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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 801-811.doi: 10.3724/SP.J.1006.2022.14077

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

通过CRISPR/Cas9技术突变BnMLO6基因提高甘蓝型油菜的抗病性

石育钦1,2(), 孙梦丹1, 陈帆1, 成洪涛1,2, 胡学志1, 付丽1, 胡琼1,2, 梅德圣1,2,*(), 李超1,*()   

  1. 1中国农业科学院油料作物研究所 / 农业农村部油料作物生物学与遗传育种重点实验室, 湖北武汉 430062
    2中国农业科学院研究生院, 北京 100081
  • 收稿日期:2021-04-25 接受日期:2021-07-12 出版日期:2022-04-12 网络出版日期:2021-08-13
  • 通讯作者: 梅德圣,李超
  • 作者简介:E-mail: shiyuqin514@163.com
  • 基金资助:
    中国农业科学院科技创新工程项目资助

Genome editing of BnMLO6 gene by CRISPR/Cas9 for the improvement of disease resistance in Brassica napus L

SHI Yu-Qin1,2(), SUN Meng-Dan1, CHEN Fan1, CHENG Hong-Tao1,2, HU Xue-Zhi1, FU Li1, HU Qiong1,2, MEI De-Sheng1,2,*(), LI Chao1,*()   

  1. 1Oil Crops Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062, Hubei, China
    2Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-04-25 Accepted:2021-07-12 Published:2022-04-12 Published online:2021-08-13
  • Contact: MEI De-Sheng,LI Chao
  • Supported by:
    Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences

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摘要：

基因编辑技术可以实现对目标基因高效准确的修饰, 为植物遗传改良开辟了新途径。霉菌抗性位点(Mildew resistance locus O, MLO)基因是植物对白粉病菌防御的主效负向调节因子, 突变后能增强植物对白粉病的抗性, 但在油菜中是否具有同样的功能尚未见报道。为解析该基因在油菜抗病中的功能, 本研究通过分析油菜接种核盘菌后基因的表达情况发现, BnMLO6基因受核盘菌诱导表达; 利用CRISPR/Cas9基因编辑技术获得了一份BnMLO6基因6个同源拷贝同时突变的材料mlo6-212。遗传分析表明, CRISPR/Cas9引起的BnMLO6基因突变能够稳定遗传; mlo6-212突变体在田间和温室条件下都表现出明显的白粉病抗性; 在接种核盘菌24 h后, 病斑面积显著低于野生型, 减小19.5%; BnMLO6基因突变能激发叶片胼胝质的自发堆积, 增强接菌后乙烯和茉莉酸抗病信号。因此, BnMLO6基因可能参与了多条抗病信号路径, 负向调控油菜对白粉病和菌核病的抗性。研究结果不仅为BnMLO6基因协同调控油菜多种病原菌抗性的研究提供了参考, 也为油菜抗病性遗传改良提供了抗性资源和技术支撑。

关键词: 油菜, 基因编辑, MLO, 白粉病, 菌核病

Abstract:

Gene editing technology can modify the target gene efficiently and accurately, which opens up a new way for crop genetic improvement. Mildew resistance locus O (MLO) gene is a key negative regulator of plant defense against powdery mildew. Mutation of MLO gene can enhance plant resistance to powdery mildew, but whether it has the same function is not reported in oilseed rape. In this study, the relative expression analysis suggested that BnMLO6 gene was induced by Sclerotinia sclerotiorum. To explore the potential role of BnMLO6 gene in pathogen resistance, six homologous copies of BnMLO6 gene mutated synchronously by CRISPR/Cas9 gene editing technology and mlo6-212 mutant line was generated for further analysis. Genetic analysis revealed that CRISPR/Cas9 induced mutagenesis of BnMLO6 gene could be stably inherited. In addition, mlo6-212 mutant line indicated obvious resistance to powdery mildew in both field and greenhouse condition. The lesion area of mlo6-212 mutant was reduced by 19.5% after 24 hours inoculation with S. sclerotiorum. Meanwhile, mutation of BnMLO6 gene could stimulate the spontaneous accumulation of callose in leaves and activate ethylene and jasmonic acid transduction pathway. Thus, BnMLO6 gene was probably involved in multiple pathogen resistance pathways to negatively regulate resistance to powdery mildew and S. sclerotiorum in oilseed rape. The results not only provide theoretical basis for the study of BnMLO6 involved resistance regulation of multiple pathogens, but also provide resistant resources and technical support for genetic improvement of disease resistance in oilseed rape.

