高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

基于Cas9/gRNA创制毛果杨COBRA3基因突变体

崔永耀 程玉祥

崔永耀, 程玉祥. 基于Cas9/gRNA创制毛果杨COBRA3基因突变体[J]. 北京林业大学学报, 2018, 40(4): 10-15. doi: 10.13332/j.1000-1522.20170421
引用本文: 崔永耀, 程玉祥. 基于Cas9/gRNA创制毛果杨COBRA3基因突变体[J]. 北京林业大学学报, 2018, 40(4): 10-15. doi: 10.13332/j.1000-1522.20170421
Cui Yongyao, Cheng Yuxiang. Cas9/gRNA-mediated COBRA3 gene mutation in Populus trichocarpa[J]. Journal of Beijing Forestry University, 2018, 40(4): 10-15. doi: 10.13332/j.1000-1522.20170421
Citation: Cui Yongyao, Cheng Yuxiang. Cas9/gRNA-mediated COBRA3 gene mutation in Populus trichocarpa[J]. Journal of Beijing Forestry University, 2018, 40(4): 10-15. doi: 10.13332/j.1000-1522.20170421

基于Cas9/gRNA创制毛果杨COBRA3基因突变体

doi: 10.13332/j.1000-1522.20170421
基金项目: 

国家自然科学基金项目 31770637

国家自然科学基金项目 31570580

详细信息
    作者简介:

    崔永耀。主要研究方向:林木遗传育种。Email:1102799243@qq.com 地址:150040黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林学院

    责任作者:

    程玉祥, 教授, 博士生导师。主要研究方向:杨树分子遗传学。Email:chengyuxiang@nefu.edu.cn 地址:同上

  • 中图分类号: S718.46; S792.119

Cas9/gRNA-mediated COBRA3 gene mutation in Populus trichocarpa

  • 摘要: 目的COBRA对植物生长发育具有重要作用,已有的研究表明,植物COBRA基因与其次生生长有关。为探究树木COBRA基因信息及部分基因功能,我们对毛果杨PtrCOBRA基因家族展开识别和分析性研究。方法分析PtrCOBRA家族成员关系时运用系统进化树,确定毛果杨PtrCOBRA家族基因的各组织转录表达时采用半定量RT-PCR,创制PtrCOBRA3基因突变体时使用Cas9/gRNA基因编辑策略,毛果杨遗传转化采用农杆菌介导组培幼苗茎段浸染法。结果我们识别毛果杨基因组上存在14个PtrCOBRA基因成员,进化树显示PtrCOBRA家族分成两个分支。半定量RT-PCR表明,PtrCOBRA3和PtrCOBRA11在毛果杨木质部高丰度、特异地转录表达,在木质化茎节中其转录水平也较高。基于Cas9/gRNA技术我们敲除了毛果杨PtrCOBRA3,获得4株ptrcobra3敲除突变体。ptrcobra3突变体植株表型:植株矮小,叶片细长且卷曲,茎干略有弯曲。结论PtrCOBRA3和PtrCOBRA11是PtrCOBRA家族中与毛果杨次生生长相关的主要成员;基因敲除遗传证据显示PtrCOBRA3基因参与毛果杨茎干次生生长,PtrCOBRA11与它存在功能的冗余。

     

  • 图  1  COBRA基因家族进化关系及基因结构图

    A. COBRA基因家族进化树;B. COBRA基因结构图。

    Figure  1.  Phylogenetic relationships and gene structure in the COBRA gene family

    A, phylogenetic tree of COBRA gene family; B, COBRA gene structures.

    图  2  PtrCOBRA3和PtrCOBRA11在不同组织内的转录表达分析

    Xy为木质部;Ph为韧皮部;Rt为根;Ap为顶芽;Pe为叶柄;YL为幼叶;ML为老叶;IN1~10为第1~10茎节;cyc.循环;A. PtrCOBRA3和PtrCOBRA11在不同组织内的表达特征;B. PtrCOBRA3和PtrCOBRA11在不同木质化程度茎节内的表达。

    Figure  2.  Analysis of PtrCOBRA3 and PtrCOBRA11gene expression in Populus trichocarpa tissues

    Xy, xylem; Ph, phloem; Rt, root; Ap, apical bud; Pe, petiole; YL, young leaf; ML, mature leaf; IN1-10, inode1-10; cyc, cycles; A, expression of PtrCOBRA3 and PtrCOBRA11 in different tissues; B, expression of PtrCOBRA3 and PtrCOBRA11 in different lignified stems.

    图  3  pHSE401-PtrCOBRA3(gRNA)表达载体构建

    M.标记DNA;a、b.两个目标片段;A.目标片段电泳检测结果;B. pHSE401-PtrCOBRA3农杆菌菌液PCR鉴定;C. Cas9/gRNA基因编辑示意图。

    Figure  3.  Construction of pHSE401-PtrCOBRA3(gRNA)expression vector

    M, DNA mark; a and b mean two target fragments; A, electrophoresis result of target fragment; B, PCR identification of pHSE401-PtrCOBRA3; C, gene editing of Cas9/gRNA.

