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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1291-1300.doi: 10.3724/SP.J.1006.2020.94169

• 研究简报 • 上一篇    

大豆生物钟基因GmLNK1/2、GmRVE4/8和GmTOC1 CRISPR/Cas9组织表达分析及敲除靶点的鉴定

甘卓然,石文茜,黎永力,侯智红,李海洋,程群,董利东,刘宝辉,芦思佳()   

  1. 广州大学生命科学学院, 广东广州 510006
  • 收稿日期:2019-11-07 接受日期:2020-03-24 出版日期:2020-08-12 网络出版日期:2020-04-07
  • 通讯作者: 芦思佳
  • 作者简介:E-mail: gggzzr@126.com
  • 基金资助:
    国家自然科学基金项目(31771815);国家自然科学基金项目(31701445);作物遗传与种质创新国家重点实验室开放课题(ZW201901)

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 Published:2020-08-12 Published online:2020-04-07
  • Contact: Si-Jia LU
  • 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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摘要：

生物钟基因能够参与调控植物的整个生命进程, 对提高作物产量具有重要的作用。LNK1 (NIGHT LIGHT- INDUCIBLE AND CLOCK-REGULATED 1)、LNK2、RVE4 (REVEILLE 4)、RVE8 (REVEILLE 8)和TOC1 (TIMING OF CAB EXPRESSION 1)是植物中重要的生物钟基因。本研究利用BLAST同源比对和进化树分析的方法分别鉴定AtLNK1、AtLNK2、AtRVE4、AtRVE8和AtTOC1在大豆中的同源基因, 通过qRT-PCR实验证明这些生物钟基因在大豆根、茎、叶等组织中均有表达。成功构建这些基因的CRISPR/Cas9敲除载体, 并利用大豆根毛转化体系成功鉴定出13个基因的CRISPR/CAS9有效靶点。为进一步获得稳定的大豆突变体材料及研究其生物钟基因的功能提供了理论基础。

关键词: 大豆, 基因敲除, 生物钟基因, CRISPR/Cas9

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

表1

基因靶点引物列表"

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

表2

萌发培养基与根诱导培养基"

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

表3

靶点检测引物"

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

图1

同源性比对 A: AtLNK1与GmLNK1a、GmLNK1b、GmLNK1c、GmLNK1d氨基酸序列对比; B: AtLNK2与GmLNK2a、GmLNK2b、GmLNK2c、GmLNK2d氨基酸序列对比; C: AtTOC1与GmTOC1a、GmTOC1b、GmTOC1c、GmTOC1d氨基酸序列对比; D: AtRVE4、AtRVE8与GmRVE4/8a、GmRVE4/8b、GmRVE4/8c、GmRVE4/8d氨基酸序列对比。"

图2

4个生物钟基因组织特异表达 A: GmLNK1a/b/c/d在W82中的组织特异表达; B: GmLNK2a/b/c/d在W82中的组织特异表达; C: GmTOC1a/b/c/d在W82中的组织特异表达; D: GmRVE4/8a/b/c/d在W82中的组织特异表达。"

图3

Cas9载体结构图 a: 靶点LNK1T1、LNK2T1、TOC1T1和RVE4/8T1; b: 靶点LNK1T2、LNK2T2、TOC1T2和RVE4/8T2; c: 靶点LNK1T3、LNK2T3、TOC1T3和RVE4/8T3; d: 靶点LNK1T4、LNK2T4、TOC1T4和RVE4/8T4。"

图4

发生编辑靶点的测序峰图 A: GmLNK1a的靶点LNK1T1、GmLNK1b的靶点LNK1T2、GmLNK1c的靶点LNK1T4和GmLNK1d的靶点LNK1T4突变情况; B: GmLNK2a的靶点LNK2T1、GmLNK2b的靶点LNK2T1、GmLNK2c的靶点LNK2T4和GmLNK2d的靶点LNK2T4突变情况; C: GmTOC1b的靶点TOC1T2、GmTOC1c的靶点TOC1T4和GmTOC1d的靶点TOC1T4突变情况; D: GmRVE4/8b的靶点RVE4/8T1和GmRVE4/8d的靶点RVE4/8T4突变情况。"

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