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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 321-331.doi: 10.3724/SP.J.1006.2023.24013

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

Genome editing of BnaMPK6 gene by CRISPR/Cas9 for loss of salt tolerance in Brassica napus L.

ZHANG Wen-Xuan(), LIANG Xiao-Mei, DAI Cheng, WEN Jing, YI Bin, TU Jin-Xing, SHEN Jin-Xiong, FU Ting-Dong, MA Chao-Zhi()   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University / National Engineering Research Center of Rapeseed / Hongshan Laboratory, Wuhan 430070, Hubei, China
  • Received:2022-01-10 Accepted:2022-06-07 Online:2022-07-08 Published:2022-07-08
  • Contact: MA Chao-Zhi E-mail:18645272827@163.com;yuanbeauty@mail.hzau.edu.cn
  • Supported by:
    Fundamental Research Funds for the Central Universities(2662020ZKPY020);National Key Research and Development Program of China(2020BBB061)

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Abstract:

Brassica napus L. is an important oil crop. MPK6 (Mitogen-Activated Protein Kinases 6) is activated by various stresses to control plant stress tolerance, which is the key gene in response to abiotic stresses. However, the function of MAPK6 in B. napus stress tolerance is still unclear. Gene structure and protein sequence analysis showed that MPK6gene structures were similar, and all proteins had the same STKc_TEY_MAPK domain in Brassica. To explore the potential role of BnaMPK6, the BnaMPK6 mutants were generated by CRISPR/Cas9 genome editing technology. Two quadruple mutants, cr-bnampk6-13-1 and cr-bnampk6-49-1, were obtained. cr-bnampk6 mutant lines were hypersensitive to salt treatment (100 mmol L-1 and 150 mmol L-1), the growth of the mutant was strongly inhibited after salt treatment, and the plant height and fresh weight were significantly lower than those of wild-type plants, but there was no significant difference in root length. In addition, reactive oxygen species (ROS), malondialdehyde (MDA), and free proline were more accumulated in cr-bnampk6 mutant lines. In conclusion, these results revealed that BnaMPK6 gene positively regulated salt tolerance in B. napus, providing theoretical and technical supports for genetic improvement of salt tolerance in Brassica napus.

Key words: Brassica napus L., BnaMPK6, CRISPR/Cas9, salt stress

Table 1

Sequence of qRT-PCR primers"

基因名称
Gene name		 引物名称
Primer name		 序列
Sequence (5'-3')
BnaC03T0189300WE BnaMPK6.C03-F GCCGGGGAAAGAGAATATTC
BnaMPK6.C03-R ATCGGAGGGTTATACTTAGCGGT
BnaA03T0226400WE BnaMPK6.A03-F CGACACTTAGCCATGGAGGGATGT
BnaMPK6.A03-R ATCGGAGGGCTATACTTGGCG
BnaC04T0043400WE BnaMPK6.C04-F GGGGTAGAGAATATTCCGGC
BnaMPK6.C04-R AACAGACGATGCCATAAGCGCCT
BnaA05T0030700WE BnaMPK6.A05-F CCGAGAGTGACTTTATGACTGA
BnaMPK6.A05-R CTTTGCGTTTTCGTTCAAGAAC
BnaActin7 actinQ7F GCTGACCGTATGAGCAAAG
actinQ7R AAGATGGATGGACCCGAC

Table 2

Sequence of HI-TOM primers"

