黄瓜类钙调蛋白CML8与性型分化主效基因编码蛋白的互作分析

Interactions between CML8 and protein encoded by major genes of sexual differentiation in Cucumis sativus L.

【目的】为探索类钙调蛋白CMLs是否参与黄瓜性型分化过程,以便进一步完善性型分化调控网络提供参考。【方法】通过克隆黄瓜CML8、ACS1G、ACS2及ACS11基因,qPCR检测CML8基因在不同花型中的表达量,酵母双杂交检测CML8与黄瓜性型分化主效基因编码蛋白间的互作情况。【结果】克隆得到黄瓜CML8基因,完整ORF 441 bp,编码146 aa,不含信号肽;不含跨膜域,为胞外蛋白,含4个具有Ca^(2+)结合能力的EF-hand结构域,不含内含子,亚细胞定位在细胞膜和细胞质;克隆得到黄瓜性型分化关键基因F(ACS1G)、M(ACS2)及A(ACS11),序列比对表明三者的基因序列及编码蛋白序列与黄瓜数据库中的一致,且在雌花、雄花和两性花3种不同花型中的表达量差异显著,在雄花中差异极显著;酵母双杂交试验表明,CML8均可与ACS1G和ACS2发生互作,而不能与ACS11发生互作。【结论】成功克隆得到黄瓜CML8基因,且CML8可与ACS1G和ACS2发生互作,可能参与黄瓜性型分化过程,本研究首次探索类钙调蛋白参与植物性型分化过程,期望为钙信号系统参与植物性型分化提供参考。

【Objective】The paper aims to explore whether calmodulin-like CMLs participate in the sexual differentiation process in Cucumis sativus L., so as to provide reference for improving the regulatory network of sexual differentiation furtherly.【Method】In the study, CML8,ACS1G,ACS2 and ACS11 genes of cucumber were cloned, qPCR was used to detect the expressions of CML8 gene in different types of the flowers, and the interactions between CML8 and proteins encoded by major genes of cucumber sex differentiation were detected by the method of yeast two-hybrid.【Result】CML8 was cloned from cucumber which contained a complete ORF of 441 bp, encoding 146 aa without signal peptide. It was an extracellular protein without transmembrane domain and introns, and contained 4 EF-hand domains with Ca^(2+) binding capacity, and subcellular was located in cell membrane and cytoplasm. ACS1G, ACS2 and ACS11, three key genes of cucumber sexual differentiation were cloned, and sequence alignment showed that the nucleotide sequences and encoded protein sequences of the three genes were consistent with those in the cucumber database. There were significant differences in the expression levels of three genes in the female flowers, male flowers and bisexual flowers, and extremely significant differences in male flowers. The yeast two-hybrid experiment showed that CML8 could interact with ACS1G and ACS2, but not with ACS11.【Conclusion】Calmodulin-like 8(CML8) was cloned, and CML8 could interact with ACS1G and ACS2, which might participate in the sexual differentiation of cucumber, and the study was the first time to explore the involvement of calmodulin in the process of plant sexual differentiation, and expect to provide reference for calcium signaling system to participate in the process of plant sexual differentiation.