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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 321-331.doi: 10.3724/SP.J.1006.2023.24013

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9技术突变BnaMPK6基因降低甘蓝型油菜的耐盐性

张文宣(), 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝()   

  1. 华中农业大学作物遗传改良全国重点实验室 / 国家油菜工程技术研究中心 / 洪山实验室, 湖北武汉 430070
  • 收稿日期:2022-01-10 接受日期:2022-06-07 出版日期:2022-07-08 网络出版日期:2022-07-08
  • 通讯作者: 马朝芝
  • 作者简介:E-mail: 18645272827@163.com
  • 基金资助:
    中央高校基本科研业务费专项基金(2662020ZKPY020);国家重点研发计划项目(2020BBB061)

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 Published:2022-07-08 Published online:2022-07-08
  • Contact: MA Chao-Zhi
  • Supported by:
    Fundamental Research Funds for the Central Universities(2662020ZKPY020);National Key Research and Development Program of China(2020BBB061)

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摘要：

甘蓝型油菜是我国一种重要的油料作物。MPK6是一种丝裂原活化蛋白激酶, MAPK级联途径可以被各种胁迫激活, 进而调节植物对胁迫的响应, 在植物响应非生物胁迫过程中发挥着重要的作用, 但在甘蓝型油菜盐胁迫响应过程中的功能还尚不清楚。基因结构和蛋白序列分析表明, MAPK6在十字花科芸薹属植物种间分化相对保守, 基因结构相似, 蛋白之间均有相同的STKc_TEY_MAPK结构域, 与胁迫和生长发育相关。为探究其功能, 本研究利用CRISPR/Cas9技术对BnaMPK6基因进行编辑, 通过农杆菌介导的方法将BnaMPK6基因转入到甘蓝型油菜中, 获得了BnaMPK6基因4个同源拷贝同时突变的材料: cr-bnampk6-13-1和cr-bnampk6-49-1。cr-bnampk6突变体表现出明显的盐敏感性: 在100 mmol L-1和150 mmol L-1浓度的NaCl溶液处理下, 功能缺失突变体长势明显缓慢, 并且株高和鲜重显著低于野生型植株, 但根长无明显差异。此外, 在盐胁迫下, 功能缺失突变体体内的活性氧和丙二醛含量的积累显著高于野生型对照, 脯氨酸含量也明显高于野生型植株。本研究结果表明, BnaMPK6基因在盐水胁迫下正向调控植株的生长和发育, 影响甘蓝型油菜的耐盐性。本研究不仅为BnaMPK6基因调控甘蓝型油菜盐胁迫的研究提供了理论基础, 而且为甘蓝型油菜耐盐性遗传改良提供了一定的技术支持。

关键词: 甘蓝型油菜, BnaMPK6, CRISPR/Cas9, 盐胁迫

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

表1

qRT-PCR引物序列"

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

表2

高通量测序引物序列"

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

图1

拟南芥、甘蓝型油菜、白菜型油菜和甘蓝MPK6基因的系统进化树及基因结构"

图2

拟南芥、甘蓝型油菜、白菜型油菜和甘蓝MPK6的蛋白序列和保守结构域"

图3

BnaMPK6基因表达模式分析 A: BnaMPK6在4个不同组织的表达模式; B、C: BnaMPK6在不同的盐水处理时间下的表达模式。数据计算采取2-ΔΔCt法, 每个数据3个重复, Actin的相对表达量设定为200。以0 h处理为对照, 使用t检验进行显著性分析, 星号(*)代表P < 0.05时显著性情况。"

图4

cr-bnampk6突变体编辑情况 A: BnaMPK6基因结构可视化和靶点位置; B: cr-bnampk6单株T1代BnaMPK6基因靶点突变情况; C: cr-bnampk6株系T0代和T1代部分基因编辑类型统计。蓝色代表变异; 红色和绿色代表靶点序列。d#: 缺失#个碱基; i#: 插入#个碱基; wt: 野生型。"

表3

不同浓度NaCl溶液处理后cr-bnampk6表型分析"

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

图5

不同浓度NaCl溶液处理后BnaMPK6基因耐盐性分析 A: WT和cr-bnampk6植株盐胁迫下的表型; B: WT和cr-bnampk6植株在盐胁迫下根长分析; C: WT和cr-bnampk6植株在盐胁迫下鲜重分析; D: WT和cr-bnampk6植株在盐胁迫下株高分析。以WT为对照, 使用t检验进行显著性分析, 星号(*)代表P < 0.05时显著性情况。"

图6

不同浓度NaCl溶液处理后cr-bnampk6植株的活性氧和脯氨酸含量分析 A: NBT染色分析cr-bnampk6植株活性氧含量; B: cr-bnampk6植株丙二醛含量分析; C: cr-bnampk6植株脯氨酸含量分析。ROS: 活性氧; MDA: 丙二醛; Pro: 脯氨酸。以WT为对照, 使用t检验进行显著性分析, 星号(*)代表P < 0.05时显著性情况。"

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