Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 80-87.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0823
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HUANG Wen-li1(), LI Xiang-xiang1, ZHOU Wen-ting1, LUO Sha1, YAO Wei-jia1, MA Jie1,2, ZHANG Fen1, SHEN Yu-sen3, GU Hong-hui3, WANG Jian-sheng3, SUN Bo1()
Received:
2022-07-04
Online:
2023-02-26
Published:
2023-03-07
HUANG Wen-li, LI Xiang-xiang, ZHOU Wen-ting, LUO Sha, YAO Wei-jia, MA Jie, ZHANG Fen, SHEN Yu-sen, GU Hong-hui, WANG Jian-sheng, SUN Bo. Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2023, 39(2): 80-87.
用途 Purpose | 引物名称 Primer name | 引物序列 Sequence of primer(5'-3') | 产物大小 Product size/bp |
---|---|---|---|
合成靶位点序列 Synthetic target site | BoZDS-CRISPR-F1 | ATTGAGGAGAGGAACGCAGTAGC | 23 |
BoZDS-CRISPR-R1 | AAACGCTACTGCGTTCCTCTCCT | 23 | |
BoZDS-CRISPR-F2 | ATTGACCTAACATGAAACCTCCG | 23 | |
BoZDS-CRISPR-R2 | AAACCGGAGGTTTCATGTTAGGT | 23 | |
潮霉素抗性基因检测 Hygromycin resistance gene detection | Hyg-F | CGATTGCGTCGCATCGACC | 558 |
Hyg-R | TTCTACAACCGGTCGCGGAG | ||
Cas9基因检测Cas9 gene detection | Cas9-F | GGCAGATCACAAAGCACGTG | 661 |
Cas9-R | ACCAGCACAGAATAGGCCAC | ||
检测转基因突变Detection of mutations in transgenic plants | BoZDS-CRISPR test-F | ATCCCACTGGCCATAGTTAGGC | 744 |
BoZDS-CRISPR test-R | CAAGTCCAGCTCCAATGATAGCTAC |
Table 1 Primers used in this study
用途 Purpose | 引物名称 Primer name | 引物序列 Sequence of primer(5'-3') | 产物大小 Product size/bp |
---|---|---|---|
合成靶位点序列 Synthetic target site | BoZDS-CRISPR-F1 | ATTGAGGAGAGGAACGCAGTAGC | 23 |
BoZDS-CRISPR-R1 | AAACGCTACTGCGTTCCTCTCCT | 23 | |
BoZDS-CRISPR-F2 | ATTGACCTAACATGAAACCTCCG | 23 | |
BoZDS-CRISPR-R2 | AAACCGGAGGTTTCATGTTAGGT | 23 | |
潮霉素抗性基因检测 Hygromycin resistance gene detection | Hyg-F | CGATTGCGTCGCATCGACC | 558 |
Hyg-R | TTCTACAACCGGTCGCGGAG | ||
Cas9基因检测Cas9 gene detection | Cas9-F | GGCAGATCACAAAGCACGTG | 661 |
Cas9-R | ACCAGCACAGAATAGGCCAC | ||
检测转基因突变Detection of mutations in transgenic plants | BoZDS-CRISPR test-F | ATCCCACTGGCCATAGTTAGGC | 744 |
BoZDS-CRISPR test-R | CAAGTCCAGCTCCAATGATAGCTAC |
靶位点编号 Target site No. | 靶位点序列 Target site sequence | sgRNA GC含量 sgRNA GC content/% | 靶位点得分 Target site score |
---|---|---|---|
1 | CCAGCTACTGC- GTTCCTCTCCTC | 61 | 0.021 1 |
2 | CCTCGGAGGTT- TCATGTTAGGTC | 52 | 0.586 5 |
Table 2 GC content and scores of target sites
靶位点编号 Target site No. | 靶位点序列 Target site sequence | sgRNA GC含量 sgRNA GC content/% | 靶位点得分 Target site score |
---|---|---|---|
1 | CCAGCTACTGC- GTTCCTCTCCTC | 61 | 0.021 1 |
2 | CCTCGGAGGTT- TCATGTTAGGTC | 52 | 0.586 5 |
