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作物学报 ›› 2024, Vol. 50 ›› Issue (8): 1961-1970.doi: 10.3724/SP.J.1006.2024.33050

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

编辑ZmCCT10、ZmCCT9、ZmGhd7基因的串联DsRed荧光表达盒的CRISPR/Cas9系统的构建及验证

曹晓晴(), 祁显涛, 刘昌林, 谢传晓()   

  1. 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2023-09-01 接受日期:2024-04-01 出版日期:2024-08-12 网络出版日期:2024-04-25
  • 通讯作者: * 谢传晓, E-mail: xiechuanxiao@caas.cn
  • 作者简介:E-mail: xiaoqingcao110@163.com
  • 基金资助:
    北京市科技计划项目(D171100007717001)

Construction and verification of the CRISPR/Cas9 system containing DsRed fluorescent expression cassette for editing of ZmCCT10, ZmCCT9, and ZmGhd7 genes in maize

CAO Xiao-Qing(), QI Xian-Tao, LIU Chang-Lin, XIE Chuan-Xiao()   

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2023-09-01 Accepted:2024-04-01 Published:2024-08-12 Published online:2024-04-25
  • Contact: * E-mail: xiechuanxiao@caas.cn
  • Supported by:
    Beijing Science and Technology Project(D171100007717001)

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摘要：

CCT家族基因影响植物开花, 在玉米中, ZmCCT10、ZmCCT9基因是光周期敏感基因, ZmGhd7基因是与开花期相关的基因。利用CRISPR/Cas9技术靶向编辑ZmCCT10、ZmCCT9、ZmGhd7基因为研究基因的功能和快速改良玉米的开花期提供了可能。本研究以玉米ZmCCT10、ZmCCT9、ZmGhd7基因为编辑对象, 以KN5585为稳定转化受体、以CML312SR、LCL-1、LCL-2为预改良的晚熟材料受体, 首先通过Sanger测序验证了3个基因靶标区域在4份玉米材料中的保守性, 其次根据sgRNA设计原则选择了1个sgRNA复合编辑3个基因, 并利用同源重组方法构建了将由胚特异性启动子Zm3896驱动的DsRed表达盒和由ZmU6-2启动子驱动的sgRNA表达盒串联的CRISPR/Cas9基因编辑敲除载体CCT-CPD, 然后采用酶切法和Sanger测序法分析T0代KN5585中3个基因的突变率和突变类型, 验证了该系统的基因编辑效果, 最后通过对稳定遗传转化植株所结籽粒在籽粒水平、组织水平进行DsRed荧光标记表型鉴定, 验证了该系统中DsRed荧光筛选标记的有效性。在此基础上, 通过杂交育种法以晚熟材料为母本、以T1代KN5585阳性株为父本获得F1并经过DsRed荧光筛选获得含有有效编辑转基因元件的晚熟材料。本研究构建的编辑ZmCCT10/ZmCCT9/ZmGhd7基因的串联DsRed荧光表达盒的CRISPR/Cas9系统为创制单基因突变体, 双基因突变体, 三基因突变体奠定了基础, 该系统中DsRed荧光筛选标记的应用可以快速筛选区分有无转基因成分的玉米籽粒, 成本低, 鉴定效率高, 具有大规模籽粒筛选的潜力, 本研究为鉴定ZmCCT10、ZmCCT9、ZmGhd7三个基因的功能和创制玉米光周期钝感材料奠定了材料基础和高效的技术基础。

关键词: CRISPR/Cas9技术, DsRed荧光, ZmCCT10、ZmCCT9、ZmGhd7基因, 玉米

Abstract:

The CCT family genes affect plant flowering time. In maize, ZmCCT10 and ZmCCT9 are photoperiod sensitive genes, and ZmGhd7 is a gene related to the flowering time. Targeted editing of ZmCCT10, ZmCCT9, and ZmGhd7 genes using CRISPR/Cas9 technology provides the possibility to study the function of three genes and to rapidly improve the flowering time of maize. In this study, maize ZmCCT10, ZmCCT9, and ZmGhd7 were used as editing objects. The inbred line KN5585 was used as a stable transforming receptor, and CML312SR, LCL-1, and LCL-2 were used as pre-modified late-flowering lines. Firstly, the conservation of the target regions of the three genes in four maize lines was verified by Sanger sequencing. Secondly, one sgRNA was selected to co-edit three genes based on sgRNA design principles. The CRISPR/Cas9 gene editing knockout vector CCT-CPD was constructed using homologous recombination, which contained the DsRed expression cassette driven by embryo-specific promoter Zm3896 and the sgRNA expression cassette driven by the ZmU6-2 promoter. Next, the mutation rate and mutation type of the three genes in T0 generation KN5585 were analyzed by enzyme digestion method and Sanger sequencing, and the gene editing effect of the CRISPR/Cas9 system was verified. Finally, the seeds produced by stable genetic transformation plants were verified by the DsRed fluorescent labeling phenotype at the kernel level and tissue level. On this basis, F1 was obtained by cross breeding using late flowering lines as female parent and T1 generation KN5585 positive plant as male parent, and late flowering lines containing effective edited transgenic elements were obtained by DsRed fluorescence screening. The CRISPR/Cas9 system for editing ZmCCT10, ZmCCT9, and ZmGhd7 genes containing DsRed fluorescent expression cassette constructed, in this study, laid a foundation for the creation of single-gene mutants, double-gene mutants, and/or triple-gene mutants. The application of DsRed fluorescent screening markers in this system can quickly screen and distinguish corn kernels with or without transgenic components, which has the potential of large-scale kernels screening with low cost and high identification efficiency. This study laid a material foundation and efficient technical basis for identifying the functions of ZmCCT10, ZmCCT9, and ZmGhd7 and creating photoperiod insensitive materials in maize.

