利用CRISPR/Cas9系统定向编辑水稻SD1基因

doi:10.16819/j.1001-7216.2018.7106

摘要/Abstract

摘要：

【目的】半矮秆水稻品种的选育和应用是水稻育种的最重大成果之一。半矮秆品种大多是半矮秆基因SD1(semi-dwarf1)功能缺失突变体,为了获得sd1突变体,本研究对SD1基因进行了定向编辑。【方法】利用CRISPR/Cas9系统,以SD1基因为靶基因,构建基因编辑载体CRISPR-SD1,用农杆菌介导的方法转化水稻恢复系申繁17和申繁24。【结果】在2个转化受体的T₀代均获得了纯合的sd1突变体,并且在T₁代株系中分离出了不含转基因序列的植株。2个品种的sd1突变体与各自的野生型相比,株高分别下降了25%左右。【结论】利用CRISPR/Cas9系统可以有效地对目的基因进行编辑,在水稻分子育种领域具有巨大的应用价值。

关键词: CRISPR/Cas9, 基因编辑, 水稻, 半矮秆基因

Abstract:

【Objective】In the last century, the usage of semi-dwarfrice varieties brought about significant improvements and great achievements inrice breeding.Most of the semi-dwarfrice varieties have loss-of-function mutations in the Semi-Dwarf1 (SD1) gene.In order to obtain sd1 mutant, targeted editing of SD1 gene was performed. 【Method】SD1 gene editing vector CRISPR-SD1 was constructed by using CRISPR/Cas9 system, and transformed into two rice restore linesShenfan17 and Shenfan24 by the Agrobacterium-mediated method.【Result】 Homozygous sd1 mutants were obtained in T₀ generation in both varieties, and vector-free sd1 mutant lines were segregated from the T₁ population. The plant heights of the two sd1 mutants were decreased by 25% as compared with those of their wild types. 【Conclusion】CRISPR/Cas9 is a powerful tool for rice target gene editing, and has enormous potential in rice molecular breeding.

Key words: CRISPR/Cas9, gene editing, rice, Semi-Dwarf1

中图分类号:

胡雪娇, 杨佳, 程灿, 周继华, 牛付安, 王新其, 张美良, 曹黎明, 储黄伟. 利用CRISPR/Cas9系统定向编辑水稻SD1基因[J]. 中国水稻科学, 2018, 32(3): 219-225.

Xuejiao HU, Jia YANG, Can CHENG, Jihua ZHOU, Fuan NIU, Xinqi WANG, Meiliang ZHANG, Liming CAO, Huangwei CHU. Targeted Editing of Rice SD1 Gene UsingCRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2018, 32(3): 219-225.

图/表 7

表1 本研究所用的引物

Table 1 Primers used in this research.

引物名称 Primer name	引物序列(5′-3′) Primer sequence (5′-3′)
Oligo-F	TGGCGGAGGATGGAGCCCAAGATCC
Oligo-R	AAACGGATCTTGGGCTCCATCCTC
M13-F SEQ1-F SEQ1-R EXON1-F EXON1-R EXON3-F EXON3-R HYG-F HYG-R CAS9-F CAS9-R	TGTAAAACGACGGCCAGT GGGTCATTGATTCGACCATC GTGCTCGGACACCTGGAAGAAC GTCATTGATTCGACCATCATGTCTGTC GAATTACTTGTTCTGTTGCTTCGAAGCA CTCTCCGTTGATGAATGATGATG CTTCTGTTCGTTCCGTTTCGTTC CGTGCTTTCAGCTTCGATGTAGGA GCATATGAAATCACGCCATGTAGTGT CTTCATCGAGCGGATGACCAACT AGATCGTGGTATGTGCCCAGGGA

图1 水稻SD1氨基酸序列的比对

Fig. 1. Comparison of rice SD1 amino acid sequence.

表2 水稻SD1基因中的CRISPR/Cas9靶位点

Table 2 Putative CRISPR/Cas9 target sites in rice SD1 gene.

靶位点编号 Targetnumber	靶位点序列 Sequence of target site	候选识别位点的毗邻基序Protospacer adjacent motif	位置 Site / bp	DNA链 DNA strand
1	GAGGATGGAGCCCAAGATCC	CGG	108	+
2	AGATCCCGGAGCCATTCGTG	TGG	122	+
3	GAGCCATTCGTGTGGCCGAA	CGG	130	+
4	GGATCTTGGGCTCCATCCTC	AGG	107	–
5	ACGAATGGCTCCGGGATCTT	GGG	120	–
6	CACGAATGGCTCCGGGATCT	TGG	121	–
7	TTCGGCCACACGAATGGCTC	CGG	129	–

图2 阳性克隆的菌落PCR筛选 M―D2000标记;N―阴性对照;1~8―克隆。

Fig. 2. Screening positive clone by colony PCR. M, D2000 marker; N, Negative control; 1 to 8, Clones.

图3 T0代转基因株系中SD1基因突变类型的分析灰色字体表示靶序列,下划线表示候选识别位点的毗邻基序(PAM)序列,方框表示插入碱基,横线表示缺失碱基,箭头表示Cas9剪切位点。

Fig. 3. Mutation type in SD1 gene of T0transgenic lines. The grey letters indicated the target sequence. The underline indicated protospacer adjacent motif(PAM) sequence. The box represented the insert base. The transverse line indicated deletion sequence. Arrow indicated the Cas9 cleavage site.

图4. T1代中不含转基因载体序列植株的PCR筛选 M―D2000标记;W―野生型;1~24―T1转基因植株;P1―引物M13F和Oligo-R;P2―引物HYG-F和HYG-R;P3―引物CAS9-F和CAS9-R。

Fig. 4. PCR screening of transgenic-free plants in T1 generation. M, D2000marker, W, Wild type;Lanes 1-24, T1 transgenic plants; P1, Primer M13F and Oligo-R;P2, Primer HYG-F and HYG-R; P3, Primer Cas9-F and Cas9-R.

图5 野生型与T2代sd1突变体的农艺性状(n=20) **表示差异极显著(P<0.01)。

Fig. 5. Agronomic traits of wild type and T2-generation sd1mutants (n=20). **Significant at 0.01 level.

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