利用CRISPR/Cas9技术靶向编辑青花菜BoZDS

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

摘要/Abstract

摘要：

ζ-胡萝卜素脱氢酶（ζ-carotene desaturase, ZDS）是类胡萝卜素合成的限速酶。本试验以青花菜多代自交系‘ZN09’为试材，以BoZDS为目标基因，在其第1个外显子上选取2个靶位点，分别构建CRISPR/Cas9载体进行稳定遗传转化。1号靶位点转化效率为0.80%，突变率为15.79%，共获得3株突变体，均为杂合突变；2号靶位点转化效率为0.84%，突变率为36.84%，共获得7株突变体，其中纯合突变2株，杂合突变2株，嵌合突变3株。突变体均出现白化或斑驳表型，突变体L*值和a*值均显著高于野生型植株，b*值均下降。本试验建立了青花菜CRISPR/Cas9基因编辑稳定遗传体系，并对BoZDS基因进行有效编辑，研究结果为利用基因编辑技术进行青花菜基因功能研究与优异性状材料创制提供了技术支撑。

关键词: 青花菜, CRISPR/Cas9, 基因编辑, 稳定遗传转化, ζ-胡萝卜素脱氢酶（ZDS）

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）

黄文莉, 李香香, 周炆婷, 罗莎, 姚维嘉, 马杰, 张芬, 沈钰森, 顾宏辉, 王建升, 孙勃. 利用CRISPR/Cas9技术靶向编辑青花菜BoZDS[J]. 生物技术通报, 2023, 39(2): 80-87.

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.

图/表 9

表1 本研究中所用的引物

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
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

表2 靶位点GC含量和得分情况

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

图1 靶位点选择（A）与载体构建（B）

Fig. 1 Selection of target sites（A）and construction of vectors（B）

图2 青花菜稳定遗传转化的过程 A：播种；B：无菌苗；C：预培养；D：共培养；E：延迟筛选；F：抗性筛选

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

图3 潮霉素抗性基因和Cas9基因检测 A：1号靶位点Hyg抗性检测（上图）和Cas9基因检测（下图）；B：2号靶位点Hyg抗性检测（上图）和Cas9基因检测（下图）。M：DL2000 marker；N：水；W：野生型；V：空载体；P：阳性质粒。1-20：1号靶位点的抗性植株编号；21-41：2号靶位点的抗性植株编号

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

图4 CRISPR/Cas9介导的青花菜BoZDS基因突变类型 A：1号靶位点突变情况；B：2号靶位点突变情况。蓝色字表示靶序列，红色下划线表示PAM序列，红色字表示突变碱基，·表示省略碱基之间的间距。d代表碱基缺失，i代表碱基插入，r代表碱基替换，序列后数字代表缺失、插入或替换的碱基数

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

图5 不同靶位点的突变体数和突变率

Fig. 5 Mutant number and mutation rate of different targets

图6 BoZDS突变体及野生型（WT）和空载植株（EV）的表型

Fig. 6 Phenotypes of BoZDS mutants, wild-type（WT）, and transgenic plant with empty vector（EV）

表3 BoZDS突变体及野生型（WT）和空载植株（EV）的颜色参数

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
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

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