Development of Fragrant Japonica Rice by CRISPR/Cas9-targeted Editing on Badh2
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摘要:
目的 利用基因编辑技术编辑水稻香味基因，改良优质粳稻香味性状。 方法 构建CRISPR/Cas9-BADH基因编辑载体，转化优质粳稻品种龙稻18、龙稻24和秀水134，测序鉴定3个优质粳稻品种的香味基因Betaine aldehyde dehydrogenase 2（Badh2）突变体并分析潜在脱靶效应，利用气相色谱-质谱联用技术测定不同遗传背景badh2突变体稻米的2-乙酰-1-吡咯啉（2-acetyl-1-pyrroline, 2AP）含量。 结果 转化获得的30株T0代中有24株为badh2突变体，其中53.33%为杂合型突变，16.67%为纯合型突变，10%为双等位突变类型。T1代非转基因植株内共鉴定获得7种纯合badh2突变基因型。在5个预测位置上未检测到脱靶事件的发生，说明设计的sgRNA具有高度特异性。所有Badh2移码突变体稻米的2AP含量都达到或高于稻花香的水平，但不同品种来源的badh2突变体间2AP含量差异极显著。 结论 本研究提供了一个能够高效诱导水稻Badh2突变的CRISPR/Cas9定向编辑靶点，改良了生产上大面积推广的3个优质水稻品种龙稻18、龙稻24和秀水134的香味性状，发现了不同遗传背景的水稻badh2突变体间2AP含量差异显著，为基于定向编辑Badh2基因的方法培育适合生产应用的香稻品种、提高育种效率提供科学依据。 -
关键词:
- 水稻 /
- 香味 /
- Badh2 /
- CRISP/CAS9 /
- 2-乙酰-1-吡咯啉
Abstract:Objective The CRISPR/Cas9 technology was applied to edit the Badh2 in rice to enrich the fragrance of current japonica varieties for marketing enhancement. Method A CRISPR/Cas9-BADH vector was constructed to transform 3 elite japonica rice, Longdao 18, Longdao 24, and Xiushui 134. The mutant badh2 was identified by sequencing, and potential off-target mutations examined. Contents of the aromatic 2-acetyl-1-pyrroline (2AP) in the mutant rice cultivars was determined by gas chromatography-mass spectrometry. Result Of the 30 T0 transgenic plants, 24 were found to contain the target mutant badh2, of which, 53.3% were heterozygous, 16.67% homozygous, and 10% bi-allelic. Seven homozygous mutation genotypes were obtained in the T1 non-transgenic mutant plants. There was no off-target mutation detected at all 5 potential sites indicating that a high specificity of the designed sgRNA for the predicted site. Interferences by the varied genome backgrounds of the rice varieties might have caused the significantly varied amounts of the aromatic 2AP detected in all badh2 frame-shift mutant rice. Conclusion The CRISPR/Cas9 technology could effectively induce desired Badh2 mutation in rice. Improvement on the fragrance for 3 japonica rice could lead to wide applications in cultivating new varieties with added commercial value. More interestingly, the significant variations on 2AP content among the mutant rice as discovered in this study would help further the understanding on the genetics associated with aroma rice breeding. -
Key words:
- rice /
- fragrance /
- Badh2 /
- CRISPR/Cas9 /
- 2AP
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图 1 秀水134转CRISPR/Cas9-BADH质粒T0代PCR检测
注：M:DL2000DNA分子量标记；1：CRISPR/Cas9-BADH质粒，2：秀水134野生型，3~15：秀水134转基因T0代植株。
Figure 1. PCR identification for CRISPR/Cas9-BADH in Xiushui134 transgenic T0 plants
Note：M: DL2000DNA marker; 1: CRISPR/Cas9-BADH construt; 2：Xiushui134 wild-type; 3–15：T0 Xiushui134 transgenic plants.
