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作物学报 ›› 2023, Vol. 49 ›› Issue (12): 3227-3238.doi: 10.3724/SP.J.1006.2023.24285

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

不同Cas9启动子对大豆CRISPR/Cas9系统效率的作用分析

牛志远1,2, 秦超1, 刘军1, 王海泽2,*(), 李宏宇1,*()   

  1. 1中国农业科学院作物科学研究所, 北京 100081
    2黑龙江八一农垦大学农学院, 黑龙江大庆 163319
  • 收稿日期:2022-12-24 接受日期:2023-05-24 出版日期:2023-12-12 网络出版日期:2023-05-30
  • 通讯作者: * 李宏宇, E-mail: lihongyu@caas.cn; 王海泽, E-mail: haizewang@163.com
  • 基金资助:
    中国农业科学院科技创新工程-作物生物信息学及应用项目(2060302-2-20);大豆藏粮于技项目(CAAS-ZDRW202003)

Function analysis of different Cas9 promoters on the efficiency of CRISPR/ Cas9 system in soybean

NIU Zhi-Yuan1,2, QIN Chao1, LIU Jun1, WANG Hai-Ze2,*(), LI Hong-Yu1,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2022-12-24 Accepted:2023-05-24 Published:2023-12-12 Published online:2023-05-30
  • Contact: * E-mail: lihongyu@caas.cn; E-mail: haizewang@163.com
  • Supported by:
    Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(2060302-2-20);Soybean Grain Storage Technology Program(CAAS-ZDRW202003)

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摘要：

CRISPR/Cas9系统作为高效基因编辑系统, 已被广泛应用于动植物中。多种Cas9基因启动子, 如RPS5A、YAO等被报道用于提高CRISPR/Cas9系统的基因编辑效率。但在大豆中, 不同Cas9启动子对CRISPR/Cas9基因编辑系统效率的影响还没有被阐明。本研究选择了6个已知功能的高效Cas9启动子(p35S、pGmRPS5Ab、pGmRPS5Ac、pAtRPS5A、pGmYAO、pZmUbiquitin)和1个大豆内源未知功能的启动子(pGmHE), 构建CRISPR/Cas9基因敲除载体。通过农杆菌介导的大豆发根系统, 检测这些Cas9启动子对大豆内源基因GmSPA1a和GmEID1的编辑效率, 结果显示大豆内源启动子pGmRPS5Ab对靶基因的编辑效率最高, pAtRPS5A、p35S、pZmUbiquitin对下游基因的编辑效率高于pGmYAO和pGmRPS5Ac。进一步对靶位点测序峰图的分析发现, 使用了pGmRPS5Ab和pAtRPS5A启动子的测序峰图中, 高峰占比较大, 分别为64.0%和58.6%; 而使用p35S的测序峰图中, 低峰占比较高, 为63.3%。这表明pGmRP5SAb和pAtRPS5A启动子不但编辑效率高, 而且编辑效果好, 更有利于在下一代中分离出纯合突变体植株。这些结果将为构建高效大豆基因编辑载体提供参考, 为提高大豆基因编辑效率提供依据。

关键词: 基因编辑, CRISPR/Cas9, 发根系统, 启动子, 大豆

Abstract:

The CRISPR/Cas9 system has been widely used in plants and animals as an efficient gene editing system. Several Cas9 promoters, such as RPS5A and YAO, have been reported to improve the efficiency of gene editing in CRISPR/Cas9 system. In soybean, the influence of different Cas9 promoters on the efficiency of the CRISPR/Cas9 gene-editing system has not been elucidated. In this study, six efficient promoters with known functions (p35S, pGmRPS5Ab, pGmRPS5Ac, pAtRPS5A, pGmYAO, and pZmUbiquitin) and one endogenous soybean promoter with unknown function (pGmHE) were selected to construct the CRISPR/Cas9 knockout vectors. The editing efficiency of the Cas9 promoters on the endogenous soybean genes GmSPA1a and GmEID1 was tested by Agrobacterium tumefaciens mediated hair roots system, which indicated that soybean endogenous promoter pGmRPS5Ab had the highest editing efficiency. The editing efficiency of pAtRPS5A, p35S, and pZmUbiquitin were higher than that of pGmYAO and pGmRPS5Ac. Further analysis of the sequencing maps of target sites showed that the high peak maps accounted for 64.0% and 58.6% in the sequencing maps of pGmRPS5Ab and pAtRPS5A promoters, respectively, while in the sequencing maps of p35S-driven hair roots, the low peak accounted for a higher proportion (63.3%). These above results indicated that pGmRP5SAb and pAtRPS5A promoters not only had high editing efficiency, but also had better editing effect, and were more conducive to the isolation of homozygous mutants in the next generation. In conclusion, this study provide the reference for the construction of efficient soybean CRISPR/Cas9 vectors and help to improve the efficiency of soybean gene editing.

