靶向BnSVP的CRISPR/Cas9基因组编辑载体的构建
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摘要
基于CRISPR/Cas9的基因组编辑技术是一种新兴的分子修饰工具,可实现基因组特定位点碱基的缺失、插入或替换,造成基因功能丧失,现已广泛应用于基因功能的研究。为深入探讨甘蓝型油菜中Bn SVP的生物学功能,以中双11号基因组(甘蓝型油菜参考基因组,v4.1)中的4个Bn SVP为靶标基因,并针对不同染色体上的Bn SVP基因,应用CRISPR-P 2.0软件共设计了12条特异sg RNA种子序列,以实现4个Bn SVP同源基因的全部或部分敲除;以p YLCRISPR-Cas9P35S-H为基本载体,以At U3b或At U3d为sg RNA转录启动子,采用Golden gate cloning技术分别组装构建了6个双靶点CRISPR/Cas9植物表达载体。测序结果表明,6个CRISPR/Cas9植物表达载体各sg RNA表达盒DNA序列正确,双靶点组装顺序无误。研究结果为进一步原生质体瞬时表达和农杆菌介导的油菜遗传转化奠定了基础,同时也为应用CRISPR/Cas9基因组编辑系统研究植物多拷贝同源基因的功能提供参考。
Targeted genome editing via CRISPR/Cas9 is emerging as a powerful tool for molecular modification in recent years,by which the target gene will lose functions because of deletion,insertion or substitution of several nucleotides in specific sites. It has been widely used to researches on gene function. To deeply elucidate the biological function of Short vegetative phase( SVP) in rapeseed( Brassica napus L.) by entire or partial knock-outs,totally twelve sg RNA seed sequences targeting four homologous copies of SVP distributed in Zhongshuang No. 11 genome( Brassica napus reference genome,v4. 1) were designed by using software CRISPR-P 2. 0,and then six dual sg RNAs CRISPR/Cas9 genome editing constructs were developed employing p YLCRISPR-Cas9 P35 S-H as backbone and At U3 b or At U3 d as promoter with Golden Gate Cloning technology. Sequencing results demonstrated that all the recombinant plasmids showed the correct nucleotides and expression cassette assembly,which lay a solid foundation for further protoplast transient expression assay and Agrobacterium-mediated transformation of rapeseed. Meanwhile,this paper provides a strategy to study functions of multiple homologous genes in plants applying CRISPR/Cas9 genome editing system.
Targeted genome editing via CRISPR/Cas9 is emerging as a powerful tool for molecular modification in recent years,by which the target gene will lose functions because of deletion,insertion or substitution of several nucleotides in specific sites. It has been widely used to researches on gene function. To deeply elucidate the biological function of Short vegetative phase( SVP) in rapeseed( Brassica napus L.) by entire or partial knock-outs,totally twelve sg RNA seed sequences targeting four homologous copies of SVP distributed in Zhongshuang No. 11 genome( Brassica napus reference genome,v4. 1) were designed by using software CRISPR-P 2. 0,and then six dual sg RNAs CRISPR/Cas9 genome editing constructs were developed employing p YLCRISPR-Cas9 P35 S-H as backbone and At U3 b or At U3 d as promoter with Golden Gate Cloning technology. Sequencing results demonstrated that all the recombinant plasmids showed the correct nucleotides and expression cassette assembly,which lay a solid foundation for further protoplast transient expression assay and Agrobacterium-mediated transformation of rapeseed. Meanwhile,this paper provides a strategy to study functions of multiple homologous genes in plants applying CRISPR/Cas9 genome editing system.
引文
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[13]Hartmann U,H9hmann S,Nettesheim K,et al.Molecular cloning of SVP:a negative regulator of the floral transition in Arabidopsis[J].The Plant Journal:for Cell and Molecular Biology,2000,21(4):351-360.
[14]潘丹丹,毛群飞,张金顺,等.油菜开花时间调控基因SVP的克隆与表达特性分析[J].华北农学报,2013,28(1):93-100.
[15]唐雨薇,刘丽萍,王若娴,等.茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建[J].茶叶科学,2016,36(4):414-426.
