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

发布时间：2021-10-02 01:41

　　油菜（Brassica napus L.）是一种非常重要的油料作物,是人类食用油、富含蛋白质的动物饲料以及工业生产原料的重要来源。纵观油菜生长发育的整个过程,开花期对生产力至关重要。因此,花期调控是油菜育种的关键目标之一。探索有效控制油菜花期的方法,对现代育种实践具有重要意义。SVP基因（short vegetative phase）通过与开花时间调控网络中的多个基因相互作用,在控制植物花期发挥着非常重要的作用。所以该基因可作为油菜早熟育种的潜在调控目标。在本研究中,我们利用CRISPR/Cas9介导的基因编辑技术对BnSVP基因的四个同源拷贝（BnaA04g12990D,BnaA09g42480D,BnaC04g35060D和BnaC08g34920D）进行靶向突变并获得了对应的突变体。含有四个sgRNAs的二元载体（pSH18）的四个sgRNA分别由Pu3b,Pu3d,Pu6＿1和Pu6＿29启动子驱动。通过农杆菌介导的遗传转化成功地将双元载体pSH18转移到甘蓝型油菜纯系J9707中。最终获得了81个独立的转化株系,其中67（82.71%）个株系携带T-DNA插入。我们检测到突变... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：110 页

【学位级别】：硕士

【文章目录】：
摘要
Abstract
Abbreviation
1 Introduction
    1.1 Literature review
        1.1.1 Classification of rapeseed based on the geographical and growing season
        1.1.2 Genetic regulation of flowering time and Interactive pathway as a basis for diversity
        1.1.3 Identification of QTLs for flowering time
        1.1.4 Genome-Wide Association study of Flowering Times
        1.1.5 New tools for flowering time regulation in B.napus
    1.2 Research Objective
2 Material and Method
    2.1 Plant materials
    2.2 Phylogenetic and bioinformatics analysis
    2.3 CRISPR/Cas9 target locus selection,construct assembly
    2.4 Agrobacterium-mediated genetic transformation in B.napus
        2.4.1 DNA extraction
    2.5 Identification of transgenic mutant plants
        2.5.1 The experimental principle of Hi-TOM method
        2.5.2 Construction of NGS library
        2.5.3 Analysis of sequencing data
        2.5.4 Preparation of barcode primers
        2.5.5 Next-generation sequencing
        2.5.6 Filtering reads and mapping reads
    2.6 Analysis of potential off-targets
    2.7 Field experiments and phenotyping
3 Results
    3.1 Bioinformatics analysis of BnSVP in J9707
    3.2 CRISPR/ Cas9 vector construction to knock out the BnSVP gene in B.napus
    3.3 Identification of mutation patterns in the transgenic plants
    3.4 Isolation of mutants without transgenic elements in T_1 and T_2 generations
    3.5 Off-target influence of CRISPR/Cas9 on transgenic plants(T_0)in B.napus
    3.6 Effectiveness of various sgRNA targets on BnSVP mutation
    3.7 Flowering phenotypes of the BnSVP mutants in B.napus
4 Discussion
    4.1 CRISPR/Cas9 system is highly efficient for the development of targeted mutations in B.napu
    4.2 Precise identification of allelic variation with Hi-tom sequencing
    4.3 SVP gene family play an important role in controlling flowering time in B.napus
Conclusions
Future prospective
References
APPENDIX
    Appendix 1
    Appendix 2
    Appendix 3
    Appendix 4
    Appendix 5
    Appendix 6
    Appendix 7
    Appendix 8
    Appendix 9
Acknowledgement


【参考文献】：
期刊论文
[1]Research progress and strategies for multifunctional rapeseed: A case study of China[J]. FU Dong-hui,JIANG Ling-yan,Annaliese S Mason,XIAO Mei-li,ZHU Long-rong,LI Li-zhi,ZHOU Qing-hong,SHEN Chang-jian,HUANG Chun-hui.  Journal of Integrative Agriculture. 2016(08)
[2]Flowering responses to light and temperature[J]. Li Li,Xu Li,Yawen Liu,Hongtao Liu.  Science China（Life Sciences）. 2016(04)



本文编号：3417755