CRISPR/Cas9 Technology-generated High-amylose Rice Varieties
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摘要:
目的 通过CRISPR/Cas9基因编辑技术遗传改良水稻种质，创制高直链淀粉新材料。 方法 以水稻品种中花11为试验材料，利用CRISPR/Cas9编辑系统对水稻淀粉分支酶（Starch branching enzymes, SBE）基因OsSBE 3进行靶向编辑，利用PCR技术鉴定无标记纯合突变体，并测定其淀粉含量。 结果 T0代获得10株突变体株系，T1代获得5个无标记纯合突变株系，其中4个株系（sbe3-22-6、sbe3-25-3、sbe3-25-4、sbe3-25-6）的直链淀粉含量和淀粉直支比显著高于野生型。 结论 本研究创制了高直链淀粉含量的水稻新种质，为水稻品质改良提供了参考。 Abstract:Objective A series of new rice germplasms with high content of amylose derived from Zhonghua 11 using the CRISPR/Cas9 technology was generated. Method The gene of starch branching enzyme in rice, OsSBE3, was targeted for the genetic editing by CRISPR/Cas9. Homozygous T-DNA-free mutants were verified by PCR with starch content measured. Result Ten mutant lines were obtained from the T0 generation. From the T1 generation, 5 homozygous T-DNA-free lines were obtained that included 4 lines, i.e., sbe3-22-6, sbe3-25-3, Sbe3-25-4, and sbe3-25-6, showing significantly increased amylose content and amylose/amylopectin ratio over the wild type. Conclusion A series of new rice germplasms with high amylose content was created. -
Key words:
- Rice /
- amylose /
- amylopectin /
- amylose/amylopectin ratio /
- genetic editing /
- CRISPR/Cas9
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图 1 YL-Hu-SBE3基因编辑载体信息
红色方框表示gRNA插入位置。
Figure 1. Information on YL-Hu-SBE3 knockout vector
Red box: gRNA insertion position.
图 2 基因编辑T0代水稻突变体的检测
WT表示野生型；黑色下划线表示靶点2序列；红色字体表示碱基插入；黑色虚线表示碱基缺失；||表示染色体正负链。
Figure 2. Detection of mutant lines in T0 generation rice
WT: Wild type; black underline: sequence of Target 2; red font: base insertion; black dotted line: base deletion; ||: plus and minus chains of chromosome.
图 3 T1代突变体无T-DNA插入原件筛选
M：DL2000 DNA 标记；WT：野生型。泳道1～55 为sbe3 T1代突变体植株，其中5、6、10、11、18、23、24、26、27、31、37、39和54分别是sbe3-6-1、sbe3-26-4、sbe3-12-4、sbe3-22-6、sbe3-26-7、sbe3-25-2、sbe3-33-1、sbe3-25-3、sbe3-25-7、sbe3-31-2、sbe-25-5、sbe3-25-4和sbe3-25-6。
Figure 3. Selection of T-DNA-free mutants in T1 generation
M: DL2000 DNA marker; WT: wild type. Lanes 1–55: sbe3 mutants in T1 generation, of which 5, 6, 10, 11, 18, 23, 24, 26, 27, 31, 37, 39, and 54 represent sbe3-6-1, sbe3-26-4, sbe3-12-4, sbe3-22-6, sbe3-26-7, sbe3-25-2, sbe3-33-1, sbe3-25-3, sbe3-25-7, sbe3-31-2, sbe-25-5, sbe3-25-4, and sbe3-25-6, respectively.
图 4 OsSBE 3基因 T1代突变体测序峰图
A：OsSBE3基因结构和靶点2示意图； B～F：sbe3-12-4、sbe3-22-6、sbe3-25-3、sbe3-25-4和sbe3-25-6测序峰图；黑色下划线：靶点2位置；箭头：突变位置。
Figure 4. Sequences and peak map of Ossbe3 mutants in T1 generation
A: Schematic diagram of gene structure and Target 2 of OsSBE3; B–F: sequences and peak map of sbe3-12-4, sbe3-22-6, sbe3-25-3, sbe3-25-4, and sbe3-25-6, respectively; black underline: position of target 2; arrow: mutation position.
图 7 OsSBE3基因编辑后代淀粉含量
A：直链淀粉含量；B：支链淀粉含量；C：淀粉直支比；****：与WT相比，差异极显著（P＜0.01）；ns：与WT相比，没有显著差异（P＞0.05）。
Figure 7. Starch content of progeny after genetic editing on OsSBE3
A: Amylose content; B: amylopectin content; C: amylose/amylopectin ratio; ****: extremely significant difference from WT (P＜0.01); ns: not significantly differ from WT (P＞0.05).
表 1 引物信息
Table 1. Information on primers applied
引物名称
Primer name引物序列（5′-3′）
Primer sequence (5′- 3′)用途
ApplicationYL-Hu-SBE3-Y1+ cagtGGTCTCatgcaGAGAGCAGCGACCGCGACGT 载体构建 Carrier construction YL-Hu-SBE3-Y1- cagtGGTCTCaaaacACGTCGCGGTCGCTGCTCTC 载体构建 Carrier construction YL-Hu-SBE3-B1+ cagtGGTCTCatgcaTTGCTCATGCGGTCTGCATTt 载体构建 Carrier construction YL-Hu-SBE3-B1- cagtGGTCTCaaaacAATGCAGACCGCATGAGCAA 载体构建 Carrier construction Pyl- ACCGGTAAGGCGCGCCGTAGT 鉴定引物 Identification primer Pbw2- GCGATTAAGTTGGGTAACGCCAGGG 鉴定引物 Identification primer Cas9-CL-F GAACGGTCGTAAGAGGATGC 无标记检测 Unmarked detection Cas9-CL-R GGTGATGGACTGGTGGATGAG 无标记检测 Unmarked detection SBE3-C-F TGAAGGTGTCACTTATCGAGAA 纯合突变检测 Homozygous mutation detection SBE3-C-R ACCACTGCGCTATACATGCGTT 纯合突变检测 Homozygous mutation detection HYG-F GGTGATGGACTGGTGGATGAG 鉴定引物 Identification primer HYG-R GGAAGTGCTTGACATTGGGGAGTTT 鉴定引物 Identification primer 32660-BA1-F GCGCACACCCACACACCGACCA 靶点1检测 Target 1 detection 32660-BA1-441Rg GCGAACGGCACCTGGACACGAGA 靶点1检测 Target 1 detection OsBE3_BA2_R ACCACTGCGCTATACATGCGTT 靶点2检测 Target 2 detection OsBE3_BA2_F TGAAGGTGTCACTTATCGAGAA 靶点2检测 Target 2 detection 黑色下划线表示Eco31 I/Bsa I酶切位点，小写斜体字母为酶切位点的保护碱基。
Black underline: Eco31 I/Bsa I cleaving site; lowercase italics: protective base of site.
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