Abstract：
Fragaria chiloensis is a Chilean native species that softens intensively during its ripening. Its softening is related to cell wall disassembly due to the participation of cell wall degrading enzymes. Softening of F. chiloensis fruit can be accelerated by ABA treatment which is accompanied by the increment in the expression of key cell wall degrading genes, however the molecular machinery involved in the transcriptional regulation has not been studied until now. Therefore, the participation of two MADS-box transcription factors belonging to different subfamilies, FchAGL9 and FchSHP, was addressed. Both TFs are members of type-II MADS-box family (MIKC-type) and localized in the nucleus. FchAGL9 and FchSHP are expressed only in flower and fruit tissues, rising as the fruit softens with the highest expression level at C3-C4 stages. EMSA assays demonstrated that FchAGL9 binds to CArG sequences of RIN and SQM, meanwhile FchSHP interacts only with RIN. Bimolecular fluorescence complementation and yeast two-hybrid assays confirmed FchAGL9-FchAGL9 and FchAGL9-FchSHP interactions. Hetero-dimer structure was built through homology modeling concluding that FchSHP monomer binds to DNA. Functional validation by Luciferase-dual assays indicated that FchAGL9 transactivates FchRGL and FchPG\'s promoters, meanwhile FchSHP transactivates those of FchEXP2, FchRGL and FchPG. Over-expression of FchAGL9 in C2 F. chiloensis fruit rises FchEXP2 and FchEXP5 transcripts, meanwhile the over-expression of FchSHP also increments FchXTH1 and FchPL; in both cases there is a down-regulation of FchRGL and FchPG. In summary, we provided evidence of FchAGL9 and FchSHP participating in the transcription regulation associated to F. chiloensis\'s softening.
摘要：
Fragariachiloensis是智利的本地物种，在成熟过程中会强烈软化。由于细胞壁降解酶的参与，其软化与细胞壁分解有关。通过ABA处理可以加速辣椒果实的软化，同时伴随着关键细胞壁降解基因表达的增加，然而，到目前为止，参与转录调控的分子机制尚未被研究。因此,两个属于不同亚家族的MADS-box转录因子的参与，FchAGL9和FchSHP,已解决。两种TF都是II型MADS盒家族（MIKC型）的成员，位于细胞核中。FchAGL9和FchSHP仅在花和果实组织中表达，随着果实在C3-C4阶段以最高表达水平软化而上升。EMSA实验证明FchAGL9与RIN和SQM的CArG序列结合,同时FchSHP仅与RIN交互。双分子荧光互补和酵母双杂交测定证实了FchAGL9-FchAGL9和FchAGL9-FchSHP相互作用。通过同源性建模建立异二聚体结构，得出结论FchSHP单体与DNA结合。通过荧光素酶双重分析的功能验证表明，FchAGL9反式激活FchRGL和FchPG的启动子，同时FchSHP激活FchEXP2、FchRGL和FchPG。在C2F.chiloensis果实中FchAGL9的过表达增加了FchEXP2和FchEXP5转录本，同时，FchSHP的过度表达也会增加FchXTH1和FchPL；在这两种情况下，FchRGL和FchPG均下调。总之,我们提供的证据表明FchAGL9和FchSHP参与了与黄曲霉软化相关的转录调控。
