中国水稻科学 ›› 2018, Vol. 32 ›› Issue (1): 35-42.DOI: 10.16819/j.1001-7216.2018.7061
汪秉琨1,2, 张慧1, 洪汝科1, 张锦文3, 杨睿2, 罗琼2,*(), 曾千春1,*()
收稿日期:
2017-05-21
修回日期:
2017-08-24
出版日期:
2018-01-10
发布日期:
2018-01-10
通讯作者:
罗琼,曾千春
作者简介:
共同第一作者:汪秉琨 张慧;
基金资助:
Bingkun WANG1,2, Hui ZHANG1, Ruke HONG1, Jinwen ZHANG3, Rui YANG2, Qiong LUO2,*(), Qianchun ZENG1,*()
Received:
2017-05-21
Revised:
2017-08-24
Online:
2018-01-10
Published:
2018-01-10
Contact:
Qiong LUO, Qianchun ZENG
About author:
These authors contributed equally to this work:WANG Bingkun, ZHANG Hui;
摘要:
【目的】 直链淀粉含量与稻米品质密切相关。Wx基因是控制水稻直链淀粉合成的主效基因,通过对Wx基因定点编辑以获得稳定遗传、直链淀粉含量适宜的突变体。【方法】 构建CRISPR/Cas9表达载体pGK03-Wx-gRNA (靶点1和2分别在Wx基因第1和第2外显子),利用工程菌EHA105遗传转化超级稻楚粳27,潮霉素筛选获得转化株系,对转化株系及其后代进行分子检测、测序、基因表达和遗传稳定性分析以及直链淀粉含量测定。【结果】 获得9个独立的T0代转化株系,靶点1(L1~L5) 5个株系,突变频率100%,靶点2(L6~L9) 4个株系,突变频率75%。由T0代突变体衍生出T1和T2代株系,测序发现T0、T1和T2代株系出现缺失(单、双、多碱基缺失)和单碱基插入两种突变类型;T0至T1代部分株系(L1、L2、L3和L6)发生再编辑,T1至T2代遗传稳定。与野生型相比,突变株系RNA水平Wx基因表达量显著下降(P<0.01),稻米直链淀粉含量显著降低(P<0.01),从17.5%降到1.93%。【结论】 利用CRISPR/Cas9系统成功编辑水稻Wx基因,获得了稳定遗传、低直链淀粉含量的突变体,为稻米品质改良提供了材料。
中图分类号:
汪秉琨, 张慧, 洪汝科, 张锦文, 杨睿, 罗琼, 曾千春. CRISPR/Cas9系统编辑水稻Wx基因[J]. 中国水稻科学, 2018, 32(1): 35-42.
Bingkun WANG, Hui ZHANG, Ruke HONG, Jinwen ZHANG, Rui YANG, Qiong LUO, Qianchun ZENG. Wx Gene Editing via CRISPR/Cas9 System in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(1): 35-42.
Oligo名称 | 序列 | 检测引物 | 序列 | ||
---|---|---|---|---|---|
Oligo name | Sequence | Test primer | Sequence | ||
Oligo-Target1-F | TGTGTGGGCGAATGGCCACAGGGTCA | G-Target1-F | GCATGAACGTCGTGTTCGTC | ||
Oligo-Target1-R | AAACTGACCCTGTGGCCATTCGCCCA | G-Target1-R | CCTCACCCTCTCGTACCTGT | ||
Oligo-Target2-F | TGTGTGGCTTGAGGCCCTGGAACCCG | G-Target2-F | TGCAGAGATCTTCCACAGCA | ||
Oligo-Target2-R | AAACCGGGTTCCAGGGCCTCAAGCCA | G-Target2-R | TTGAAGACGACGACGGTCAG |
表1 寡核苷酸序列及检测引物序列
Table 1 Oligo sequence and test primer sequence.