图 2 CRISPR/Cas9系统介导的Badh2位点特异性突变
注：（A）Badh2基因结构的示意图。黑色矩形代表Badh2的15个外显子，红色字符表示sgRNA-Cas9的靶序列，下划线处为PAM序列。（B）T1代突变植株badh2纯合突变测序峰图。红色和黑色三角形分别表示碱基插入和缺失；WT代表野生型；LD18badh2-#、LD24badh2-# 和 XS134badh2-#分别代表龙稻18、龙稻24和秀水134遗传背景的突变体材料。
Figure 2. CRISPR/Cas9-mediated genome editing on Badh2
Note：(A) Schematic Badh2 gene structure showing 15 exons in black rectangular frames; red-colored characters indicate target sequences of sgRNA-Cas9; PAM sequences are underlined. (B) Examples of sequencing chromatograms of T1 lines with homozygous mutant badh2. Deletions are indicated by black triangle, and insertions by red triangle. WT: wild-type sequence. LD18badh2-#, LD24badh2-#, and XS134badh2-# represent mutations in Longdao 18, Longdao 24, and Xiushui 134 genome backgrounds, respectively.
图 3 badh2突变体及对照2AP总离子流图峰面积
Figure 3. Total ion chromatograms of 2AP in badh2 mutant and control lines
图 4 不同品种badh2突变体稻米中的2AP含量
注：2AP含量的数值为平均值±标准差表示，不同大写字母表示各材料间的2AP含量经LSD法多重比较存在极显著性差异（P < 0.01）。
Figure 4. 2AP contents in badh2 mutants with different genome backgrounds
Note: 2AP levels are means ± SD of three replications，different capital letters indicate the significant differences of 2AP content according to LSD multiple range test at P < 0.01.
表 1 引物信息
Table 1. Primers information
引物名
Primer序列（5′- 3′）
Sequence（5′- 3′）引物用途
ApplicationBADH2-S CAGGAAGAGGAGGGTACCGATGG sgRNA构建 sgRNA Construction BADH2-A AACCCATCGGTACCCTCCTCTTC sgRNA构建 sgRNA Construction BADHF TCGCTTTCCACCTCAACG Badh2基因扩增、测序 Badh2 PCR and Sanger sequence BADHR GCTTGAAACGAATCACCACA Badh2基因扩增、测序 Badh2 PCR and Sanger sequence HptF AAGCTGCATCATCGAAATTG 转基因阳性株检测 Identify transgenic plants HptR TCGTTATGTTTATCGGCACT 转基因阳性株检测 Identify transgenic plants BOTF1 TGACAACAGCAAAGATGAGAAATGG 潜在脱靶位点BS1的正向PCR引物 F primer for the putative off-target site of BS1 BOTR1 AGGAAGACGCCGACAACACG 潜在脱靶位点BS1的反向PCR引物 R primer for the putative off-target site of BS1 BOTF2 TTCATCAAAGCAGACAACTACCATT 潜在脱靶位点BS2的正向PCR引物 F primer for the putative off-target site of BS2 BOTR2 ACCACGACGGGAAGCACC 潜在脱靶位点BS2的反向PCR引物 R primer for the putative off-target site of BS2 BOTF3 AGATCGCAAGAACGCAACAAG 潜在脱靶位点BS3的正向PCR引物 F primer for the putative off-target site of BS3 BOTR3 TGAAGTAGTGGATGGAGACGGAG 潜在脱靶位点BS3的反向PCR引物 R primer for the putative off-target site of BS3 BOTF4 GGCGGCACCTCTCCAAG 潜在脱靶位点BS4的正向PCR引物 F primer for the putative off-target site of BS4 BOTR4 TCCTCAATACCATAAATCTCACCC 潜在脱靶位点BS4的反向PCR引物 R primer for the putative off-target site of BS4 BOTF5 CGCATTTCAGCGAGTTCCA 潜在脱靶位点BS5的正向PCR引物 F primer for the putative off-target site of BS5 BOTR5 GCAGGCAGAGGTGTAGGGTAA 潜在脱靶位点BS5的反向PCR引物 R primer for the putative off-target site of BS5 
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表 2 转基因T0代植株Badh2突变分析
Table 2. Mutation of Badh2 in T0 transgenic plants
品种
Variety再生苗数
Regenerated plants转基因阳性苗数
Transgenic plantsT0代Badh2突变 Mutations in Badh2 of T0 generation 总突变数
Total plants with mutations纯合突变数
Homozygote双等位突变数
Bi-allele杂合突变数
Heterozygote秀水134 XS134 13 11 9（81.81） 2（18.18） 1（9.09） 6（54.55） 龙稻18 LD18 10 8 7（87.50） 1（12.50） 0（0.00） 6（65.000） 龙稻24 LD24 12 11 8（72.72） 2（18.18） 2（18.18） 4（36.36） 平均 Average 10 8（80.00） 1.67（16.67） 1（10.00） 5.33（53.33） 注：*括号内为转基因植株内所含突变植株数的百分率，%。
Note：*Percentages were calculated based on the number of plants with mutations over the total number of transgenic plants, %.
