APETALA1基因启动子的克隆与功能分析

Cloning and Functional Analysis of APETALA1 Promoter

为了分析AP1的表达调控模式,本研究克隆了拟南芥花异常株系AFDL的AP1启动子,启动子元件预测结果表明:AP1启动子中含有3个结合MADS调控因子的CArG box(从5'依次编号为CArG1、CArG2、CArG3),通过删减AP1启动子长度以及改变CArG box数量构建了5个GUS表达载体并转化野生型拟南芥。测序结果显示:AFDL的AP1启动子在核苷酸序列上与野生型拟南芥完全一致,这表明AP1在AFDL中的表达显著降低并不是启动子序列突变引起的;转基因植株的GUS表达模式说明了CArG1在花发育早期及后期激活基因的表达,CArG2在整个后期都对基因的表达有抑制作用,而CArG3在花发育初期就能抑制基因的表达,并且在中后期仍然保持了对下游基因的抑制作用,CArG box1、2、3对AP1的表达有显著但非决定性的影响。此外,还推测在AP1启动子0-3 579 bp范围之外存在影响AP1在第4轮花器官表达的调控元件,-3 579 bp至-1 752 bp区域可促进AP1的表达,而AP1启动子-1 759 bp至-1 359 bp区域除CArG2外的其它元件对调节其表达无明显作用。

APETALA1 is an important MADS box gene involved in the regulatory pathway of flowering. The expres- sion of the AP1 decreases dramatically in the Abnormal Flower Development Line （AFDL） of Arabidopsis. AFDL is absent of petals and with a stacked inflorescence meristems. In order to analysis the matically in the Abnormal Flow- er Development Line （AFDL） of Arabidopsis. AFDL is absent of petals and with a stacked inflorescence meristems. In order to analysis the regulation of the promoter of AP1, the - 3 579 bp promoter region of AP1 was cloned from AFDL. The functional element predication indicated that three CArG boxes, the binding sites for the MADS regula- tor, were located in the promoter （the three boxes were identified by progressive number as CArG1, CArG2, CArG3 form）. 5 vectors were constructed with the promoters of AP1 to drive GUS in wild-type, which with different length and number of the CArG boxes. The sequence of AP1 in AFDL was the same as that of wild-type, that indicated that the significant down-regulation of AP1 was not associated with the CArG boxes as well as its sequenced; And the GUS staining of the transformed plants showed that the CArG1 was the binding site for positively acting factor （s） during the early stage and the later stage of the flower development, and the deletion of the CArG2 and the mutations in CArG3 resulted in an increase in the level of reporter gene activity during the early or later floral stages, sugges- ting that CArG2 and CArG3 are the binding sites for negatively regulators. Therefore the three CArG boxes in the promoter of AP1 are functional on the regulation the AP1 expression rather than determinant expression of AP1. The ectopic GUS expression in the whorl 4 indicated that there were unidentified elements that played a role on the ex- pression specificity outside the 0 - -3 579 bp upstream of the transcriptional start site. In addition, there may be some elements located in the region from - 3 579 bp to - 1 752 bp but the region from - 1 752 bp to - 1 359 bp was nonfunctional on the regulation of AP1 except the CArG2 box.