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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (8): 1961-1970.doi: 10.3724/SP.J.1006.2024.33050

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

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 Online:2024-08-12 Published:2024-04-25
  • Contact: * E-mail: xiechuanxiao@caas.cn
  • Supported by:
    Beijing Science and Technology Project(D171100007717001)

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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

Table 1

Primers used in this study"

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

Fig. 1

PCR amplification and sequence alignment of ZmCCT10, ZmCCT9, and ZmGhd7 target regions of four maize materials A: PCR amplification of ZmCCT10, ZmCCT9, and ZmGhd7 target regions of 4 maize materials. M: DNA marker (DM2000); 1: KN5585; 2: LCL-1; 3: LCL-2; 4: CML312SR; 5: H2O; B: Sequence alignment of ZmCCT10, ZmCCT9, and ZmGhd7 target regions of 4 maize materials. The red boxes indicate target sequences containing PAM; the numbers on the right represent the number of bases."

Fig. 2

Schematic diagram of target sites design and CCT-CPD vector A: Gene structure and schematic diagram of the target sites of ZmCCT10, ZmCCT9, and ZmGhd7; B: Sequence information of the target sites of ZmCCT10, ZmCCT9, and ZmGhd7. The purple letters are the bases with different target sequences from sgRNA; the red arrowhead is the expecting cleavage site; C: Schematic diagram of CCT-CPD vector."

Fig. 3

Mutation identification of ZmCCT10, ZmCCT9, and ZmGhd7 genes in T0 plants A: BstX I digestion validation of ZmCCT10 target region in T0 plants. M: DNA marker (DM2000); 1-45: T0 plants; B: the mutation types of ZmCCT10 in T0 plants; C: the mutation types of ZmCCT9 in T0 plants; D: the mutation types of ZmGhd7 in T0 plants; E: sequencing profiles of ZmCCT10 target regions of #1,2,20. The purple letters are the bases with different target sequences from sgRNA; the red letters are PAM; the bold red letters are the inserted bases; the red dashes are the deleted bases; the red arrowhead indicates the location of mutation."

Table 2

Number of mutants among transgenic plants"

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)

Fig. 4

Phenotypes of Zm3896-driven DsRed expression cassettes in corn kernels A: the fluorescence observation of corn kernels’ embryo (by camera). Bar: 0.5 cm; WT: wild type; B: the fluorescence observation of corn kernels’ embryo (by nightsea stereomicroscope). Bar: 2 mm; WT: wild type; C: the fluorescence observation of embryo and endosperm of positive kernel (by laser scanning confocal microscopy). Bar: 200 μm; Em: embryo; En: endosperm."

Table 3

Results of F1 seeds fluorescence screening"