Key words: CRISPR/Cas9 technology, DsRed fluorescence, ZmCCT10, ZmCCT9, and ZmGhd7 genes, maize

表1

本研究所用引物"

扩增产物及长度
Amplification
product and length		 引物名称
Primer name		 引物序列
Primer sequence
(5'-3')		 退火温度
Annealing temperature (℃)
ZmCCT10-517 bp ZmCCT10F1 CTCTATCGATCAACAGCGGC 62
ZmCCT10R1 CGGGAGCAATACTTACGATG
ZmCCT9-468 bp ZmCCT9F1 AAGGGCTCAAGCTCAAGAGAGAGCG 67
ZmCCT9R1 CAGCTGGCCGTACTGAGC
ZmGhd7-638 bp ZmGhd7F1 AGGAGGAAGAGGGGTACGTC 67
ZmGhd7R1 TTTTGGAACCGAAGCGCAAG
Zm3896-1976 bp Zm3896-F cacgctgcactgcacaagctGGGTAGAGAAAGCAAGGGAGAC 68
Zm3896-R acgttctcggaggaggccatGGCGCCCGTCGTCTGTGG
DsRed-NOS-950 bp DsRed-NOS-F ATGGCCTCCTCCGAGAACGTCATCAC 68
DsRed-NOS-R gacggccagtgccaagctTGATCTAGTAACATAGATGACAC
U6-sgRNA-434 bp U6-F tatgttactagatcaagctCTAATTGGCCCTTACAAAATAG 68
U6-R aactggaactcgtgcagcGGAGCGGTGGTCGCAGCTGAAC
sgRNA-sgRNA scaffold-123 bp sgRNA-F gctgcacgagttccagttcttGTTTTAGAGCTAGAAATAGCAAG 68
sgRNA-R gacggccagtgccaagctTAAAAAAAGCACCGACTCGGTGCCAC
Cas9-568 bp Cas9-F CAACCGGAAAGTGACCGTGA 62
Cas9-R CACCACCTTCACTGTCTGCA
U6-Sg-829 bp CPB-U6-SG-F CCGCCACCACCTGTTCCT 64
CPB-U6-SG-R GGGTTTTCCCAGTCACGA
ZmCCT10-390 bp (102/288) ZmCCT10-MQ-F TCTTCTCCGTCTTCCCTGTC 62
ZmCCT10-MQ-R CGGGAGCAATACTTACGATG

图1

4份玉米材料ZmCCT10、ZmCCT9、ZmGhd7靶标区域PCR扩增和序列比对 A: 4份玉米材料ZmCCT10、ZmCCT9、ZmGhd7靶标区域PCR扩增。M: DNA marker (DM2000); 1: KN5585; 2: LCL-1; 3: LCL-2; 4: CML312SR; 5: H2O; B: 4份玉米材料ZmCCT10、ZmCCT9、ZmGhd7靶标区域序列比对。红色框内为含PAM的靶点序列; 右侧数字代表碱基数目。"

图2

靶位点设计和CCT-CPD载体示意图 A: ZmCCT10、ZmCCT9、ZmGhd7基因结构和靶位点位置示意图; B: ZmCCT10、ZmCCT9、ZmGhd7基因靶位点序列信息。紫色字母为靶点序列与sgRNA存在差异的碱基; 红色箭头为预期的切割位点; C: CCT-CPD载体示意图。"

图3

T0代植株ZmCCT10、ZmCCT9和ZmGhd7基因突变鉴定 A: T0代植株ZmCCT10基因靶标区域BstX I酶切验证。M: DNA marker (DM2000); 1~45: T0代植株; B: T0代植株ZmCCT10基因靶位点的突变类型; C: T0代植株ZmCCT9基因的突变类型; D: T0代植株ZmGdh7基因的突变类型; E: #1,2,20植株ZmCCT10基因靶位点测序峰图。紫色字母为靶点序列与sgRNA存在差异的碱基; 红色字母为PAM序列; 加粗红色字母为插入的碱基; 红色-为缺失的碱基; 红色箭头指的是突变的位置。"

表2

转基因植株突变体数量统计"

基因
Gene name		 突变植株
Number of mutant plants		 突变率
Mutation ratio
(%)		 纯合突变体
Homozygous mutant		 纯合突变或双等位突变比率
Ratio of homozygous or double allele mutations
(%)
ZmCCT10 17 65.4% (17/26) 15 88.2% (15/17)
ZmCCT9 3 11.5% (3/26) 2 66.7% (2/3)
ZmGhd7 11 42.3% (11/26) 11 100.0% (11/11)

图4

Zm3896驱动的DsRed表达盒在玉米籽粒中的表型 A: 玉米籽粒胚部荧光观察(相机)。标尺为0.5 cm; WT: 野生型; B: 玉米籽粒胚部荧光观察(体式显微镜)。标尺为2 mm; WT: 野生型; C: 阳性籽粒胚及胚乳组织荧光观察(激光共聚焦显微镜)。标尺为200 μm; Em: 胚; En: 胚乳。"

表3

F1荧光筛选结果"

种子来源
Seed source		 阳性种子数(粒)
Number of positive seeds		 阴性种子数(粒)
Number of negative seeds		 种子总数(粒)
Total number of seeds		 阳性率
Positive rate (%)
165×8-7 98 55 153 64.1
165×8-14 41 44 85 48.2
166×8-14 11 13 24 45.8
166×8-16 17 13 30 56.7
167×8-14 77 79 156 49.4
167×8-14 50 32 82 61.0
167×8-17 16 11 27 59.3
167×30-1 29 23 52 55.8
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