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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1291-1300.doi: 10.3724/SP.J.1006.2020.94169

• RESEARCH NOTES • Previous Articles    

Identification of CRISPR/Cas9 knockout targets and tissue expression analysis of circadian clock genes GmLNK1/2, GmRVE4/8, and GmTOC1 in soybean

GAN Zhuo-Ran,SHI Wen-Qian,LI Yong-Li,HOU Zhi-Hong,LI Hai-Yang,CHENG Qun,DONG Li-Dong,LIU Bao-Hui,LU Si-Jia()   

  1. School of Life Sciences, Guangzhou University, Guangzhou 510006, Guangdong, China
  • Received:2019-11-07 Accepted:2020-03-24 Online:2020-08-12 Published:2020-04-07
  • Contact: Si-Jia LU E-mail:lusijia@gzhu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31771815);National Natural Science Foundation of China(31701445);State Key Laboratory of Crop Genetics & Germplasm Enhancement (ZW201901)(ZW201901)

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Abstract:

Circadian clock genes play an important role in improving crop yield. LNK1, LNK2, RVE4, RVE8, and TOC1 are important circadian clock genes in plants. Homologous genes of AtLNK1, AtLNK2, AtRVE4, AtRVE8, and AtTOC1 in soybean were found by evolutionary tree analysis. These genes were expressed in soybean roots, stems and leaves. The knockout vectors of these genes were successfully constructed by using CRISPR/Cas9 gene editing technology. Transformation system of soybean root hair and RT-PCR were used to identify 13 genes targets effectively. The results of this study provide important target informations for further obtaining soybean mutant materials, and a foundation for further studying the function of circadian clock genes.

Key words: soybean, gene knockout, clock gene, CRISPR/Cas9

Table 1

List of gene target primers"

靶点引物
Target primer		 引物序列
Primer sequence (5°-3°)		 靶点对应基因
Target of gene
LNK1T1F gtcaGGAGACAAGTGTGTGGTGG GmLNK1a, GmLNK1b
LNK1T1R aaacCCACCACACACTTGTCTCC GmLNK1a, GmLNK1b
LNK1T2F gtcaAGAGGAGTTCTGCTGGCTC GmLNK1a, GmLNK1b
LNK1T2R aaacGAGCCAGCAGAACTCCTCT GmLNK1a, GmLNK1b
LNK1T3F attgCTTGGGAGACAAGTGTGTGG GmLNK1c, GmLNK1d
LNK1T3R aaacCCACACACTTGTCTCCCAAG GmLNK1c, GmLNK1d
LNK1T4F attgAGACTTTGAAGATGTTGAC GmLNK1c, GmLNK1d
LNK1T4R aaacGTCAACATCTTCAAAGTCT GmLNK1c, GmLNK1d
LNK2T1F gtcaACATAATATGGGGTGAAGG GmLNK2a, GmLNK2b
LNK2T1R aaacCCTTCACCCCATATTATGT GmLNK2a, GmLNK2b
LNK2T2F gtcaAAACTGATCAGGGTTCCCT GmLNK2c, GmLNK2d
LNK2T2R aaacAGGGAACCCTGATCAGTTT GmLNK2c, GmLNK2d
LNK2T3F attgTTTGATTGGAACGACGAAG GmLNK2a, 2b, 2c, 2d
LNK2T3R aaacCTTCGTCGTTCCAATCAAA GmLNK2a, 2b, 2c, 2d
LNK2T4F attgTCATATTGTGCCTTATCCGG GmLNK2c, GmLNK2d
LNK2T4R aaacCCGGATAAGGCACAATATGA GmLNK2c, GmLNK2d
RVE48T1F gtcaCTTCCCTGCTGATGAATGC GmRVE4/8b, GmRVE4/8c
RVE48T1R aaacGCATTCATCAGCAGGGAAG GmRVE4/8b, GmRVE4/8c
RVE48T2F gtcaTCATCCCATGTGACATACCC GmRVE4/8a, GmRVE4/8d
RVE48T2R aaacGGGTATGTCACATGGGATGA GmRVE4/8a, GmRVE4/8d
RVE48T3F attgCAGCTTTGCGCTTTGGACG GmRVE4/8b, GmRVE4/8c
RVE48T3R aaacCGTCCAAAGCGCAAAGCTG GmRVE4/8b, GmRVE4/8c
RVE48T4F attgAAGCTTTGCGCTTAGGCCG GmRVE4/8a, GmRVE4/8d
RVE48T4R aaacCGGCCTAAGCGCAAAGCTT GmRVE4/8a, GmRVE4/8d
TOC1T1F gtcaCGATTCCAAGAGTTCTCAAG GmTOC1a, GmTOC1b
TOC1T1R aaacCTTGAGAACTCTTGGAATCG GmTOC1a, GmTOC1b
TOC1T2F gtcaGTGATGTCCGCACAAGATG GmTOC1a, GmTOC1b
TOC1T2R aaacCATCTTGTGCGGACATCAC GmTOC1a, GmTOC1b
TOC1T3F attgGTGGGAATAATAGTAAGAG GmTOC1c, GmTOC1d
TOC1T3R aaacCTCTTACTATTATTCCCAC GmTOC1c, GmTOC1d
TOC1T4F attgTTGTAAAGTGCTTGAGGCT GmTOC1c, GmTOC1d
TOC1T4R aaacAGCCTCAAGCACTTTACAA GmTOC1c, GmTOC1d

Table 2

Germination medium and root medium"