基因名称
Gene name		 引物名称
Primer name		 序列
Sequence (5'-3')
BnaC03T0189300WE sgRNA1-C3-F ggagtgagtacggtgtgcACTGCGGCGATAGATGTTTGG
sgRNA1-C3-R gagttggatgctggatggACAGCATCAAATAGAGCTTCAAAGGT
BnaA03T0226400WE sgRNA1-A3-F ggagtgagtacggtgtgcACTGCGGCGATAGATGTTTGG
sgRNA1-A3-R gagttggatgctggatggCAGCAAAATACGAGTCTTTGTTGGT
BnaC04T0043400WE sgRNA2-C4-F ggagtgagtacggtgtgcGCTTGAGTTCTTGAACGAAAACGC
sgRNA2-C4-R gagttggatgctggatggTCAAACGTCAGCATCTTCTCGATAAG
BnaA05T0030700WE sgRNA2-A5-F ggagtgagtacggtgtgcGCTTGAGTTCTTGAACGAAAACGC
sgRNA2-A5-R gagttggatgctggatggTCAAACGTCAGCATCTTCTCGATAAG

Fig. 1

Phylogenetic relationships and gene structure of MPK6 genes in Arabidopsis thaliana, Brassica napus, Brassica rapa, and Brassica oleracea"

Fig. 2

Protein sequence and conserved domain of MPK6 in Arabidopsis thaliana, Brassica napus L., Brassica rapa, and Brassica oleracea"

Fig. 3

Expression patterns of BnaMPK6 gene A: the relative expression patterns of BnaMPK6 genes in four different tissues; B, C: the relative expression pattern of BnaMPK6 genes under different salt treatment time. The data was calculated in the light of the 2-ΔΔCt method and the mRNA levels of Actin genes set to 200. 0 hour is used as control, the asterisk (*) are significantly different (t-test: *, P < 0.05)."

Fig. 4

Editing results of cr-bnampk6 mutants A: the visualization of BnaMPK6 gene structure and the target sites; B: the mutation of targeted sites of BnaMPK6 homoulogous in cr-bnampk6 T1 plants; C: the partial editing results of T0 and T1 generation of cr-bnampk6. The blue parts indicate mutations; the red and green parts indicate target sites. d#: # of bp deleted from target site; i#: # of bp inserted from target site; wt: wild type."

Table 3

Analysis of phenotypic of cr-bnampk6 under different concentrations of NaCl solution"

处理
Treatment		 表型
Phenotype		 野生型
WT		 13-1 49-1
0 mmol L-1 NaCl 株高 Stem length (cm) 21.307±0.488 a 22.145±0.485 a 20.925±0.641 a
根长 Root length (cm) 30.547±1.649 a 27.855±0.805 a 29.300±1.199 a
鲜重 Fresh weight (g) 6.925±0.141 a 7.014±0.197 a 6.629±0.178 a
100 mmol L-1 NaCl 株高 Stem length (cm) 16.190±0.569 a 13.610±0.549 b 11.620±0.53 c
根长 Root length (cm) 24.428±1.018 a 25.155±0.683 a 26.700±0.837 a
鲜重 Fresh weight (g) 4.425±0.095 a 2.302±0.101 b 2.476±0.077 b
150 mmol L-1 NaCl 株高 Stem length (cm) 12.013±0.649 a 7.300±0.399 b 7.685±0.301 b
根长 Root length (cm) 22.265±0.646 a 21.630±0.578 a 21.905±0.668 a
鲜重 Fresh weight (g) 2.928±0.149 a 1.511±0.067 b 1.317±0.065 b

Fig. 5

Salt tolerance of BnaMPK6 genes under different concentrations of NaCl solutions A: WT and cr-bnampk6 plants phenotypes under salt stress; B: root length of WT and cr-bnampk6 plants under salt stress; C: fresh weight of WT and cr-bnampk6 plants under salt stress; D: plant height of WT and cr-bnampk6 plants under salt stress. WT are used as control, the asterisk (*) are significantly different (t-test: *, P < 0.05)."

Fig. 6

Accumulation of ROS and proline of cr-bnampk6 plants under different concentrations of NaCl solutions A: the accumulation of ROS of cr-bnampk6 plants by NBT; B: the accumulation of MDA of cr-bnampk6 plants; C: the accumulation of proline of cr-bnampk6 plants. ROS: reactive oxygen species; MDA: malondialdehyde; WT are used as control, the asterisk (*) are significantly different (t-test: *, P < 0.05)."

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