Cloning And Functional Analysis Of WUS Gene In Betula Platyphylla

Betula platyphylla Suk is a deciduous tree in the genus Betulaceae.As a major regulator of plant growth signals,the WUS gene is necessary to maintain the stem cell niche in the bud tip meristem,the differentiation of side units,totipotency of plant cells,and other diverse cellular processes.In order to further study the role of WUS gene in Betula platyphylla,Bp WUS gene was analyzed by bioinformatics method,Bp WUS gene was cloned and the Bp WUS-PA7-GFP fusion expression vector was constructed,which was successfully transferred into onion epidermal cells by vacuum osmosis method and found to be located in the nucleus.The overexpression vector of Bp WUS gene was constructed by homologous recombination method and transferred into the zygotic embryos of Betula platyphylla infected by Agrobacterium tumefaciens.The transgenic was successfully obtained by PCR identification and q RT-PCR identification,and a transgenic phenotype of leaf crimpy was found.A total of 273 differentially expressed genes were screened out,including 106 up-regulated genes and 167 down-regulated genes.These differentially expressed genes were analyzed by GO and KEGG enrichment,and the differentially expressed genes were verified by q RT-PCR.In order to further explore the molecular mechanism of WUS gene in Betula platyphylla and understand the role of WUS gene in the growth and development of Betula platyphylla.The results are as follows:1.Bioinformatics method was used to analyze the Bp WUS gene of Betula platyphylla.Bp WUS gene encoded 274 amino acid sequences,molecular weight was 30476.39,theoretical isoelectric point was 6.02,lipid index was 46.64,hydrophobicity was-0.912,instability index was 67.83,belonging to unstable protein.Bp WUS protein was predicted to have one HOX domain and the number of transmembrane helices was predicted to be 0,so it was not a transmembrane helices protein.And its subcellular localization was predicted to be in the nucleus.A highly conserved HOX domain and WUS-Box functional domain were found in both Bp WUS and At WUS by amino acid sequence alignment,and a phylogenetic tree of was constructed.2.Birch RNA was extracted and Bp WUS gene was cloned.The Bp WUS-PA7-GFP fusion expression vector was constructed by homologous recombination method,and the plasmid was infiltrated into onion epidermal cells by vacuum osmosis method.3.The overexpression vector of Betula platyphylla Bp WUS gene was constructed by homologous recombination method and transferred into Agrobacterium tumefaciens.The zygotic embryos of Betula platyphylla were infected by agrobacterium tumefaciens mediated genetic transformation.After hygromycin screening,four transgenic lines were identified by PCR and q RT-PCR.The development of the transgenic bud was observed,the cotyledon embryo stage was found,the transgenic line was compared with the wild type phenotype,and a transgenic plant with curly leaves was found.4.Transcriptome sequencing was performed on the transgenic lines,and a total of 273 differentially expressed genes were screened,including 106 up-regulated genes and 167 downregulated genes.GO functional enrichment analysis and KEGG enrichment analysis were performed on the differentially expressed genes.At the same time,the expression of ethylene response factor affecting leaf development was up-regulated,and the expression of zinc finger protein involved in ethylene synthesis was down-regulated.Ten different genes such as ethylene response factor and zinc finger protein were verified by q RT-PCR,and the results were basically consistent with RNA-seq results.These results will further lay a foundation for exploring the regulatory role of WUS gene in Betula platyphylla.