Function Analysis Of The Unisexual-determination Gene M In Cucumber Plant

Due to its diversity in sex types and to the extensive physiological and genetics studies, cucumber (Cucumis sativus L.) is becoming a model plant for sex-determination research. Recently, the gynoecious-influence gene F and unisexual-determination gene M have been cloned, and showed that they both encode homologies of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which is related with phytohormone ethylene. However, the function and regulation pattern of the M gene (CsACS2) haven't been elucidated very well.In this study, alignment analysis between CsACS2-M (encoded by wild M gene) and CsACS2-m (encoded by mutant m gene) reveals that the mutant amino residue (Gly33Cys) may reside in a putative conserved domain. With the E. coli expression system, we study the original enzymology activity of different isoforms of CsACS-M mutant. The results indicated that CsACS2-M has ACS activity, and it can synthesize ACC in E. coli strain JAde6, which can use ACC as its C source. Nevertheless, CsACS2-m has lost its original activity. Meanwhile, the mutagenic isoforms, CsACS2-A (Gly33Ala) and CsACS2-S (Gly33Ser) also show the disordered activity. Combined with some previous studies, we think there is a new putative conserved domain (S-YF-GW) at the 28~34 position (in CsACS2), which may influence the final enzymology activity by folding anαhelix.To study the CsACS2 gene function in plant, we transform tobacco plants with CsACS2 gene controlled by two kinds of promoters (one of which is cucumber CsACS2 gene original promoter, and another is CaMV 35S promoter). Then, Southern, dot blotting and RT-PCR analysis are used to identify the positive TB0B transformation plants. The results reveal that the expression of CsACS2 in PBMB-CsACS2 transformation plants can not been indicated, which is identity with wild tobacco plant. However, after incubating with ethylene, the expression can be found in the floral buds and young leaves of PBMB-CsACS2 transformation plants. On the other hand, PB35SB-CsACS2 transformation plants show constitutive expression pattern. At the different growth stages, some of PB35SB-CsACS2 transformation plants reveal etiolated seedling, shorter section spacing, smaller leaves, later flowering and shorter filament. All of these reveal close relationship between CsACS2 gene and ethylene.