Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology

doi:10.13560/j.cnki.biotech.bull.1985.2022-0823

Abstract

Abstract:

ζ-Carotene desaturase（ZDS）is one of the key enzymes regulating carotenoids biosynthesis. Here having the high-generation inbred broccoli（Brassica oleracea var. italica）line ‘ZN09’ as testing materials and BoZDS as target gene, then two target sites on the first exon of BoZDS were selected to construct CRISPR/Cas9 vector for stable genetic transformation. For the target site 1, the transformation efficiency and the mutation rate were 0.80% and 15.79%, respectively, and three heterozygous mutants were generated. For the target site 2, the transformation efficiency was 0.84%, and the mutation rate was 36.84%, and seven mutants were generated, including two homozygous, two heterozygous, and three chimeric mutants. All of the mutant plants showed albino or mottled phenotype. The L* and a* values of mutant plants were significantly higher than those of wild-type plants, while the b* value decreased. To sum up, a stable genetic system for CRISPR/Cas9-mediated gene editing in broccoli was established, and BoZDS was effectively edited. Our results provide the technical support for gene function study and germplasm innovation of broccoli by using gene editing technology.

Key words: broccoli, CRISPR/Cas9, gene editing, stable genetic transformation, ζ-carotene desaturase（ZDS）

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.

Figures/Tables 9

References 34

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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
潮霉素抗性基因检测 Hygromycin resistance gene detection	Hyg-R	TTCTACAACCGGTCGCGGAG	558
Cas9基因检测Cas9 gene detection	Cas9-F	GGCAGATCACAAAGCACGTG	661
Cas9基因检测Cas9 gene detection	Cas9-R	ACCAGCACAGAATAGGCCAC	661
检测转基因突变Detection of mutations in transgenic plants	BoZDS-CRISPR test-F	ATCCCACTGGCCATAGTTAGGC	744
检测转基因突变Detection of mutations in transgenic plants	BoZDS-CRISPR test-R	CAAGTCCAGCTCCAATGATAGCTAC	744

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
潮霉素抗性基因检测 Hygromycin resistance gene detection	Hyg-R	TTCTACAACCGGTCGCGGAG	558
Cas9基因检测Cas9 gene detection	Cas9-F	GGCAGATCACAAAGCACGTG	661
Cas9基因检测Cas9 gene detection	Cas9-R	ACCAGCACAGAATAGGCCAC	661
检测转基因突变Detection of mutations in transgenic plants	BoZDS-CRISPR test-F	ATCCCACTGGCCATAGTTAGGC	744
检测转基因突变Detection of mutations in transgenic plants	BoZDS-CRISPR test-R	CAAGTCCAGCTCCAATGATAGCTAC	744

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

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

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
纯合突变 Homozygous	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