    图  4  pHSE401-PtrCOBRA3(gRNA)遗传转化及转基因植株鉴定

    A. pHSE401-PtrCOBRA3(gRNA)载体遗传转化,Bars=3 cm;B.转基因植株鉴定。

    Figure  4.  Transformation of pHSE401-PtrCOBRA3(gRNA) and identification of the transgenic plants

    A, transformation of pHSE401-PtrCOBRA3(gRNA), Bars=3 cm; B, identification of the transgenic plants.

    图  5  转基因植株PtrCOBRA3基因编辑分析

    A. PtrCOBRA3基因靶位点位置; B.转基因植株PtrCOBRA3基因编辑情况; C.编辑后PtrCOBRA3编码推测氨基酸。

    Figure  5.  Analysis of PtrCOBRA3 gene editing in transgenic Populus trichocarpa

    A, the target site of PtrCOBRA3; B, PtrCOBRA3 gene editing in transgenic Populus trichocarpa; C, amino acids are concluded from the edited PtrCOBRA3.

    图  6  毛果杨ptrcobra3突变体表型分析

    WT为野生型;cobra3为突变体;Bars=5 cm;A.野生型与突变体植株比较;B.野生型与突变体叶形比较;C.野生型与突变体不同茎节长度比较。

    Figure  6.  Phenotypic analysis of ptrcobra3 mutants

    WT, wide type; cobra3 is mutant; Bars=5 cm; A, comparison of WT and cobra3 mutant; B, comparison of leaf between WT and cobra3 mutant; C, comparison of inode length between WT and cobra3 mutant.

    表  1  所用引物及序列

    Table  1.   Primer sequences used in this study

    基因名称
    Gene name
    引物用途
    Primer use
    引物序列(5′~3′)
    Primer sequence(5′~3′)
    PtrCOBRA3 半定量RT-PCR
    Semi-quantitative RT-PCR
    GCTCTCTTCTGCAATGCACACAC;GCCCTGCTTCCATTAATACGTCATT
    PtrCOBRA11 半定量RT-PCR
    Semi-quantitative RT-PCR
    CAGGATCCTACAGCTTCAGTCTC;GCACAGGTTGGGCAAGGAGTGAT
    PtrCOBRA3 靶位点gRNA-Ⅰ DT1-BsF:ATATATGGTCTCGATTGTCCATCAATGGTGCTCAAGGTT
    (C3A) DT1-F0:TGTCCATCAATGGTGCTCAAGGTTTTAGAGCTAGAAATAGC
    Target site gRNA-Ⅰ DT2-R0:AACTCAGCTGCTGTCTCTCAATCAATCTCTTAGTCGACTCTAC
    (C3A) DT2-BsR:ATTATTGGTCTCTAAACTCAGCTGCTGTCTCTCAATC
    靶位点gRNA-Ⅱ DT1-BsF:ATATATGGTCTCGATTGCTCCTTTCAACCAGCAATTGTT
    (C3B) DT1-F0:TGCTCCTTTCAACCAGCAATTGTTTTAGAGCTAGAAATAGC
    Target site gRNA-Ⅱ DT2-R0:AACAAGAGCTTCACCTTGTTAGCAATCTCTTAGTCGACTCTAC
    (C3B) DT2-BsR:ATTATTGGTCTCTAAACAAGAGCTTCACCTTGTTAGC
    PtrCOBRA3 位点编辑鉴定
    Identifying editing site
    GGGATATTATGTCTTGGACGACAG;GAAAGAGATACACAGCAACTTGGG
    zCas 转基因鉴定
    Transgenic identification
    TGAGAACATCGTCATTGAGATGG;TCAGCTTGTCATTCTCATCGTAC
    PtrActin2 内参基因
    Reference gene
    AACATGGGATTGTTAGCAACTGG;TCCATCACCAGAATCCAGCACA
    下载: 导出CSV