培养基类型
Medium type		 药品名称
Name of the medicine		 药品用量
Medicine
dosage
萌发培养基
Germination medium		 B5盐 B5 salt mixture 1×
蔗糖 Sucrose (g L-1) 20
琼脂 Agar (g L-1) 8
发根培养基
Rooting medium		 MS合成盐 MS salt mixture 1×
蔗糖 Sucrose (g L-1) 30
2-(4-吗啉)乙磺酸 MES 0.6
琼脂 Agar (g L-1) 8
头孢霉素 Cef (mg L-1) 250
羧苄青霉素 Car (mg L-1) 250

Table 3

Target primer"

靶点检测引物
Target primer		 引物序列
Primer sequence (5°-3°)		 靶点检测引物
Target primer		 引物序列
Primer sequence (5°-3°)
SP3 GTCGTGCTCCACATGTTGACCGG LNK2cT4&2R TCCTGAGGTTAGTAGTTCTCCACT
SP1 GAAGTTATTGCATCTATGTCGGG LNK2dT3F CTCTCCGTCGCCGTTATAGCA
LNK1aF TGACGCCAGGGTATCTTAAA LNK2dT3R ACCAAACGAAGCACGAACA
LNK1aR TCTCCATGTGTGTGTTTTGGTA LNK2dT4&2F GGTGGTGGAGGGAGAAGATGAG
LNK1bF CTCAGGGTAGGGAGGACTTG LNK2dT4&2R AGACACTTATTGCCGCTACAACTG
LNK1bR CGGTAAAGTTGAGCCTTGGT RVE4/8aF ACAGCTCTTCAGCTAGGTGTT
LNK1cF TCATATAGTGCCCCATGCCA RVE4/8aR GAGGAGAGGGGGTATGGGTT
LNK1cR AGTTCTATAGCAGCTCATGACA RVE4/8bF AGCGAAGAACTCTGCAATCCA
LNK1dF GCCCGATCATTGCTTCAAGAG RVE4/8bR CTACCACCTTGGGCCGAAAT
LNK1dR AGTTCTATAGCAGCTCGTGGC RVE4/8cF TTCGAAGCCATGCTCAGAAG
LNK2aT3F GAATTCGGCGATGTGTGAGC RVE4/8cR CCAGCAACAAGGTTCGTAGT
LNK2aT3R ACAGCTACACAAAGACACACA RVE4/8dF TTCGTTGGTCATCTTGCTGGT
LNK2aT1F CCGTCCAAGGAGATTGTCACTGA RVE4/8dR CTGGACATGGCCTTCTGTGT
LNK2aT1R TCCTGAGGTAGGTAGTTCTCCACT TOC1aF TCCCTCAACGATGCTG
LNK2bT3F TGATGGAGTGCGTTTCTCTG TOC1aR GCCTCCGTCTCCACAT
LNK2bT3R CCCTGATTTTCCTGGCGTAA TOC1bF TTGAGCAAGTCCAGGGTT
LNK2bT1F CCGTCCAAGGAGATTGTCACTGA TOC1bR ATGGCTGTGATGGTAACTCG
LNK2bT1R TCCTGAGGTAGGTAGTTCTCCACT TOC1cF CTCTAACTAACTATCCAGACCCTA
LNK2cT3F TTCTCCGTCGATCAGTGAAGTG TOC1cR ATGGCTGGTGGGTTGA
LNK2cT3R GCTAAGAGTCACGCCTCCTTG TOC1dF TACGCCCTCCCTCTTT
LNK2cT4&2F CGTGATGCCAAATTAGTTGGGTAT TOC1dR GGGACTTGGGAAATACA

Fig. 1

Homology comparison A: amino acid sequences comparison of AtLNK1, GmLNK1a, GmLNK1b, GmLNK1c, and GmLNK1d; B: amino acid sequences comparison of AtLNK2, GmLNK2a, GmLNK2b, GmLNK2c, and GmLNK2d; C: amino acid sequences comparison of AtTOC1, GmTOC1a, GmTOC1b, GmTOC1c, and GmTOC1d; D: amino acid sequences comparison of AtRVE4, AtRVE8, GmRVE4/8a, GmRVE4/8b, GmRVE4/8c, and GmRVE4/8d."

Fig. 2

Expression of four clock genes in soybean tissues A: the expression of GmLNK1a/b/c/d in soybean W82 tissues; B: the expression of GmLNK2a/b/c/d in soybean W82 tissues; C: the expression of GmTOC1a/b/c/d in soybean W82 tissues; D: the expression of GmRVE4/8a/b/c/d in soybean W82 tissues."

Fig. 3

Schematic diagram of the Cas9 vector construction a: target of LNK1T1, LNK2T1, TOC1T1, and RVE4/8T1; b: target of LNK1T2, LNK2T2, TOC1T2, and RVE4/8T2; c: target of LNK1T3, LNK2T3, TOC1T3, and RVE4/8T3; d: target LNK1T4, LNK2T4, TOC1T4, and RVE4/8T4."

Fig. 4

Detailed sequence of the targets site in the transgenic soybean hairy roots A: mutations were found in target LNK1T1 of GmLNK1a, target LNK1T2 of GmLNK1b, target LNK1T4 of GmLNK1c and target LNK1T4 of GmLNK1d; B: mutations were found in target LNK2T1 of GmLNK2a, target LNK2T1 of GmLNK2b, target LNK2T4 of GmLNK2c and target LNK2T4 of GmLNK2d; C: mutations were found in target TOC1T2 of GmTOC1b, target TOC1T4 of GmTOC1c and target TOC1T4 of GmTOC1d; D: mutations were found in target RVE4/8T1 of GmRVE4/8b and target RVE4/8T4 of GmRVE4/8d."

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