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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 100-109.doi: 10.3724/SP.J.1006.2024.34056

• 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技术编辑GmBADH1基因改变大豆耐盐性

石宇欣1,2(), 刘欣玥1,2, 孙建强1,2, 李晓菲1,2, 郭潇阳2, 周雅2, 邱丽娟1,2,*()   

  1. 1东北农业大学农学院, 黑龙江哈尔滨 150030
    2农作物基因资源与遗传改良国家重大科学工程 / 农业农村部种质资源利用重点实验室 / 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2023-03-17 接受日期:2023-06-29 出版日期:2024-01-12 网络出版日期:2023-07-26
  • 通讯作者: *邱丽娟, E-mail: qiulijuan@caas.cn
  • 作者简介:石宇欣, E-mail: 2279790926@qq.com

    **同等贡献

  • 基金资助:
    国家重点研发计划项目: “北方大豆高产优质耐密新种质创制与应用项目”(2021YFD1201104)

Knockout of GmBADH1 gene using CRISPR/Cas9 technique to reduce salt tolerance in soybean

SHI Yu-Xin1,2(), LIU Xin-Yue1,2, SUN Jian-Qiang1,2, LI Xiao-Fei1,2, GUO Xiao-Yang2, ZHOU Ya2, QIU Li-Juan1,2,*()   

  1. 1College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
    2National Key Facility for Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture and Rural Affairs / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2023-03-17 Accepted:2023-06-29 Published:2024-01-12 Published online:2023-07-26
  • Contact: *E-mail: qiulijuan@caas.cn
  • About author:**Contributed equally to this study
  • Supported by:
    National Key Research and Development Program of China: "the Development and Application of High Yield, High Quality and Dense Tolerance New Germplasm of Soybean in Northern China"(2021YFD1201104)

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摘要：

耐盐基因功能鉴定对于大豆品种改良以及盐碱地的开发利用至关重要。盐害胁迫下作物通过合成和积累甜菜碱作为渗透保护剂, 减小盐害对作物产量的影响。BADH基因已在多种植物中被证实调节植物对逆境胁迫的响应, 但在大豆中的调控机制尚不清晰。本研究克隆了GmBADH1基因, qRT-PCR显示该基因在根、茎、叶中均有表达, 尤其在根中的表达丰度最高。通过农杆菌介导的大豆遗传转化技术将CRISPR/Cas9系统构建的表达载体转入到大豆品种JACK中, 产生了3种可以调控大豆耐盐性状的靶向突变, 均发生在编码区, 分别为缺失19 bp、插入1 bp、插入9 bp替换2 bp。前两者, 过早出现的终止密码子, 使其均产生截断的BADH1蛋白, 后者发生移码突变。在出苗期和苗期分别对突变纯合植株进行盐处理, 结果表明, 出苗期耐盐性与野生型相比显著下降, 苗期与野生型相比无明显差异。这说明GmBADH1基因可能主要调控出苗期的耐盐性状。本研究为深入挖掘大豆耐盐基因和培育大豆耐盐品种提供了依据。

关键词: 大豆, GmBADH1, CRISPR/Cas9, 耐盐性

Abstract:

Functional identification of salt-tolerant genes is crucial for the improvement of soybean varieties and the development and utilization of saline-alkali land. Under salt stress, crops reduce the effect of salt damage on crop yield by synthesizing and accumulating betaine as osmotic protective agent. BADH gene is been proved to regulate plant response to stress in many plants, but the regulation mechanism in soybean is not clear. In this study, GmBADH1 gene was cloned, and qRT-PCR showed that the gene was expressed stems and leaves, especially in roots. The expression vector constructed by CRISPR/Cas9 system was transferred into soybean variety JACK by Agrobacterium-mediated soybean genetic transformation technology, and three targeted mutations that could regulate the salt tolerance of soybean were produced. They all occurred in the coding region, which had three difference of deletion 19 bp, insertion 1 bp, and insertion 9 bp to replace 2 bp. The first two, premature termination codons, resulting in truncated BADH1 protein, the latter occurred frameshift mutation. The mutant homozygous plants were treated with salt at the emergence stage and seedling stage, respectively. The results showed that the salt tolerance of the mutant homozygous plants was significantly lower than that of the wild type at seedling stage, and there was no significant difference compared with the wild type at seeding stage, which indicating that GmBADH1 gene may mainly regulate the salt tolerance of seedling stage. This study provides a basis for further excavation of soybean salt-tolerant genes and cultivation of soybean salt-tolerant varieties.