    表  2  毛果杨COBRA基因

    Table  2.   COBRA genes in Populus trichocarpa

    基因名称
    Gene name
    基因座位
    Gene locus
    氨基酸数目
    Amino acid
    信号肽
    Signal peptide
    PtrCOBRA1 Potri.001G419000 664 1-31
    PtrCOBRA2 Potri.004G117100 453 1-33
    PtrCOBRA3 Potri.004G117200 433 1-21
    PtrCOBRA4 Potri.004G167100 454 1-24
    PtrCOBRA5 Potri.004G219200 431 1-23
    PtrCOBRA6 Potri.010G001100 650 1-22
    PtrCOBRA7 Potri.011G135200 647
    PtrCOBRA8 Potri.012G062200 384
    PtrCOBRA9 Potri.014G125300 640 1-22
    PtrCOBRA10 Potri.015G060000 457 1-36
    PtrCOBRA11 Potri.015G060100 404
    PtrCOBRA12 Potri.015G060200 439 1-25
    PtrCOBRA13 Potri.017G098600 412 1-25
    PtrCOBRA14 Potri.017G098700 414 1-25
    下载: 导出CSV
  • [1] Niu E L, Shang X G, Cheng C Z, et al. Comprehensive analysis of the COBRA-like (COBL) gene family in Gossypium identifies two COBLs potentially associated with fiber quality[J/OL]. PLoS One, 2015, 10(12): e0145725[2017-03-10]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692504/?report=reader.
    [2] Roudier F, Schindelman G, DeSalle R, et al. The COBRA family of putative GPI-anchored proteins in Arabidopsis: a new fellowship in expansion[J]. Plant Physiology, 2002, 130(2): 538-548. doi: 10.1104/pp.007468
    [3] Li Y H, Qian Q, Zhou Y H, et al. BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants[J]. The Plant Cell, 2003, 15(9): 2020-2031. doi: 10.1105/tpc.011775
    [4] Brady S M, Song S, Dhugga K S, et al. Combining expression and comparative evolutionary analysis:the COBRA gene family[J]. Plant Physiology, 2007, 143(1): 172-187. http://d.old.wanfangdata.com.cn/NSTLQK/NSTL_QKJJ021162845/
    [5] Brown D M, Zeef L A H, Ellis J, et al. Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics[J]. The Plant Cell, 2005, 17(8): 2281-2295. doi: 10.1105/tpc.105.031542
    [6] Persson S, Wei H, Milne J, et al. Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(24): 8633-8638. doi: 10.1073/pnas.0503392102
    [7] Parker J S, Cavell A C, Dolan L, et al. Genetic interactions during root hair morphogenesis in Arabidopsis[J]. The Plant Cell, 2000, 12(10): 1961-1974. doi: 10.1105/tpc.12.10.1961
    [8] Jones M A, Raymond M J, Smirnoff N. Analysis of the root-hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root-hair development in Arabidopsis[J]. The Plant Journal, 2006, 45(1): 83-100. doi: 10.1111/j.1365-313X.2005.02609.x
    [9] Ching A, Dhugga K S, Appenzeller L, et al. Brittle stalk 2 encodes a putative glycosylphosphatidylinositol-anchored protein that affects mechanical strength of maize tissues by altering the composition and structure of secondary cell walls[J]. Planta, 2006, 224(5): 1174-1184. doi: 10.1007/s00425-006-0299-8
    [10] Sindhu A, Langewisch T, Olek A, et al. Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity[J]. Plant Physiology, 2007, 145(4): 1444-1459. doi: 10.1104/pp.107.102582
    [11] Tuskan G A, Difazio S, Jansson S, et al. The genome of black cottonwood, Populus trichocarpa (Torr. &Gray)[J]. Science, 2006, 313: 1596-1604. doi: 10.1126/science.1128691
    [12] Miao J, Guo D, Zhang J, et al. Targeted mutagenesis in rice using CRISPR-Cassystem[J]. Cell Research, 2013, 23(10): 1233. doi: 10.1038/cr.2013.123
    [13] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337: 816-821. doi: 10.1126/science.1225829
    [14] Mao Y F, Zhang H, Xu N F, et al. Application of the CRISPR-Cas system for efficient genome engineering in plants[J]. Molecular Plant, 2013, 6(6): 2008-2011. doi: 10.1093/mp/sst121
    [15] Feng Z Y, Zhang B T, Ding W N, et al. Efficient genome editing in plants using a CRISPR/Cas system[J]. Cell Research, 2013, 23(10): 1229. doi: 10.1038/cr.2013.114
    [16] Liang Z, Zhang K, Chen K L, et al. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system[J]. Journal of Genetics and Genomics, 2014, 41(2): 63-68. doi: 10.1016/j.jgg.2013.12.001
    [17] Fan D, Liu T T, Li C F, et al. Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation[J]. Scientific Reports, 2015, 5:12217 doi: 10.1038/srep12217
    [18] Letunic I, Doerks T, Bork P. SMART 7: recent updates to the protein domain annotation resource[J]. Nucleic Acids Research, 2011, 40: 302-305. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_3245027
    [19] Tamura K, Dudley J, Nei M, et al. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8): 1596-1599. doi: 10.1093/molbev/msm092
    [20] Xing H L, Dong L, Wang Z P, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC Plant Biology, 2014, 14(1): 327.
    [21] Li S J, Zhen C, Xu W J, et al. Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree[J]. Scientific Reports, 2017, 7:2638 doi: 10.1038/s41598-017-02651-x
    [22] Gritsch C, Wan Y, Mitchell R A C, et al. G-fibre cell wall development in willow stems during tension wood induction[J]. Journal of Experimental Botany, 2015, 66(20): 6447-6459. doi: 10.1093/jxb/erv358
  • 加载中
图(6) / 表(2)
计量
  • 文章访问数:  1099
  • HTML全文浏览量:  281
  • PDF下载量:  96
  • 被引次数: 0
出版历程
  • 收稿日期:  2017-11-27
  • 修回日期:  2018-01-15
  • 刊出日期:  2018-04-01

目录

    /

    返回文章
    返回