Key words: gene editing, CRISPR/Cas9, hair roots system, promoter, soybean

图1

7个CRISPR/Cas9载体构建示意图 绿色箭头代表7个启动子, 红色方框代表终止子。黄色方框代表gRNA序列。"

表1

PCR引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物名称
Primer name		 引物序列
Primer sequence (5'-3')
Glyma19g29210-F GAAGGAGGAGGAGCATAAG Glyma20g27950-F TACAACATTCAGAAGGAGAG
Glyma19g29210-R TGTTCACCATGATGCTCTC Glyma20g27950-R GGTGAGTGTCTTAACGAAG
Glyma06g42071-F GAGTATGTGCATATTCGTGTA Glyma20g24280-F GAAGAAGATGATAGCGACTC
Glyma06g42071-R GCAACAGAACTCTTTCTTGAC Glyma20g24280-R ACTTGTTGAGAGGGATGAG
Glyma19g39070-F ACAAGTGACCCAGGTTAGAGT Glyma09g24070-F CTGAAAGAGATCCAGAAGGTTCC
Glyma19g39070-R AATCTTCTTGCTTCCCTTTC Glyma09g24070-R GAGCCTGTGCTTGGTGAAG
Glyma10g39780-F ATCTTCGTCAAGACCCTTAC Glyma08g11070-F TTAGCGAATCTCTACCAGGA
Glyma10g39780-R TTCGCCTTCACATTGTCAA Glyma08g11070-R TTGGCAACGATGTTGGAAG
Glyma08g23830-F CGAATCTGGTCAGGAATTG Glyma15g17920-F TACATCAAGAAGGCGATTCA
Glyma08g23830-R TTGGAGAGAGCAAAATGGA Glyma15g17920-R CAACGAGGTAGGAGAAGAT
Glyma08g46860-F GAAAGAACTTCTGAATGGG GmActin-qF CGGTGGTTCTATCTTGGCATC
Glyma08g46860-R TACTTCATCTTCTGCCTGA GmActin-qR GTCTTTCGCTTCAATAACCCTA

表2

启动子序列扩增引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5'−3')		 产物长度
Product length (bp)
GmRPS5Ac-F tcggcacacgtgagatctATTTAGTTGGGTTGCATACTCCT 2767
GmRPS5Ac-R GAGCATGGTGGCAGATCTCATCATCGTCATTTTAGTTTCTT
ZmUbi1-F tcggcacacgtgagatctTGTGCGGAGGCGTGAGCCCGTTTA 2048
ZmUbi1-R GAGCATGGTGGCAGATCTAACAGGGTGACTATCAACAAAA
35S-F tcggcacacgtgagatctTACTCCAAAAATGTCAAAGATAC 727
35S-R GAGCATGGTGGCAGATCTCTCTCCAAATGAAATGAACTTCC
GmRPS5Ab-F tcggcacacgtgagatctTCATACACATGACCGATACAAAT 2582
GmRPS5Ab-R GAGCATGGTGGCAGATCTCGGCTGCACAAGCAATTGAATAG
GmHE-F tcggcacacgtgagatctCCGCATTCCACGCTACCCTAATT 2590
GmHE-R GAGCATGGTGGCAGATCTTATAAAATAAAAACATCTTTTTT
AtRPS5A-F tcggcacacgtgagatctGATCGATCCCTCAACTTTTGATT 1664
AtRPS5A-R GAGCATGGTGGCAGATCTCGGCTGTGGTGAGAGAAACAGA
GmYAO-F tcggcacacgtgagatctAACTTTTAATTACTCAAACTTAT 2522
GmYAO-R GAGCATGGTGGCAGATCTGATTCAGATTCAAACGTTTGCG

表3

引物序列"

引物用途
Primer purpose		 引物名称
Primer name		 引物序列
Primer sequence (5-3')
sgRNA构建引物 U6-Xba I-F GGAAGCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTC
Primers for sgRNA construction U6-R CAATCCATGTGGTGGCACAT
sgRNA-Xba I-R GCTCGGCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGT
sgRNA-SPA1a-F AATGTGCCACCACATGGATTGTGTTGAAGAGTTGCCTGTT GTTTTAGAGCTAGAAATAGCAA
sgRNA-EID1-F CTAGAGTCGAAGTAGTGATTGCTAAGCGAGCCCAGTGGCG GTTTTAGAGCTAGAAATAGCAA
验证编辑载体的引物序列 Cas9-F TCTGGACATTGGGACGAA
Primer for verifying the editing vectors Cas9-R ACGCGGAGAATATCACTC
U6-F ACATTTAATACGCGATAGAAAAC
U6-R TGCAAGGCGATTAAGTTGGGTAA
突变检测PCR引物 target-SPA1a-F ATTGAAGGGACTTTTGTGA
PCR primers for mutation detection target-SPA1a-R TCTCATCCTCTTGGCTGTT
target-EID1-F AAGAACCTGTTTTCGGAACTAA
target-EID1-R ACATCCACGCCCGCTCTTACGC