Key words: soybean, GmBADH1, CRISPR/Cas9, salt tolerance

表1

本研究所用引物"

引物名称
Primer name		 正向序列
Forward sequence (5°-3°)		 反向序列
Reverse sequence (5°-3°)
D-GmBADH1-F/R AGCAAGAACAAGACGTAGCCT AGCGATGGCTCGAAGATAGC
zCAS9-F/R AAGAACCTGCCAAATGAGA CCTTGAATGTGAGGCTGTC
J-CDS-F/R ATGGCAATCTCCATACCCA TCACAGCTTTGAAGGCGAC
GmBADH1-off target-F1/R1 AGTTCTTCTGCCTCATTCC AGAATAGCCACATAATCCC
GmBADH1-off target-F2/R2 GCGTTTGTTTCACCAGTTT ATAGATGTAAGGAGGAGGG
GmBADH1-off target-F3/R3 AGATCAGTTCGCCTTGTAG AAATTATAGCAGGACCCAC
GmBADH1-off target-F4/R4 AGGAGGTCTGCTATTTATT CACTTTATAGAAGCCAAATC
Actin-18g290800 GGTGGTTCTATCTTGGCATC CTTTCGCTTCAATAACCCTA
qPCR-BADH1-F/R GAAGCACTGGCAGACCTGG GGGCTCCTTGAGAACATAA

表2

盐胁迫后大豆出苗期和苗期的盐害症状"

时期
Stage		 类别
Category		 表型特征
Symptom
出苗期
Emergence stage		 I 植株凋亡, 子叶干枯(类别数值为1)
Plant deed, cotyledons were dry (category value is 1)
II 植株生长受到严重抑制, 子叶未展开(类别数值为2)
Plant groeth was severly inhibited (category value is 2)
III 植株生长受到抑制, 具有生长点, 但真叶未展开(类别数值为3)
Plant growth was inhibited, with shoot apical meristem, but true leaves were not unfolded (category value is 3)
IV 植株生长基本正常, 真叶未完全展开(类别数值为4)
Plant growth was basically normal, true leaves were not fully expanded (category value is 4)
V 植株生长正常, 真叶完全展开(类别数值为5)
The plant grow was normal,the true leaves were fully expanded (category value is 5)
苗期
Seedling stage		 1 健康的绿叶, 没有观察到损伤
Healthy green leaves, no damage observed
2 轻度坏死, 真叶轻微发黄
Slight chlorosis, light yellowish color observed in ture leaves
3 中度坏死, 三出复叶发黄
Moderate chlorosis, chlorosis observed in trifoliate leaves
4 严重坏死, 超过75%的叶面发黄
Severe chlorosis,more than 75% of the leaf area showed chlorosis
5 凋亡, 植物完全枯萎
Dead, plants were completely withered

图1

GmBADH1基因表达模式分析 A: GmBADH1基因在3个不同组织中的表达模式(水处理)。不同小写字母表示之间差异显著(P < 0.05); B: GmBADH1基因在不同组织、不同盐水处理时间下的表达模式, 2 h、8 h、24 h表示盐胁迫下植物取样的时间节点, 盐处理浓度为150 mmol L-1, 误差线表示标准误差。差异显著性分析采用t检测方法。*: P < 0.05; **: P < 0.01; ***: P < 0.001。"

图2

GmBADH1的gRNA靶点和pBSE401-GmBADH1-g6表达载体重组示意图 A: GmBADH1的gRNA靶点位置; B: 靶点gRNA表达盒与pBSE401重组示意图。"