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
[1] 徐雷, 贾飞飞, 王利琳. 拟南芥开花诱导途径分子机制研究进展. 西北植物学报, 2011, 31: 1057-1065.
Xu L, Jia F F, Wang L L. Progresses on molecular mechanisms of flowering transition in Arabidopsis. Acta Bot Boreali-Occident Sin, 2011, 31: 1057-1065 (in Chinese with English abstract).
[2] Hung H Y, Shannon L M, Tian F, Bradbury P J, Chen C, Garcia S A F, McMullen M D, Ware D, Buckler E S, Doebley J F, Holland J B. ZmCCT and the genetic basis of day-length adaptation underlying the post domestication spread of maize. Proc Natl Acad Sci USA, 2012, 109: 1913-1921.
[3] Jin M L, Liu X G, Jia W, Liu H J, Li W Q, Peng Y, Du Y F, Wang Y B, Yin Y J, Zhang X H, Liu Q, Deng M, Li N, Cui X Y, Hao D Y, Yan J B. ZmCOL3, a CCT gene represses flowering in maize by interfering with the circadian clock and activating expression of ZmCCT. J Integr Plant Biol, 2018, 60: 465-480.
[4] Xue W Y, Xing Y Z, Weng X Y, Zhao Y, Tang W J, Wang L, Zhou H J, Yu S B, Xu C G, Li X H, Zhang Q F. Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet, 2008, 40: 761-767.
[5] Meng X, Muszynski M G, Danilevskaya O N. The FT-like ZCN8 gene functions as a floral activator and is involved in photoperiod sensitivity in maize. Plant Cell, 2011, 23: 942-960.
[6] Yang Q, Li Z, Li W Q, Ku L X, Wang C, Ye J R, Li K, Yang N, Li Y P, Zhong T, Li J S, Chen Y H, Yan J B, Yang X H, Xu M L. CACTA-like transposable element in ZmCCT attenuated photoperiod sensitivity and accelerated the post domestication spread of maize. Proc Natl Acad Sci USA, 2013, 110: 16969-16974.
doi: 10.1073/pnas.1310949110 pmid: 24089449
[7] Huang C, Sun H Y, Xu D Y, Chen Q Y, Liang Y M, Wang X F, Xu G H, Tian J G, Wang C L, Li D, Wu L S, Yang X H, Jin W W, Doebley J F, Tian F. ZmCCT9 enhances maize adaptation to higher latitudes. Proc Natl Acad Sci USA, 2018, 115: 334-341.
[8] Jamann T M, Sood S, Wisser R J, Holland J B. High-throughput resequencing of maize landraces at genomic regions associated with flowering time. PLoS One, 2017, 12: e0168910.
[9] 郭栋, 杜媚, 周宝元, 高卓晗, 曹哲统, 赵明. 玉米CCT基因家族的鉴定与生物信息学分析. 植物遗传资源学报, 2019, 20: 1001-1010.
doi: 10.13430/j.cnki.jpgr.20181107001
Guo D, Du M, Zhou B Y, Gao Z H, Cao Z T, Zhao M. Identification and bioinformatic analysis of maize CCT gene family. J Plant Genet Resour, 2019, 20: 1001-1010 <br (in Chinese with English abstract).
[10] Zhang H, Zhang J S, Wei P L, Zhang B T, Gou F, Feng Z Y, Mao Y F, Yang L, Zhang H, Xu N F, Zhu J K. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J, 2014, 12: 797-807.
doi: 10.1111/pbi.12200 pmid: 24854982
[11] Zeng D C, Liu T L, Ma X L, Wang B, Zheng Z Y, Zhang Y L, Xie X R, Yang B W, Zhao Z, Zhu Q L, Liu Y G. Quantitative regulation of Waxy expression by CRISPR/Cas9-based promoter and 5'UTR-intron editing improves grain quality in rice. Plant Biotechnol J, 2020, 18: 2385.
[12] Wang Y P, Cheng X, Shan Q W, Zhang Y, Liu J X, Gao C X, Qiu J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol, 2014, 32: 947-953.
doi: 10.1038/nbt.2969 pmid: 25038773
[13] Zhang S J, Zhang R Z, Gao J, Gu T T, Song G Q, Li W, Li D D, Li Y L, Li Y G. Highly efficient and heritable targeted mutagenesis in wheat via the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system. Int J Mol Sci, 2019, 20: 4257.
[14] Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol, 2015, 15: 16.
doi: 10.1186/s12896-015-0131-2 pmid: 25879861
[15] Wang L W, Sun S, Wu T T, Liu L P, Sun X G, Cai Y P, Li J C, Jia H C, Yuan S, Chen L, Jiang B J, Wu C X, Hou W S, Han T F. Natural variation and CRISPR/Cas9-mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean. Plant Biotechnol J, 2020, 18: 1869-1881.
doi: 10.1111/pbi.13346 pmid: 31981443
[16] Li C X, Liu C L, Qi X T, Wu Y C, Fei X H, Mao L, Cheng B J, Li X H, Xie C X. RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnol J, 2017, 15: 1566-1576.
[17] Dong L, Qi X T, Zhu J J, Liu C L, Zhang X, Cheng B J, Mao L, Xie C X. Supersweet and waxy: meeting the diverse demands for specialty maize by genome editing. Plant Biotechnol J, 2019, 17: 1853.
doi: 10.1111/pbi.13144 pmid: 31050154
[18] Yifhar T, Pekker I, Peled D, Friedlander D, Pistunov A, Sabban M, Wachsman G, Alvarez J P, Amsellem Z, Eshed Y. Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome. Plant Cell, 2012, 24: 3575-3589.
[19] Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 2015, 8: 1274-1284.
doi: 10.1016/j.molp.2015.04.007 pmid: 25917172
[20] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157: 1262-1278.
doi: S0092-8674(14)00604-7 pmid: 24906146
[21] Zhang H, Zhang J S, Lang Z B, Botella J R, Zhu J K. Genome editing-principles and applications for functional genomics research and crop improvement. Crit Rev Plant Sci, 2017, 36: 291-309.
[22] Soyk S, Müller N A, Park S J, Schmalenbach I, Jiang K, Hayama R, Zhang L, Eck J V, Jiménez-Gómez J M, Lippman Z B. Variation in the flowering gene SELF PRUNING 5G promotes day- neutrality and early yield in tomato. Nat Genet, 2017, 49: 162-168.
[23] Cai Y P, Chen L, Liu X J, Guo C, Sun S, Wu C X, Jiang B J, Han T F, Hou W S. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soybean. Plant Biotechnol J, 2018, 16: 176-185.
[24] Liu X Q, Tian J, Zhou X J, Chen R M, Wang L, Zhang C Y, Zhao J, FanY L. Identification and characterization of promoters specifically and strongly expressed in maize embryos. Plant Biotechnol J, 2014, 12: 1286-1296.
doi: 10.1111/pbi.12227 pmid: 25052028
[25] Kalla R, Shimamoto K, Potter R, Nielsen P S, Linnestad C, Olsen O A. The promoter of the barley aleurone-specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell-specific expression in transgenic rice. Plant J, 1994, 6: 849-860.
pmid: 7849757
[26] Dong L, Li L N, Liu C L, Liu C X, Geng S F, Li X H, Huang C L, Mao L, Chen S J, Xie C X. Genome editing and double- fluorescence proteins enable robust maternal haploid induction and identification in maize. Mol Plant, 2018, 11: 1214-1217.
doi: S1674-2052(18)30218-1 pmid: 30010025
[27] 李荣华, 夏岩石, 刘顺枝, 孙莉丽, 郭培国, 缪绅裕, 陈健辉. 改进的CTAB提取植物DNA方法. 实验室研究与探索, 2009, 28(9): 14-16.
Li R H, Xia Y S, Liu S Z, Sun L L, Guo P G, Miao S Y, Chen J H. CTAB-improved method of DNA extraction in plant. Res Explor Lab, 2009, 28(9): 14-16 (in Chinese with English abstract).
[28] 马兴亮, 刘耀光. 植物CRISPR/Cas9基因组编辑系统与突变分析. 遗传, 2015, 38: 118-125.
Ma X L, Liu Y G. CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants. Hereditas (Beijing), 2015, 38: 118-125 (in Chinese with English abstract).
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