Expression Characteristics And Genetic Transformation Of BpCUC2 In Betula Platyphylla

CUC2 (CUP-SHAPED COTYLEDON 2), a member of the plant-specific NAC family NAM superfamily, is required for the shoot apical meristem formation, organ primordia formation and border establishment, leaf margin morphogenesis establishment and flower growth and development. Besides, CUC2 is regulated by miR164. In the past, the study of the CUC2 gene almost focused on the model plants Arabidopsis, rice and some other plants. So far, it has not been reported on woody plants. In this study, birch was used as genetic transformation receptor for studying expression characteristic of BpCUC2 and genetic transformation to lay the foundation for people to learn more about the gene function.The results included:(1) 1497bp sequence of BpCUC2 upstream were cloned and the cis-acting element were analysed. The results showed that this sequence contained TATA-box, CAAT-box, hormonal response, environmental stress response and transcription factor binding sites. We selected the auxin-responsive element CATATGGMSAUR, its core sequence CATATG, built report vector and adopting yeast one-hybrid technology got 20 proteins interaction with this element. These proteins all had protein structure and function domains. We constructed PromCUC3::LUC vector for genetic transformation, using Agrobacterium tumefaciens mediated zygotic embryo method. PCR detection proved that three transgenic lines have been successfully obtained. The results of real-time quantitative PCR of transgenic lines organization parts showed that PromCUC2 had promoter activity and specificity of temporal and spatial expression. The apical bud owned the highest expression of LUC gene, followed by leaves, lowest expression in roots. Under MeJA, ABA, IAA, GA hormone treatment, the results of LUC relative expression in transgenic lines showed that different tissues inconsistently responses to four hormones.(2) BpCUC2 gene, cloned from birch, was proved belong to NAC family NAM superfamily via multiple sequence alignment analysis. This gene have a higher degree of similarity with Arabidopsis AtCUC2, grapes VvCUC2, which are related to the development of shoot apical meristem and leaves. According to regulatory relationship between BpCUC2 and miR164 gene, BpCUC2-m gene was cloned by overlap extension PCR. Enzyme cutting technique was used to build overexpression, suppression and anti-miR164 regulated BpCUC2-m overexpression vector for genetic transformation using Agrobacterium tumefaciens mediated zygotic embryo method. Transgenic lines were identified by PCR, real-time quantitative PCR and Northern blot analysis. The results indicated that BpCUC2 has been successfully integrated into the plant genome and expressed in mRNA levels. Compared with control plants, 35S::BpCUC2 transgenic lines were lower 34.03%,47.53%, leaf spacing mainly in 0-1.2cm, but control plants mainly in 0.8～2.0cm.