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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (1): 100-109.doi: 10.3724/SP.J.1006.2024.34056

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

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 Online:2024-01-12 Published: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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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

Table 1

Primers used in this study"

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

Table 2

Symptoms of soybean at emergence and seeding stages under salt stress"

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

Fig. 1

Relative expression pattern of GmBADH1 genes A: the relative expression pattern of GmBADH1 genes in three different tissues (water treatment). Different lowercase letters are significantly different at the 0.05 probability level; B: GmBADH1 gene expression patterns in different tissues and saline treatment times, 2 h, 8 h, and 24 h represent time nodes of plant sampling under salt stress, and the error line represents standard error. The significant difference was evaluated by the Studuent’s t-test.*: P < 0.05; **: P < 0.01; ***: P < 0.001."

Fig. 2

Target sites of the gRNA in the GmBADH1 gene and recombining diagram of the pBSE401-GmBADH1-g6 vector A: the position of gRNA targets in the GmBADH1 gene locus; B: the recombining diagram of gRNA casaettes and pBSE401-GmBADH1-g6 vector."

Fig. 3

Screening for homozygous mutation in T1 and T2 generation A: nucleotide variation; B: the corresponding sequencing peak map; C: amino acid sequence alignment."

Table 3

Editing genotypes of homozygous mutants"

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

Table 4

The number of CRISPR-mediated T1-T2 targeted mutant plants of BADH1 gene"

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

Table 5

Potential miss site prediction of BADH1-Target"

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

Table 6

Transgene-clenn homozygous mutants"

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

Fig. 4

Positive identification and identification of transgenic components A: PAT/Bar strip test selection marker screening, red arrow indicates that Bar gene is positive; B is the gel image of PCR products in the T-DNA region, Cas9 is a part of the coding sequence, lane 1-5 is L1. Lane 6-10 is L2; Lane 11-14 is L3; Lane 15 was the negative control. Lane 16 was the positive control, and the fragment length was 619 bp. C is for sgRNA detection, the sample is the same as Cas9 detection, and the fragment length is 512 bp."

Table 7

Salt tolerance of GmBADH1 homozygous plants"

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

Fig. 5

Identification of salt tolerance phenotype of mutant at seedling stage a: wild-type plant; b-d: the mutant plant; e: negative control: salt-sensitive material wild bean NY27-38; f: positive control: soybean ZH39 cultivated with salt-tolerant materials; the salt concentration was 150 mmol L-1 at the emergence stage and was rated on the 15th day."

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