Cloning And Function Identification Of Related Genes Of Flavonoids Synthesis In Cucumis Sativus L.

Cucumber spine is one of the highly valuable external quality traits of cucumber (Cucumis sativus L.). The fruit spines play important roles in protecting plants from biotic attack by producing defensive compounds. The previous study has revealed that the cucumber black spine is the dominant trait, and controlled by a pair of alleles. Meanwhile, the orange mature fruit colors are controlled by the same gene. CsMYB60 is regarded as the best candidate gene conditioning black spine and orange mature fruit colors of cultivated cucumber. However, the biological function of CsMYB60 and the coloring mechanism of the black spine controlled by it are still unknown. In addition, what secondary metabolites accumulated in black spine has not been reported yet. Our team has found that the main secondary metabolites in black spine are flavonoids. Meanwhile, flavonoids are also accumulated in pericarp of the black-spined cucumber. Flavonoids are beneficial to plants and human health. Therefore, the research on the function of genes related to flavonoid synthesis in cucumber has important theoretical significance and potential application value.Our team constructed F2 segregant populations from a cross between V05A0071 (black spine) and V05A0672 (white spine), and two sample pools were taken for high-throughput sequencing based on BSA (Bulked Segregant Analysis) method. On the basis of previous research, this study selected some genes obviously differentially expressed from the transcriptome data, including Cs4CL, CsCHS, CsF3H, CsLAR, CsGST. These genes are enzyme genes related to flavonoid synthesis. Firstly, we confirmed these genes differentially expressed by semi-quantitative and real time fluorescence quantitative PCR, and also analyzed their expression pattern during developmental period of cucumber fruit. The results showed that the expression of these genes in the pericarp of the black-spined cucumber was obviously higher than that in the pericarp of the white-spined cucumber.Then, we cloned these genes and constructed their overexpression vectors. By the transient expression system in spine, we found that only Cs4CL overexpression (OE) could cause flavonoids accumulated in white spine. Despite our team found CsMYB60 involved in conditioning black spine color by the transient expression system in spine, the data from Yeast One Hybrid and transient expression experiments showed that the candidate gene CsMYB60 could not directly regulate the expression of Cs4CL.At the same time, we performed Cs4CL genetic transformation in cucumber, tomato and Arabidopsis, but only gained transgenic tomato(Solanum lycopersicum L.) and Arabidopsis thaliana. After testing the transgenic lines, we found that Cs4CL OE could improve the lignin content in tomato leaf, and the content of total phenol and flavonoid in the tomato leaf and fruit. Also, Cs4CL OE could improve the content of total phenol, flavonoid and anthocyanin in the Arabidopsis leaf. In addition, Cs4CL OE could accelerate germination rate of Arabidopsis seeds. But, the lignin content in Arabidopsis inflorescence stems was not obviously affected.The above results showed that Cs4CL gene played a key role in the process of flavonoids synthesis and accumulation in the black-spined cucumber, and provided the basis for further research on regulatory mechanism of flavonoids synthesis and accumulation in cucumber.