利用CRISPR/Cas9技术敲除水稻Pi21基因的效率分析

doi:10.16819/j.1001-7216.2016.6009

摘要/Abstract

摘要：

利用CRISPR/Cas9技术,针对Pi21基因的两个靶位点(靶位1和靶位2),构建基因敲除载体,转化水稻品种南粳9108。经PCR鉴定,获得了28株T₀代阳性转基因植株。对靶位点酶切检测发现,靶位 1突变效率为78.57%,靶位 2突变效率为92.86%;靶位 1和靶位 2同时突变的效率为78.57%,突变效率较高。通过对靶位点进行测序,发现靶位点突变类型较多,包括碱基缺失、碱基插入、碱基缺失后插入其他碱基和大片段DNA缺失等类型。对突变株系进行氨基酸预测,发现大部分株系都存在移码突变现象而使基因突变彻底,少数株系表现为部分氨基酸的缺失或变异。成功敲除了水稻Pi21基因,并对突变效率和类型进行了分析,为进一步验证Pi21基因功能、培育广谱抗稻瘟病的水稻新品系奠定了基础。

关键词: CRISPR/Cas9, 稻瘟病, Pi21, 基因敲除

Abstract:

A CRISPR/Cas9 vector, which contains two targets (Target 1 and Target 2) of Pi21gene, was constructed and transformed to rice variety Nanjing 9108. Twenty-eight T₀ transgenic lines were obtained and confirmed by T-DNA specific PCR. Restriction digestion analysis revealed 78.57% mutant ratio in Target 1 site, 92.86% mutant ratio in Target 2 site, 78.57% ratio of lines containing both Target 1 and Target 2 mutant in transgenic lines. Through sequencing analysis, we found the mutant types of targets, such as deletion of bases, insertion of bases, insertion behinds deletion of bases, and long fragment deletion of DNA, etc. Moreover, except amino acid deletion, most transgenic lines displayed frameshift mutation,which caused function loss of Pi21. In this way, a series of Pi21-knock-out rice lines were obtained, and further, could be used for functional analysis of Pi21 and rice blast broad-spectrum resistant breeding.

Key words: CRISPR/Cas9, rice blast, Pi21, gene knock-out

中图分类号:

Q755
S511.01

王芳权, 范方军, 李文奇, 朱金燕, 王军, 仲维功, 杨杰. 利用CRISPR/Cas9技术敲除水稻Pi21基因的效率分析[J]. 中国水稻科学, 2016, 30(5): 469-478.

Fang-quan WANG, Fang-jun FAN, Wen-qi LI, Jin-yan ZHU, Jun WANG, Wei-gong ZHONG, Jie YANG. Knock-out Efficiency Analysis of Pi21 Gene Using CRISPR/Cas9 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(5): 469-478.

图/表 10

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')
Pi21g-F	ACCAAAGCCTGTCTATCTGC
Pi21g-R	CACATCGATCAGCCTCGTG
Pi21UT1-F	GGCAGAAGCTGTGCAAGAAGATC
Pi21UT1-R	AAACGATCTTCTTGCACAGCTTC
Pi21UT2-F	GCCGCCGCTGCGATGCCAAGATC
Pi21UT2-R	AAACGATCTTGGCATCGCAGCGG
pU-F	CTCCGTTTTACCTGTGGAATCG
gRNA-R	CGGAGGAAAATTCCATCCAC
Pi21T1L-F	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
Pi21T1L-R	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Pi21T2L-F	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
Pi21T2L-R	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
SP1	CCCGACATAGATGCAATAACTTC
SP2	GCGCGGTGTCATCTATGTTA
Pi21T1-F	AGGCTAATCAGCAGTGTTCCT
Pi21T1-R	CAGCTTGCACTCCGGCTTCG
Pi21T2-F	ATTGGTAACATTCGGCAAATT
Pi21T2-R	GTTCTTCACGTCGTACTCCA

图1 Pi21基因编码序列比对南粳9108感稻瘟病, 为Pi21感病基因型; Owarihatamochi抗稻瘟病, 为pi21抗病基因型。

Fig. 1. Coding sequences co-linearity analysis of Pi21 gene. Nanjing 9108 is a blast sensitive variety, containing the susceptible allele Pi21. Owarihatamochi is a blast resistant variety, containing the resistant allele pi21.

图2 Pi21基因结构及gRNA靶点位置加粗的碱基为PAM,下划线标出的碱基为限制性内切酶识别位点,黑色区域为外显子,白色区域为内含子,灰色方框标识区域为富脯氨酸元件。

Fig. 2. Schematic diagram of Pi21 gene structure and gRNA targets. The base in bold indicates PAM; The restriction endonuclease recognized site is underlined; The black region indicates exon; The white region indicates intron; The gray box region indicates proline-rich motifs.

图3 CRISPR/Cas9-Pi21载体结构

Fig. 3. Schematic diagram of the CRISPR/Cas9-Pi21 vector construction.

图4 PCR鉴定转基因阳性植株 M-DL 2000 DNA标记; 1~42-T0代转基因株系; WT-南粳9108(野生型)。

Fig. 4. PCR identification of the positive transgenic plants. M, DL 2000 DNA marker; 1-42, T0 transgenic lines; WT, Nanjing 9108 (wild-type).

图5 酶切鉴定靶位 1突变情况 A-酶切前; B-酶切后。 1~42-T0代转基因株系; WT-南粳9108(野生型)。

Fig. 5. Mutation identification at Target 1 by enzyme digestion. A, Before digestion; B, After digestion. 1~42, T0 transgenic lines; WT, Nanjing 9108 (wild-type).

图6 酶切鉴定靶位 2突变情况 A-酶切前; B-酶切后。 1~42-T0代转基因株系; WT-南粳9108(野生型)。

Fig. 6. Mutation identification at Target 2 by enzyme digestion. A, Before digestion; B, After digestion. 1~42, T0 transgenic lines; WT, Nanjing 9108 (wild-type).

图7 野生型、转基因株系16和21靶位测序结果

Fig. 7. Sequencing results of target sites from wild-type, transgenic lines 16 and 21.

图8 靶位点突变序列分析 “-1”和“-2”表示不同突变类型,括号内的数值为测序的克隆数,箭头所指的位置为Cas9剪切位点,加粗碱基为前间区序列邻近基序(PAM),红色字体的碱基为靶序列,蓝色字体的碱基为插入碱基。WT, 野生型;#, 转基因株系; d, 缺失; i, 插入。

Fig. 8. Analysis of mutation sequences of targets. “-1” and “-2” indicate different mutation type; The numbers in brackets are the numbers of sequenced clone; The arrows indicate the digestion sites of Cas9; The base in bold indicates protospacer adjacent motif(PAM); The base in red is the target sequence; The base in blue is the insert base; WT, Wild-type; #, Transgenic lines; d, Deletion; i, Insert.

图9 突变株系的氨基酸序列共线性分析

Fig. 9. Co-linearity analysis of amino acid sequences of mutant lines.

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