發(fā)布時(shí)間：2021-10-02 01:41

　　油菜（Brassica napus L.）是一種非常重要的油料作物,是人類(lèi)食用油、富含蛋白質(zhì)的動(dòng)物飼料以及工業(yè)生產(chǎn)原料的重要來(lái)源�？v觀油菜生長(zhǎng)發(fā)育的整個(gè)過(guò)程,開(kāi)花期對(duì)生產(chǎn)力至關(guān)重要。因此,花期調(diào)控是油菜育種的關(guān)鍵目標(biāo)之一。探索有效控制油菜花期的方法,對(duì)現(xiàn)代育種實(shí)踐具有重要意義。SVP基因（short vegetative phase）通過(guò)與開(kāi)花時(shí)間調(diào)控網(wǎng)絡(luò)中的多個(gè)基因相互作用,在控制植物花期發(fā)揮著非常重要的作用。所以該基因可作為油菜早熟育種的潛在調(diào)控目標(biāo)。在本研究中,我們利用CRISPR/Cas9介導(dǎo)的基因編輯技術(shù)對(duì)BnSVP基因的四個(gè)同源拷貝（BnaA04g12990D,BnaA09g42480D,BnaC04g35060D和BnaC08g34920D）進(jìn)行靶向突變并獲得了對(duì)應(yīng)的突變體。含有四個(gè)sgRNAs的二元載體（pSH18）的四個(gè)sgRNA分別由Pu3b,Pu3d,Pu6＿1和Pu6＿29啟動(dòng)子驅(qū)動(dòng)。通過(guò)農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化成功地將雙元載體pSH18轉(zhuǎn)移到甘藍(lán)型油菜純系J9707中。最終獲得了81個(gè)獨(dú)立的轉(zhuǎn)化株系,其中67（82.71%）個(gè)株系攜帶T-DNA插入。我們檢測(cè)到突變... 

【文章來(lái)源】：華中農(nóng)業(yè)大學(xué)湖北省 211工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：110 頁(yè)

【學(xué)位級(jí)別】：碩士

【文章目錄】：
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


【參考文獻(xiàn)】：
期刊論文
[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)



本文編號(hào)：3417755