CsACO4, an ACC oxidase gene regulating male differentiation in cucumber

Abstract

Ethylene has been implicated as a sex-determining hormone in cucumber: Its exogenous application increases femaleness and gynoecious genotypes were reported to produce more ethylene. 1- aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) is the key enzyme in ethylene biosynthesis. In this study, a 1,188 base pair (bp) fragment was amplified from cucumber (Jinyan-4) genome with degenerated primers derived from the ACO amino acid consensus sequence among different plant species. The putative new gene was analyzed by bioinformatic tools. Phylogenic analysis result of the new gene (CsACO4, GenBank accession number AY450356) was in accordance with the evolution relationship of genetics among various plant species. The result of BLAST showed the sequence presented a very high match with the ACO genes from other plants; the homologue was from 80 to 99%. Using the sequence, a RNA interference (RNAi) transformation vector was constructed through the way of BP cloning. Eleven transgenic plants were obtained. The transgene integrated into cucumber genome was proved with PCR and southern blotting. The morphological exploration showed that the inserted RNAi target fragments could inhibit the endogenous CsACO4 gene expression and that it could regulate male differentiation in the lower nodes in cucumber.

Keywords: Cucumber, ACC oxidase gene, RNAi, male differentiation