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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 801-811.doi: 10.3724/SP.J.1006.2022.14077

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-04-12 Published:2021-08-13
  • Contact: MEI De-Sheng,LI Chao E-mail:shiyuqin514@163.com;meidesheng@caas.com;lichao01@caas.cn
  • Supported by:
    Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences

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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

Table 1

List of PCR primers"

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

Table 2

Primers of disease resistance related genes"

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

Fig. 1

Induced expression of BnMLO6 genes by S. sclerotiorum"

Fig. 2

Design of sgRNA and construction of vector"

Fig. 3

PAGE-based genotyping of the BnMLO6-C01 T0 generation plants M: DNA marker; the triangle arrow represents the edit strip."

Fig. 4

Mutation of targeted sites of MLO homologous in mlo6-212 T0 plants A: mutation of DNA sequence, the underlined parts represent PAM sites; B: change of amino acid sequence, and the red parts indicate mutations."

Table 3

Partial editing results of T1 generation of mlo6-212"

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

Fig. 5

Infection of powdery mildew in T1 generation A: field powdery mildew infection; B: indoor powdery mildew infection, bar: 1 cm; C: field susceptibility of mlo6-212 lines to powdery mildew in T2 generation."

Fig. 6

Microscopic observation and analysis of callose in T1 generation A: microscopic observation of callose, the black arrow indicates callose, bar: 30 mm; B: the number of callose deposits per field area."

Fig. 7

Assessment of stem rot resistance by inoculation of S. sclerotiorum in T2 generation A: 24 hours after S. sclerotiorum infection; B: 36 hours after S. sclerotiorum infection; bars: 1 cm; C: analysis of lesion area. *: significant difference at P < 0.05 between the two groups."

Fig. 8

Expression patterns of JA/ET signaling pathway genes before and after inoculation Each row represents the expression change of a homologous copy of the related gene; from top to bottom, the three copies of BnERF2 are BnaA09G0212800ZS, BnaC02G0416900ZS, and BnaC09G0247600ZS; the two copies of BnERF1 are BnaA01G0293300ZS, and BnaC01G0362300ZS; the four copies of BnPR4 are BnaA03G0296000ZS, BnaA03G0296200ZS, BnaC03G0354600ZS, and BnaC03G0354700ZS; the two copies of BnOAR059 are BnaA10G0042700ZS and BnaC05G0044000ZS; samples of the order from left to right is wild-type uninfected, mutant infected, wild-type infected 24 hours, mutant infected 24 hours, wild-type infected 36 hours, and mutant uninfected 36 hours."

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