[16]Taylor G P.Current Protocols in Molecular Biology[M]//Ma X,Liu Y G.CRISPR/Cas9-based multiplex genome editing in monocot and dicot plants.New Jersey:John Wiley&Sons,Inc.,2016,115:31.6.1-31.6.21.
[17]Ding Y,Li H,Chen L L,et al.Recent advances in genome editing using CRISPR/Cas9[J].Frontiers in Plant Science,2016,7:703.
[18]Lysak M A,Koch M A,Pecinka A,et al.Chromosome triplication found across the tribe Brassiceae[J].Genome Research,2005,15(4):516-525.
[19]Lu H P,Liu S M,Xu S L,et al.CRISPR-S:an active interference element for a rapid and inexpensive selection of genome-edited,transgene-free rice plants[J].Plant Biotechnology Journal,2017,15(11):1371-1373.
[20]Ren B,Yan F,Kuang Y,et al.Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive h AID mutant[J].Molecular Plant,2018,11(4):623-626.
[2]Schiml S,Fauser F,Puchta H.The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny[J].The Plant Journal:for Cell and Molecular Biology,2014,80(6):1139-1150.
[3]Hilton I B,D'ippolito A M,Vockley C M,et al.Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[J].Nature Biotechnology,2015,33(5):510-517.
[4]Gao J,Wang G,Ma S,et al.CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum[J].Plant Molecular Biology,2015,87(1/2):99-110.
[5]Fan D,Liu T,Li C,et al.Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation[J].Scientific Reports,2015,5:12217.
[6]Zhou H,Liu B,Weeks D P,et al.Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J].Nucleic Acids Research,2014,42(17):10903-10914.
[7]Endo M,Mikami M,Toki S.Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice[J].Plant&Cell Physiology,2015,56(1):41-47.
[8]Upadhyay S K,Kumar J,Alok A,et al.RNA-Guided genome editing for target gene mutations in wheat[J].G3-Genes Genomes Genetics,2013,3(12):2233-2238.
[9]Zhang Y,Liang Z,Zong Y,et al.Efficient and transgenefree genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA[J].Nature Communications,2016,7:12617.
[10]Svitashev S,Young J K,Schwartz C,et al.Targeted mu-tagenesis,precise gene editing,and Site-Specific gene insertion in maize using Cas9 and guide RNA[J].Plant Physiology,2015,169(2):931-945.
[11]Yang H,Wu J J,Tang T,et al.CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sg RNA in Brassica napus[J].Scientific Reports,2017,7(1):7489.
[12]Braatz J,Harloff H J,Mascher M,et al.CRISPR-Cas9targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape(Brassica napus)[J].Plant Physiology,2017,174(2):935-942.
[13]Hartmann U,H9hmann S,Nettesheim K,et al.Molecular cloning of SVP:a negative regulator of the floral transition in Arabidopsis[J].The Plant Journal:for Cell and Molecular Biology,2000,21(4):351-360.
[14]潘丹丹,毛群飞,张金顺,等.油菜开花时间调控基因SVP的克隆与表达特性分析[J].华北农学报,2013,28(1):93-100.
[15]唐雨薇,刘丽萍,王若娴,等.茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建[J].茶叶科学,2016,36(4):414-426.
[16]Taylor G P.Current Protocols in Molecular Biology[M]//Ma X,Liu Y G.CRISPR/Cas9-based multiplex genome editing in monocot and dicot plants.New Jersey:John Wiley&Sons,Inc.,2016,115:31.6.1-31.6.21.
[17]Ding Y,Li H,Chen L L,et al.Recent advances in genome editing using CRISPR/Cas9[J].Frontiers in Plant Science,2016,7:703.
[18]Lysak M A,Koch M A,Pecinka A,et al.Chromosome triplication found across the tribe Brassiceae[J].Genome Research,2005,15(4):516-525.
[19]Lu H P,Liu S M,Xu S L,et al.CRISPR-S:an active interference element for a rapid and inexpensive selection of genome-edited,transgene-free rice plants[J].Plant Biotechnology Journal,2017,15(11):1371-1373.
[20]Ren B,Yan F,Kuang Y,et al.Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive h AID mutant[J].Molecular Plant,2018,11(4):623-626.