Oligo名称 | 序列 | 检测引物 | 序列 | ||
---|---|---|---|---|---|
Oligo name | Sequence | Test primer | Sequence | ||
Oligo-Target1-F | TGTGTGGGCGAATGGCCACAGGGTCA | G-Target1-F | GCATGAACGTCGTGTTCGTC | ||
Oligo-Target1-R | AAACTGACCCTGTGGCCATTCGCCCA | G-Target1-R | CCTCACCCTCTCGTACCTGT | ||
Oligo-Target2-F | TGTGTGGCTTGAGGCCCTGGAACCCG | G-Target2-F | TGCAGAGATCTTCCACAGCA | ||
Oligo-Target2-R | AAACCGGGTTCCAGGGCCTCAAGCCA | G-Target2-R | TTGAAGACGACGACGGTCAG |
图2 pGK03-Wx-gRNA表达载体 LB–左边界;U6–水稻U6启动子;SG–向导RNA;UBI–UBI启动子;NOS Ter–NOS终止子;35S–35S启动子;Hpt–潮霉素基因;PolyA Ter–PolyA终止子;RB–右边界。
Fig. 2. Vector map of pGK-Wx-gRNA. LB, Left border; U6,Rice U6 promoter; SG, Guide RNA; UBI, UBI promoter; NOS Ter, NOS terminator; 35S, 35S promoter; Hpt, Hygromycin gene; PolyA Ter, PolyA terminator; RB, Right border.
图4 野生型与9个T0转化株系突变位点序列比对下划线表示gRNA靶序列;阴影表示PAM序列;“-”示碱基缺失;a示碱基插入。–1表示单碱基缺失;+1表示单碱基插入。
Fig. 4. DNA sequence alignment for wild type and the nine transformants in T0 generation. sequences are shown by black hyphens. One base deletion is recorded by minus one and the one base insertion by plus one. The targeted sequence is highlighted with underline and the PAM sequence in shadow. Mutation with 1 bp insertion is represented by a, the deleted
图5 野生型与9个T0转化株系氨基酸序列比对“-”表示氨基酸缺失;L1~L5和L6~L8翻译提前终止。
Fig. 5. Amino acid sequence alignment for wild type and nine transformants in T0 generation. The deleted amino acids are shown by black hyphens, the translation were terminated earlier in mutants L1-L5 and L6-L8.
图6 野生型与L1~L8株系T1和T2代突变位点序列比对下划线表示gRNA靶序列;阴影表示PAM序列;“-”示碱基缺失;a示碱基插入。
Fig. 6. DNA sequence alignment for the wild type and the L1-L8 in T1 and T2 generations. The targeted sequence is highlighted with underline and the PAM sequence in shadow. Mutation with 1 bp insertion is represented by a, the deleted sequences are shown by black hyphens.
图7 野生型与T1突变体Wx的表达量(平均值±标准差) **表示野生型与T1突变体在P<0.01下差异显著(t检验)。
Fig. 7. Relative expression level of Wx gene in wild type and transformants in T1 generation(Mean±SD). **indicates significant difference at P<0.01(t test).
株系 Line | WT | L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | 平均 Average |
---|---|---|---|---|---|---|---|---|---|---|
直链淀粉含量Amylose content/% | 17.5 | 1.78** | 1.82** | 1.76** | 2.15** | 2.06** | 2.04** | 1.93** | 1.87** | 1.93 |
表2 野生型与转化株系L1~L8的直链淀粉含量
Table 2 Amylose content comparison of the wild type and transformants L1-L8.
株系 Line | WT | L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | 平均 Average |
---|---|---|---|---|---|---|---|---|---|---|
直链淀粉含量Amylose content/% | 17.5 | 1.78** | 1.82** | 1.76** | 2.15** | 2.06** | 2.04** | 1.93** | 1.87** | 1.93 |
图8 野生型和突变体的糙米表型 WT为楚粳27的糙米表型;L1~L8为突变株系T1代的糙米表型;比例尺,1 cm
Fig. 8. Brown rice of wild type and mutants. WT, Brown rice of Chujing 27; L1-L8, Brown rice of mutants in T1 generation and the bar represents one centimeter.
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