Key words: oilseed rape, genome editing, MLO, powdery mildew, Sclerotinia sclerotiorum

表1

引物列表"

引物名称
Primer name		 引物序列
Primer sequence (5¢-3¢)		 扩增产物
Product length (bp)
Bnactin-RT-F TCTGGCATCACACTTTCTACAACGAGC 688
Bnactin-RT-R CAGGGAACATGGTCGAACCACC
MLO6-A03-RT-F2 ATCAGAAGAGTTGCAAGAGTATCC 603
MLO6-A03-RT-R2 GCCACAAACCAGATCACAGGAC
MLO6-C03-RT-F2 GCAGTAAATCCGAAGAGTTGAAAG 617
MLO6-C03-RT-R3 TGGTTGTGAGGAGGAATAGCAC
MLO6-A01-RT-F1 AGAACAAAAAAGCACTGTATGAAGG 1096
MLO6-A01-RT-R1 CACAGTTCTTGAGCTTGAACTCATA
MLO6-C01-RT-R1 TTCTTGTGGAAGCAGTTCTTGAG 952
MLO6-C01-RT-F2 CTCCGAGAGCATTGCAGCATCA
MLO6-A09-RT-F2 AGTGTGCAGAGAAGGGAAAAGTC 846
MLO6-A09-RT-R2 AGAGTTTCTTGGAGACAGCGTGT
MLO6-C09-RT-R1 TGTGTGTGGAACTTACCTGAGTCA 835
MLO6-C09-RT-F2 TGCAGAGAAGGGAAAGGTTGCT
NPTII-F GATGGATTGCACGCAGGT 1050
NPTII-R TCGTCAAGAAGGCGATAGA
MLO6-A03-F2 ATTTGGGTTTTTGTTACATATACCAATGTATA 669
MLO6-A03-R2 CTCTTTGTGTGTATGTGTTTGTGACTTACC
MLO6-C03-F2 TTCGGTTTTTTTTTTTTTTACATGAACC 678
MLO6-C03-R2 GAAAGATGTTAGTTTGATGTTTTCTCTCTG
MLO6-A01-F1 GGTCTTAACGGCATAGCAAAACGAT 677
MLO6-A01-R2 CGTGGAATTATTTTGTTTGCTTACCGT
MLO6-C01-F1 TTCTTACCGCATTGCAAAATGATTCTT 680
MLO6-C01-R2 TGTGGGATACTACTTTGCTTGCTTACC
MLO6-A09-F1 CTCAGAATGTTTCTGCATCCGATATCT 510
MLO6-A09-R3 CTTTTACTTACCCAACTGAAATTCTGATGAC
MLO6-C09-R1 GAATTGCTCTGCTTGCTTACCGTTG 619
MLO6-C09-F2 CAACAATCCCAGAACTAATATATTTGCTCT

表2

防御相关基因引物"

