以水稻品种‘日本晴’(Oryza sativa ‘Nipponbare’)为实验材料,根据 GenBank 上公布的同品种水稻的基因组DNA 序列设计 1 对引物,对水稻 Xa21 基因启动子进行克隆并测序,通过 PCR 扩增获得的 Xa21 基因启动子序列长1 982 bp,其中除包含启动子基本元件外,还包含一些与逆境信号相关的元件( GCC-box、A-box、TC-rich repeats、MBS、LTR 和 W-box 等)。 利用 GUS 组织化学染色和定量分析方法,研究了转基因水稻 T1 代株系不同器官和发育阶段 Xa21 基因启动子的表达特异性及其在不同逆境和激素处理条件下的表达特征,结果显示:在转基因水稻的叶、茎和根部均能检测到 GUS 活性,但根部 GUS 活性最高,特别是在根尖的中柱区活性最强;随苗龄增长(3 叶期、5叶期和 8 至 9 叶期)叶片中 GUS 活性逐渐增加,8 至 9 叶期 GUS 活性最高;机械损伤和 100μmol· L^-1茉莉酸甲酯(MeJA)处理可使叶片中 GUS 活性显著或极显著提高,而干旱、500 μmol· L^-1水杨酸(SA)和 100 μmol· L^-1脱落酸(ABA)处理则对叶片中 GUS 活性无明显影响。 研究结果表明:外界逆境胁迫对水稻 Xa21 基因启动子的表达有诱导作用;该启动子的表达受水稻发育阶段的调控并具有一定的器官组织特异性,在根中的表达量最高;其介导的抗病反应依赖于茉莉酸(JA)信号通路。

 

Taking cultivar‘ Nipponbare’ of rice ( Oryza sativa L.) as the experimental materials, Xa21 gene promoter was cloned and sequenced by a pair of primers designed on the basis of genomic DNA sequence of same cultivar published on GenBank. Xa21 gene promoter with sequence length of 1 982 bp is obtained by PCR amplification, in which, except with promoter basic elements, it also contains some elements related to stress signals, such as GCC-box, A-box, TC-rich repeats, MBS, LTR and W-box, etc. By means of GUS histochemical staining and quantitative assay methods, expression specificity of Xa21 gene promoter in different organs and developmental stages of T1 strain of transgenic rice and its expression characteristics under different conditions with stress and hormone treatments were researched. The results indicate that GUS activity in leaf, stem and root of transgenic rice can be detected but that is the highest in root, especially in stele of root tip. With increasing of seedling age (3-, 5- and 8- to 9-leaf stages), GUS activity in leaf increases gradually and reaches the highest at 8- to 9-leaf stage. Treatments with mechanical damage or 100 μmol· L^-1 methyl jasmonate (MeJA) can lead GUS activity to increase significantly or extremely significantly, respectively, but treatments with drought, 500 μmol · L^-1 salicylic acid (SA) or 100 μmol· L^-1 abscisic acid (ABA) have no obvious effect on GUS activity. It is suggested that external stress has an induction to expression of Xa21 gene promoter in rice, and the expression of promoter is controlled by developmental stages and possesses the specificity in organ or tissue with the highest expression in root. Disease resistance response mediated by Xa21 gene promoter depends on the jasmonate acid (JA) signal pathway.