子(5'untranslated region intron,5UI)。本研究利用重叠PCR,构建了TaSAP1的2个5UI缺失突变的表达载体,并转化二穗短柄草(Brachypodium distachyon),通过分析GUS活性,研究TaSAP1启动子及其5UI的生物学功能。结果表明TaSAP1启动子P1可受干旱、低温和外源ABA胁迫上调表达,表达活性分别是对照的10、6和4倍。TaSAP1基因2个5UI对启动子活性至关重要,2个5UI同时突变可致P1失活;Intron-1缺失突变导致P1活性降低约2.7倍(P<0.05),但不影响P1对逆境胁迫的响应;Intron-2缺失突变导致P1失去对干旱、低温和外源ABA的响应能力。本研究结果为深入研究TaSAP1 5UI的生物学功能奠定了基础,也为改善作物抗逆性提供了重要元件。

Stress associated proteins (SAPs),a group of A20/AN1 zinc-finger domain-containing proteins,are mainly involved in responding to abiotic stresses in plant.TaSAP1 involved in the responses of wheat to several abiotic stresses has two introns in the 5' untranslated region intron (5UIs).In this study,the two 5UIs were removed respectively by using overlapping PCR,and the expression vectors were constructed and transformed into Brachypodium distachyon via Agrobacterium-mediated transformation.The functions of TaSAP1 promoter and 5UIs were analyzed according to GUS activity.The activity of TaSAP1 promoter (P1) was up-regulated under the stresses of drought,cold and exogenous abscisic acid (ABA) by 10,6,and 4 folds,respectively.The 5UIs were absolutely necessary for the promoter activity that lost in the double mutants of 5UIs.Intron-1 deletion led to 2.7-fold decrease (P<0.05) in GUS activity,whereas Intron-2 deletion resulted in that P1 was not able to respond to drought,low temperature and exogenous abscisic acid.These results provide basic information for further study in biological function of TaSAP1 5UIs.