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表 3 T1代植株Badh2基因纯合突变类型
Table 3. Homozygous mutation genotype of Badh2 in T1 plants
T1植株编号
T1 plant ID纯合突变基因型①
Genotype of homozygous mutant突变类型②
Mutation type/bpXS134badh2-#1 gagtcccccat-ggtaccctcctcttcacc −1 XS134badh2-#2 gagtcccccatAcggtaccctcctcttcac +1 XS134badh2-#3 gagtcccccat-ggtaccctcctcttcacc −1 XS134badh2-#4 gagtcccccat----accctcctcttcac −4 XS134badh2-#5 gagtcccccat-ggtaccctcctcttcacc −1 XS134badh2-#6 gagtccccca------ccctcctcttcacc −6 XS134badh2-#7 gagtcccccat-ggtaccctcctcttcacc −1 XS134badh2-#8 gagtcccccat-ggtaccctcctcttcacc −1 XS134badh2-#9 gagtcccccat----accctcctcttcac −4 LD24 badh2-#1 gagtcccccat-ggtaccctcctcttcacc −1 LD24 badh2-#2 gagtcccccat--gtaccctcctcttcacc −2 LD24 badh2-#3 gagtcc-------gtaccctcctcttcacc −7 LD24 badh2-#4 gagtcccccat---taccctcctcttcacc −3 LD24 badh2-#5 gagtcccccat-ggtaccctcctcttcacc −1 LD24 badh2-#6 gagtcccccat-ggtaccctcctcttcacc −1 LD24 badh2-#7 gagtcccccat----accctcctcttcac −4 LD24 badh2-#8 gagtcccccat-ggtaccctcctcttcacc −1 LD18 badh2-#1 gagtcccccatAcggtaccctcctcttcac +1 LD18 badh2-#2 gagtcccccat-ggtaccctcctcttcacc −1 LD18 badh2-#3 gagtcccccat----accctcctcttcac −4 LD18 badh2-#4 gagtcccccat-ggtaccctcctcttcacc −1 LD18 badh2-#5 gagtcccccat-ggtaccctcctcttcacc −1 LD18 badh2-#6 gagtcccccat-ggtaccctcctcttcacc −1 LD18 badh2-#7 gagtcccccat-ggtaccctcctcttcacc −1 注：①短划线和黑色加粗字母分别表示缺失和插入碱基。②+：插入，−：缺失，具体数字表示所涉及的核苷酸数。
Note: ①Deletions and insertions are indicated by dashes and black bold letters, respectively; ②+: insertion, −: deletion, the numbers indicate the number of nucleotides involved.
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表 4 T1代badh2突变体植株的潜在脱靶检测
Table 4. Examination for possible off-target mutations in T1 badh2 mutant lines
潜在脱靶位点
Potential off-target site潜在脱靶位点的序列
Sequence of the potential
off-target site①潜在脱靶位置
Potential off-target locus基因
Gene在基因上
所处区域
Region错配碱基数
No. of mismatching
bases测序植
株数
No. of plants sequenced②突变植株数
No. of plants
with mutationsBADH GAAGAGGAGGGTACCGATGGGGG BS1 GGAGAGGAGGGTGCCGATGGTGG Chr6:+574034 Os06g0109801 exon 2 18 0 BS2 GGAGAGGAGGGTGCCGATGGTGG Chr2:+20036025 Os02g0540000 exon 2 18 0 BS3 GGAGAGGAGGGTGCCGATGGTGG Chr10:+22435422 Os10g0566400 exon 2 18 0 BS4 CGTGAGGAGGGCACCGATGGTGG Chr 4:-19800287 Os04g0400000 exon 4 18 0 BS5 GAGGAGGAAGGTGCCGATGGAGG Chr 7:-88557 Os07g0101500 exon 3 18 0 注：①黑色加粗字母为错配碱基，PAM序列用下划线表示。②来自龙稻18、龙稻24及秀水134背景的18株T1代非转基因植株被用来进行脱靶检测。
Note: ①The mismatching bases were shown in bold letters, and PAM sequences are underlined. ②Eighteen transgene-free T1mutant plants in Longdao18、Longdao24 and XS134 backgrounds were evaluated.
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