花生小GTP结合蛋白基因AhRabG3f启动子的盐胁迫响应元件分析

Analysis of the salt-stress responsive element of the promoter of peanut small GTP binding protein gene AhRabG3f

摘要:

为研究花生小GTP结合蛋白基因AhRabG3f对盐胁迫响应的分子机制，文中克隆了花生AhRabG3f基因起始密码子上游1 914 bp的启动子片段(3f-P)。将该启动子5'末端截短获得5个片段(3f-P1-3f-P5)，长度分别为1 729、1 379、666、510、179 bp。构建了将这6个启动子片段与gus基因融合的植物表达载体，利用农杆菌介导法转化烟草。对转基因烟草进行GUS表达分析和酶活性检测，结果表明，在转入各启动子片段的烟草中，都能检测到gus基因的表达，其中全长启动子3f-P的驱动活性最弱，而截短片段3f-P3的驱动活性最强。对转基因烟草进行盐胁迫处理后，3f-P、3f-P1、3f-P2和3f-P3所驱动GUS酶活性是未经盐诱导的3.3、1.2、1.9、1.2倍，表明AhRabG3f启动子是盐诱导型的，在3f-P至3f-P3之间可能存在对盐响应的正调控元件。通过对盐胁迫处理后各启动子片段驱动的GUS活性分析，推测在AhRabG3f启动子上游1 930–1 745 bp、682–526 bp之间存在可能对盐响应的正调控元件MYB、GT1和富含TC的重复序列，1 395–682 bp之间存在可能对盐响应的负调控元件MYC。研究结果可为利用诱导型启动子调控花生的耐盐性提供指导。

关键词:花生;小GTP结合蛋白;启动子;盐胁迫响应元件

Abstract:

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.

Key words:peanut;small GTP binding protein;promoter;salt stress-responsive element