玉米淀粉合成酶基因GBSS启动子的克隆与鉴定

曾礼华1,*, 汪瀚宇2,*, 谢程程2, 曹墨菊2,**
1四川师范大学生命科学学院, 成都610101; 2四川农业大学玉米研究所, 农业部西南玉米生物学与遗传育种重点实验室, 成都611130

通信作者:曾礼华;E-mail: caomj@sicau.edu.cn;Tel: 13882439529

摘 要:

以玉米自交系‘18红’的基因组DNA为模板采用PCR技术对玉米淀粉合成酶基因GBSS (ZmGBSS)的启动子进行克隆, 获得了1 884 bp的扩增片段(PZmGBSS), 应用启动子分析软件PlantCARE进行分析, 发现该片段含有多个不同的调控元件。通过半定量RT-PCR分析表明, 在授粉15 d的胚乳中ZmGBSS的表达量最高, 其次为胚, 在根和叶中的表达量较低。通过不同诱导培养基对胚乳进行培养, 发现脱落酸(ABA)诱导后ZmGBSS的表达量明显提高, 葡萄糖和赤霉素(GA)诱导后的表达量有所降低。通过构建启动子PZmGBSS瞬时表达载体, 并利用基因枪对不同受体进行转化分析, 结果发现胚乳中PZmGBSS的启动活性最高, 其次为胚, 根和叶中最弱。对转化后的胚乳进行诱导培养, 发现ABA诱导可使报告基因LUC的表达明显增强。以上结果表明, 启动子PZmGBSS为胚乳特异启动子且能够被ABA正向调控。

关键词:玉米; GBSS基因; 启动子; 克隆

收稿:2015-06-19   修定:2015-08-03

资助:“十二五”国家“863”项目(2011AA10A103和2012AA-101202-4)。 * 共同第一作者。

Clone and Identification of Granule-Bound Starch Synthase Gene Promoter in Maize

ZENG Li-Hua1,*, WANG Han-Yu2,*, XIE Cheng-Cheng2, CAO Mo-Ju2,**
1College of Life Sciences of Sichuan Normal University, Chengdu 610101, China; 2Key Laboratory of Maize Biology and Genetic Breeding on Southwest, Ministry of Agriculture, Maize Research Institute of Sichuan Agricultural University, Chengdu 611130, China

Corresponding author: ZENG Li-Hua; E-mail: caomj@sicau.edu.cn; Tel: 13882439529

Abstract:

The genome DNA of maize inbred line ‘18 Hong’ was used as template for the promoter isolation of maize GBSS gene and a fragment with length of 1 844 bp was obtained. Based on the analysis with the Plant CARE software, several cis-regulation elements were found within the sequence of PZmGBSS. The semi-quantitative RT-PCR for ZmGBSS showed that 15 day endosperm after pollination with the highest expression, the embryo second, both root and leaf with relatively low expression. Detached maize 15 day endosperms after pollination were incubated in different treatments, it suggested the expression of GBSS gene was up-regulated significantly with abscisic acid (ABA) induction, the expression was little down-regulated with glucose and gibberellin (GA). Transcriptional analysis revealed that it is mainly expressed in endosperm and is induced by ABA. The 1 884 bp promoter region of the GBSS gene (PZmGBSS) was fused with the LUC reporter gene to get the transient expression vector. Bombardment transformations were conducted with different receptors, such as leaf, root, endosperm and embryo. Transient expression assay showed that PZmGBSS could drive the LUC gene and it was highly expressed in the endosperm relative to the embryo, the leaf and root with lowest expression. The activity of 1 884 bp promoter PZmGBSS was relative low compared with ubiquitin promoter. The PZmGBSS may be a promoter specific to endosperm. These results suggested that the LUC activity drove by PZmGBSS increased significantly when treated with ABA. So it was concluded that the promoter PZmGBSS is an endosperm specific promoter and can be activated by ABA induction.

Key words: maize; GBSS gene; promoter; clone

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