通过CRISPR_Cas9介导的SVP基因的靶向突变来调节甘蓝型油菜的开花期.pdfVIP

Regulation of Flowering Time by Targeted mutation of the SVP gene with CRISPR/Cas9-Mediated gene editing in Brassica napus L. Table of Contents 摘 要 i Abstract iii Abbreviation v 1 Introduction 1 1.1 Literature review 2 1.1.1 Classification of rapeseed based on the geographical and growing season 2 1.1.2 Genetic regulation of flowering time and Interactive pathway as a basis for diversity 4 1.1.3 Identification of QTLs for flowering time 14 1.1.4 Genome-Wide Association study of Flowering Times 18 1.1.5 New tools for flowering time regulation in B. napus 18 1.2 Research Objective 22 2 Material and Method 23 2.1 Plant materials 23 2.2 Phylogenetic and bioinformatics analysis 23 2.3 CRISPR/Cas9 target locus selection, construct assembly 23 2.4 Agrobacterium-mediated genetic transformation in B. napus 23 2.4.1 DNA extraction 25 2.5 Identification of transgenic mutant plants 26 2.5.1 The experimental principle of Hi-TOM method 26 2.5.2 Construction of NGS library 27 2.5.3 Analysis of sequencing data 28 2.5.4 Preparation of barcode primers 29 2.5.5 Next-generation sequencing 29 2.5.6 Filtering reads and mapping reads 30 2.6 Analysis of potential off-targets 30 2.7 Field experiments and phenotyping 30 3 Results 32 3.1 Bioinformatics analysis of BnSVP in J9707 32 3.2 CRISPR / Cas9 vector construction to knock out the BnSVP gene in B. napus 33 3.3 Identification of mutation patterns in the transgenic plants 35 I Huazhong Agricultural University 2020 Masters Degree Thesis 3.4 Isolation of mutants without transgenic elements in T and