图2

热图分析11个基因在根、茎、复叶、单叶和茎端分生组织中的表达量 以大豆GmActin-1基因作为内参, 使用2-ΔΔCt计算各基因的相对表达量。将计算所得的相对表达量通过GraphPad Prism 7软件绘制热图, 相对表达水平以蓝色(高)到白色(低)的色阶表示。"

图3

发根检测不同启动子对GmSPA1a基因的编辑效率 A: 使用GmRPS5Ab启动子, 在发状根中PCR检测单根U6与Cas9序列电泳图。M: 分子量marker; 空白对照: 水, 阴性对照: 天隆1号基因组DNA; 阳性对照: 带有Cas9、U6序列质粒。同时检测到Cas9序列与U6序列的阳性根编号用红色数字标识, U6序列大小为950 bp, Cas9序列大小为864 bp。B: 发状根检测所取单根数量、阳性未编辑根数量和被编辑的根数量。3个柱形图为3次重复结果, 其中蓝色柱为检测根总数量, 红色柱为PCR检测出的阳性根数, 绿色柱为GmSPA1a位点被编辑的根的数量, 具体取样根的数量见表4。"

附图1

PCR检测U6与Cas9序列电泳图 使用CAMV35S、GmHE、ZmUbi、GmRPS5Ac、AtRPS5A、 GmYAO启动子，在发状根中PCR检测单根U6与Cas9序列电泳图。M: 分子量 marker；空白对照：水，阴性对照：天隆1号基因组DNA；阳性对照：带有Cas9、U6序列质粒。同时检测到Cas9序列与U6序列的阳性根编号用红色数字标识，U6序列大小为950 bp，Cas9序列大小为864 bp。"

表4

大豆发状根检测中所取发状根数量、阳性数量及编辑效率统计"

载体名称
Vector name		 第1次重复
Repeat for the first time		 第2次重复
Repeat for the second time		 第3次重复
Repeat for the third time
检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)		 检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)		 检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)
GmHE-pro:Cas9 42 28 5 18 42 24 5 21 42 28 7 25
GmYAO-pro:Cas9 42 32 10 31 42 22 7 32 42 26 7 27
GmRP5SAb-pro:Cas9 90 58 40 69 90 43 24 56 90 46 28 61
AtRPS5A-pro:Cas9 90 44 28 64 90 38 18 47 90 50 27 54
35S-pro:Cas9 90 52 30 58 42 22 10 46 42 28 12 43
GmRPS5Ac-pro:Cas9 90 74 23 37 90 46 14 30 90 44 16 36
ZmUbi1-pro:Cas9 90 56 26 46 90 56 33 59 90 56 31 55

图4

使用不同Cas9启动子的编辑效率 A: 7个启动子对GmSPA1a基因的编辑效率。B: 3个启动子对GmEID1基因的编辑效率。*表示差异显著(t-test P<0.05), **表示差异极显著(t-test P < 0.01), ns表示无差异。"

表5

大豆发状根检测中所取发状根数量、阳性数量及编辑效率统计"

载体名称
Vector name		 第1次重复
Repeat the first time		 第2次重复
Repeat the second time		 第3次重复
Repeat the third time
检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)		 检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)		 检测
根数
NTHR		 阳性
数量
NPHR		 编辑
数量
NPHR		 编辑
效率
EE (%)
GmRP5SAb-pro:Cas9 42 18 8 44 42 24 15 63 42 30 16 53
AtRPS5A-pro:Cas9 42 20 8 40 42 20 11 55 42 29 14 48
35S-pro:Cas9 42 22 5 23 42 30 9 30 42 27 7 26

图5

GmSPA1a编辑位点的测序峰图分析 A: 测序峰图高峰示例; B: 测序峰图低峰示例; C: 统计分析3次发根检测中7个启动子对GmSPA1a编辑峰图中高峰和低峰比例。柱形图黑色部分代表测序峰图中高峰所占比例, 柱形图灰色部分代测序峰图中低峰所占比例。"

附图2

统计第1次、第2次、第3次发根转化中7个启动子对GmSPA1a编辑峰图中高峰和低峰比例 黑色柱代表测序峰图中高峰比例，灰色代表测序峰图中低峰比例。"

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