Preliminary Investigation Of Mechanism Of TCP Gene Regulating The Development Of Cucumber Tendrils

Cucumber is one of the important vegetable crops worldwide. The plant type of cucumber is an important agronomic traits, and tendrils affect the plant type. A novel organ key gene, TEN, that controls the development of tendrils, was identified and cloned by a rare SNP before. TEN belongs to CYC/TB1 subclass of Class II TCP genes. TCP(TEOSINTE BRANCHED1/CYCLOIDEA/PCF1, 2) transcription factors are a class of plant-specific transcription factors, mainly involved in the differentiation and growth of plant cells. TCP plays an important role in plant growth and development, morphogenesis and the response of the external environment. But research about the function mechanism of this subclass is little. In this study, SAAB assay was carried out to identify the binding elements of TEN. Ch IP-Seq and RNA-seq were performed to identify the candidates of downstream genes. These candidates probably control the development of cucumber tendrils, thus preliminary explaining the molecular mechanisms of the development of tendrils. The main results are as follows: 1. The total protein of the cucumber roots, stems, leaves, female flowers, male flowers, fruits and tendrils was detected using the TEN antibody by Western Blot. The result showed that TEN was specially expressed in cucumber tendrils. 2. Identify the content of protein TEN in three of different stage of tendrils- young tendrils; straight tendrils and coiled tendrils. It turned out that the content of total protein was increasing from young tendrils, and reached the highest level in the straight tendrils, then reduced at the coiled period. The distribution of TEN in tendrils cells indicated the protein mostly concentrated in the cytoplasm, but little in the nucleus. 3. The analysis result of RNA-seq data of normal and abnormal tendrils showed that 1271 genes were up-regulated in abnormal tendrils, which were significantly enriched associated with the development of lateral branch. 433 genes were down-regulated, and those genes were significantly enriched related to the tropism movement. 4. After sequencing the DNA from Ch IP assay, the data analyzed by bioinformatics showed that the core binding site of TEN was probably AAGAAG/A or its reverse complement sequence. 5. After optimizing the inducing conditions, we could obtain more soluble fusion protein of GST-TEN in the condition of 0.1 m M IPTG, 22℃, 180 rpm, 8 h. The molecular size of the fusion protein was 71 k Da, which was consistent with the predicted size. 6. The result of SAAB assay in vitro showed the binding element of TEN appeared to be GGNCCC and ACAC.