Fig. 2 Process of stable genetic transformation of broccoli A: Sowing; B: sterile seedlings; C: pre-culture; D: co-culture; E: delayed selection;F: resistance selection
Fig. 3 Detection of the hygromycin-resistant gene and Cas9 gene A: Hyg resistance test of target 1(above)and Cas9 gene detection(below); B: Hyg resistance test of target 2(above)and Cas9 gene detection(below). M: DL2000 marker; N: water as negative control; W: wild type; V: empty vector; P: positive plasmid. 1-20: the number of target 1 resistant plants; 21-41: the number of target 2 resistant plants
Fig. 4 Type of CRISPR/Cas9 system-mediated BoZDS mutation in broccoli A: Mutation of target 1; B: mutation of target 2. Target sequence is indicated in blue, PAM sequence(NGG)is underlined in red, mutated bases were indicated in red font, and the points indicate the spacing between bases. d indicate the number of bases deletion. i indicate the number of bases insertion. r indicate the number of bases replacement
突变类型 Mutation type | 植株 Plant line | 亮度值 L* | 红绿值 a* | 黄蓝值 b* | |||
---|---|---|---|---|---|---|---|
野生型Wild-type | WT | 46.33 | c | -12.67 | e | 23.78 | a |
空载Empty vector | EV | 46.73 | c | -11.65 | e | 23.47 | a |
纯合突变 Homozygous | 2-19 | 79.71 | a | 1.64 | cd | 10.38 | bc |
2-23 | 77.41 | a | 4.48 | bc | 11.80 | bc | |
杂合突变 Heterozygote | 1-1 | 79.86 | a | 1.33 | cd | 11.95 | bc |
1-5 | 67.69 | b | 8.31 | a | 23.76 | a | |
1-19 | 79.60 | a | 5.76 | ab | 21.97 | a | |
2-2 | 68.26 | b | 8.46 | a | 19.19 | ab | |
2-16 | 80.19 | a | 1.21 | cd | 12.14 | bc | |
嵌合突变 Chimera | 2-1 | 80.47 | a | 1.15 | cd | 11.09 | bc |
2-7 | 77.86 | a | 2.88 | d | 7.24 | c | |
2-12 | 80.27 | a | 0.95 | cd | 12.47 | bc |
Table 3 Color parameters of BoZDS mutants, wild-type(WT), and transgenic plant with empty vector(EV)
突变类型 Mutation type | 植株 Plant line | 亮度值 L* | 红绿值 a* | 黄蓝值 b* | |||
---|---|---|---|---|---|---|---|
野生型Wild-type | WT | 46.33 | c | -12.67 | e | 23.78 | a |
空载Empty vector | EV | 46.73 | c | -11.65 | e | 23.47 | a |
纯合突变 Homozygous | 2-19 | 79.71 | a | 1.64 | cd | 10.38 | bc |
2-23 | 77.41 | a | 4.48 | bc | 11.80 | bc | |
杂合突变 Heterozygote | 1-1 | 79.86 | a | 1.33 | cd | 11.95 | bc |
1-5 | 67.69 | b | 8.31 | a | 23.76 | a | |
1-19 | 79.60 | a | 5.76 | ab | 21.97 | a | |
2-2 | 68.26 | b | 8.46 | a | 19.19 | ab | |
2-16 | 80.19 | a | 1.21 | cd | 12.14 | bc | |
嵌合突变 Chimera | 2-1 | 80.47 | a | 1.15 | cd | 11.09 | bc |
2-7 | 77.86 | a | 2.88 | d | 7.24 | c | |
2-12 | 80.27 | a | 0.95 | cd | 12.47 | bc |
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