图3

T1和T2纯合突变体筛选 A: 核苷酸变异; B: 对应测序峰图; C: 氨基酸序列比对。"

表3

纯合突变体编辑基因型"

材料名称
Materials name		 编码序列CDS sequence 氨基酸序列
Sequence of amino acid
长度Length 变异Variation
JACK CDS:1512 bp no 504个氨基酸
504 amino acids
BADH1-L1 1513 bp +1 bp 提前终止, 剩余32个氨基酸
Premature termination, residue 32 amino acids
BADH1-L3 1521 bp +9/replace2 bp 缺失3个氨基酸, 替换4个氨基酸
Miss 3 amino acids and substitude for 4 amino acids
BADH1-L4 1493 bp -19 bp 提前终止, 剩余29个氨基酸
Premature termination, residue 29 amino acids

表4

CRISPR介导BADH1基因T1~T2靶向突变植株数"

突变位点
Site of
mutation		 T1 T2
纯合植株数目
No. of
homozygous plants		 杂合植株数目
No. of
heterzygous plants		 野生型植株数目
No. of
mutant
plants		 纯合植株数目
No. of
homozygous plants		 杂合植株数目
No. of
heterzygous plants		 野生型植株数目
No. of
mutant
plants
L1 2 5 35 3 6 23
L3 2 10 56 3 3 8
L4 0 8 9 4 11 47
总数Total 4 23 100 10 20 78

表5

BADH1-Target的潜在的脱靶位点预测"

潜在靶点
Potential target		 物理位置
Physical position		 靶点序列
Target sequence		 位置
Position
Off-target-1 Glyma.08G210000 Chr08:16998801-16998823 CCTTTGCA[TAGTTGTCTACT] TGG CDS
Off-target-2 Glyma.07G032500 Chr07:2553714-2553736 CCTTTGCA[TAGTTGTCTACT] TGG CDS
Off-target-3 Glyma.18G016000 Chr18:1148893-1148915 GCTATTAA[AAATAGTCTACT] TGG CDS
Off-target-4 Glyma.17G194300 Chr17:29066147-29066169 TTGATGAA[ATGTTGTCTTCT] TGG CDS

表6

无转基因检测"

株系
Line		 检测植株数
No. of plant
detection		 无T-DNA植株数量
No. of plants without T-DNA
L1 5 2
L3 5 5
L4 4 4
总数Total 14 11

图4

阳性鉴定与转基因成分鉴定 A: PAT/Bar试纸条检测选择标记筛选, 红色箭头表示Bar基因为阳性; B: T-DNA区域PCR产物的凝胶图像, Cas9是编码序列的一部分, 泳道1~5为L1; 泳道6~10为L3; 泳道11~14为L4; 泳道15为阴性对照; 泳道16为阳性对照, 片段长度619 bp。C: sgRNA检测, 样品同Cas9检测, 片段长度512 bp。"

表7

GmBADH1纯合植株耐盐鉴定"

遗传材料
Genetic
materials		 突变位点
Site of mutation		 出苗期耐盐鉴定
Identification of salt tolerance at seedling stage		 苗期耐盐鉴定
Identification of salt tolerance at seedling stage
耐盐等级
Salt tolerance rating		 等级描述
Description of rank		 耐盐等级
Salt tolerance rating		 等级描述
Rank description
野生型WT CDS:1512 bp 1 高度耐盐High salt-tolerant 3 盐敏感Salt sensitivity
BADH1-L1 +1 bp 4 敏感型Salt-sensitive 3 盐敏感Salt sensitivity
BADH1-L3 +9/replace 2 bp 3 中度耐盐Moderate salt-tolerant 3 盐敏感Salt sensitivity
BADH1-L4 -19 bp 2 耐盐Salt-tolerant 3 盐敏感Salt sensitivity

图5

出苗期突变体耐盐表型鉴定 a为野生型植株; b~d为突变植株; e为阴性对照: 盐敏感材料野生豆NY27-38; f为阳性对照: 耐盐材料栽培豆中黄39; 出苗期处理盐浓度为150 mmol L-1, 于第15天进行评级。"

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