基因名称
Gene name		 油菜中基因号
Gene name of B. napus		 引物名称
Primer name		 引物序列
Primer sequence (5¢-3¢)		 扩增产物
Product length (bp)
BnORA059 BnaA10G0042700ZS ORA59-2-F ATCAATCCTTCCTCTCAGTTAGC 517
ORA59-2-R TCTTGATCCGTAACAATACTCTG
BnaC05G0044000ZS ORA59-3-F CAATCATTCCTCTCAGCTGTTAGC 553
ORA59-3-R CTTGATTCGTAACAACACTTTGTT
BnPR4 BnaA03G0296000ZS PR4-1-F CGCGTTTGCGGCTAAAACA 130
PR4-1-R TGGTTTCCTTTCCACGTTGAG
BnaA03G0296200ZS PR4-3-F CAGACTTAGCATAGCCATCATAT 214
PR4-3-R GCTCTCTCCAGAGCCGCTT
BnaC03G0354600ZS PR4-5-F AGCCGCTCAATCCGCTAATA 194
PR4-5-R TACCGCAAGAATCACGACCA
基因名称
Gene name		 油菜中基因号
Gene name of B. napus		 引物名称
Primer name		 引物序列
Primer sequence (5¢-3¢)		 扩增产物
Product length (bp)
BnPR4 BnaC03G0354700ZS PR4-6-F CAGACTTAGCATAACCCTCGTA 213
PR4-6-R CTCTCTCCGGAACCGCTTG
BnERF1 BnaA01G0293300ZS ERF1-1-F TCGTCCAACAACTACTCTCTCC 147
ERF1-1-R GTACGACTTCTCTGGCTTTCTTG
BnaC01G0362300ZS ERF1-2-F GGAATCTTTCTCCTCCTCGTCTT 153
ERF1-2-R TGACTTCTCTGGTTTTCTCGATG
BnERF2 BnaA09G0212800ZS ERF2-3-F AGATTTAGAACCGTCGTCGAAG 409
ERF2-3-R TATTTCTCCTCCGTTTCGGTGT
BnaC02G0416900ZS ERF2-4-F GAGTTGTATCACCGAATTTGTAG 320
ERF2-4-R ACGAAGACGATGAAGACGAAGTA
BnaC09G0247600ZS ERF2-6-F TAGATAAGCCCCCGGCTAATC 396
ERF2-6-R TCCGTTTCGGCGTCCCGTTA

图1

BnMLO6基因的核盘菌诱导表达分析"

图2

sgRNA设计以及载体构建"

图3

BnMLO6-C01拷贝基因PAGE电泳分型部分结果 M: DNA marker; 三角箭头表示编辑条带。"

图4

mlo6-212单株T0代BnMLO6基因靶点突变情况 A: DNA序列突变情况, 下画线部分表示PAM位点; B: 氨基酸序列变化情况, 红色表示变异。"

表3

mlo6-212株系T1代部分编辑情况"

T1代编号
Number of T1 generation		 MLO6-A03 MLO6-C03 MLO6-A01 MLO6-C01 MLO6-A09 MLO6-C09
mlo6-212-1 -15 bp -13 bp +1 bp -12 bp -3 bp -2 bp
mlo6-212-3 -15 bp -13 bp -2 bp 无No mutation -3 bp -2 bp
mlo6-212-4 -15 bp -2 bp -2 bp -12 bp -3 bp -2 bp
mlo6-212-7 -15 bp -13 bp +1 bp 无No mutation 无No mutation 无No mutation
mlo6-212-8 -15 bp -13 bp -2 bp -12 bp -3 bp -2 bp
mlo6-212-9 -15 bp -13 bp -2 bp -12 bp -3 bp -2 bp
mlo6-212-10 -15 bp -13 bp +1 bp -12 bp 无No mutation 无 No mutation

图5

T1代编辑植株白粉病感病情况 A: 田间白粉病感染情况; B: 室内白粉病感染情况, 标尺为1 cm; C: mlo6-212的4个株系T2代白粉病田间感病情况。"

图6

T1代胼胝质观察及分析 A: 在显微镜下的胼胝质, 黑色箭头表示胼胝质, 标尺为30 mm; B: 每单个视野胼胝质沉积的数量。"

图7

T2代核盘菌接种抗性鉴定结果 A: 核盘菌侵染24 h后; B核盘菌侵染36 h后; 标尺为1 cm; C: 叶片菌斑面积分析; *表明在P < 0.05水平差异显著。"

图8

JA/ET信号通路基因接种前后的表达分析 每一行代表相关基因一个同源拷贝的表达量变化, 每个从上到下, BnERF2的3个拷贝为BnaA09G0212800ZS、BnaC02G0416900ZS、BnaC09G0247600ZS; BnERF1的2个拷贝为BnaA01G0293300ZS、BnaC01G0362300ZS; BnPR4的4个拷贝为BnaA03G0296000ZS、BnaA03G0296200ZS、BnaC03G0354600ZS、BnaC03G0354700ZS; BnOAR059的2个拷贝为BnaA10G0042700ZS、BnaC05G0044000ZS; 从左到右样品依次为野生型未接菌、突变体未接菌、野生型接菌24 h、突变体接菌24 h、野生型接菌36 h、突变体接菌36 h。"

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