利用CRISPR/Cas9技术编辑AFP1基因提高水稻耐逆性

doi:10.16819/j.1001-7216.2021.0503

摘要/Abstract

摘要： 目的为鉴定水稻AFP1在非生物胁迫响应中的作用,创制非生物胁迫抗性的水稻新材料。方法以优异籼稻恢复系华占为转化受体,利用CRISPR/Cas9技术创制afp1突变体,并对afp1突变体的耐逆性进行初步鉴定。结果 AFP1靶点1和靶点2的编辑效率分别为66.67%和75.00%。所有突变株系中,突变类型仅有插入和缺失突变,90%突变株系的突变长度为小片段突变（<5bp）。获得了6种无转基因成分的afp1纯合突变体。正常条件下,afp1突变体株高和结实率降低,有效分蘖增加,穗长显著升高,单株产量在-4.06%和11.75%之间变化。和野生型相比,afp1突变体的ABA敏感性和叶片水分散失率降低,耐干旱、热和渗透胁迫能力提高。结论编辑AFP1基因可提高水稻多种非生物胁迫抗性。

关键词: 水稻, CRISPR/Cas9基因编辑技术, AFP1, 耐逆性

Abstract:

【Objective】It is important to reveal the functions of rice AFP1 in response to abiotic stress, and createnewstress-resistant rice germplasm in innovative ways.【Method】Excellent indica restoring line Huazhan was used as transgenic receptor material to generate afp1 mutants usingCRISPR/Cas9 system, and abiotic stress tolerances of afp1 knockout lines were preliminaryanalyzed. 【Results】The editing efficiency of AFP1 in two targets were 66.67% and 75.00%, respectively.In all mutant plants, only insertions and deletions occurred.About 90% of mutant bases were small fragments,with a lengthless than 5bp.Six homozygous mutants without genetically modified ingredients were generated.Under normal conditions, plant height and seed setting rate of afp1 mutants were reduced, but effective tiller number per plant and panicle length were significantly increased. Interestingly, the yield per plant of mutants changed between-4.06%and +11.75%. Compared with the wild type, the ABA sensitivity and water loss rate of afp1 mutants were obviously decreased. Drought, heat and osmotic stress resistances were significantly improved.【Conclusion】MutationofAFP1genecouldimprovemultiple abiotic stress resistances in rice.

Key words: rice, CRISPR/Cas9 gene editing technology, AFP1, abiotic stresstolerance

周天顺, 余东, 刘玲, 欧阳宁, 袁贵龙, 段美娟, 袁定阳. 利用CRISPR/Cas9技术编辑AFP1基因提高水稻耐逆性[J]. 中国水稻科学, 2021, 35(1): 11-18.

Tianshun ZHOU, Dong YU, Ling LIU, Ning OUYANG, Guilong YUAN, Meijuan DUAN, Dingyang YUAN. CRISPR/Cas9-mediatedEditing of AFP1Improves Rice Stress Tolerance[J]. Chinese Journal OF Rice Science, 2021, 35(1): 11-18.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study.

名称 Name	正向引物 Forward primer (5' - 3')	反向引物 Reverse primer (5' - 3')
AFP1-T₁	ggcaCTGTGCGGCATCGGCAAGGG	aaacCCCTTGCCGATGCCGCACAG
AFP1-T₂	gccgCTGTCGCTCGGCGGCCGGTT	aaacAACCGGCCGCCGAGCGACAG
AFP1-J	GGCGGATTACTCGCTCGGTCAGTTCT	TGGAGAGAAGCGATGACGCACCTTCAAG
Hpt	GACAGCGTCTCCGACCTGAT	CATCGCCTCGCTCCAGTCAAT
Cas9	CTCTTCCTTCCAAGTACGTG	GAAAGGTCGATACGAGTCTC
OT₁-1	CAAACGCCAAGGCGGGAATGCAG	GTGCGCCGCGGCGCAGCTGTTG
OT₁-2	GATCCATGTGGGCCCCACATGTC	CGCTGGTCCATCCTCTCGATC
OT₁-3	GAAGTTCTTGCCGATGACCTCG	GACGAGTGAAGAGGAGGAGAC
OT₂-1	CAATCAGTGTAAGCTTGCTCTAG	GGTGAAGTCACACAATTACTG
OT₂-2	CTTCTTGCTTGGAGGAGCATG	CTCAGCTGCAACCTGTTAACG
OT₂-3	CAAGGAGCGCCGTGTACCTCG	GTAGTCCAGCTTCAGCTTCAC

表2 T0代突变体突变基因型比率

Table 2 Mutant genotype ratios of T0 mutations.

靶位点 Target	阳性植株Positive plants	突变率 Mutant ratio/%	突变基因型比率Mutant genotype ratio/%
靶位点 Target	阳性植株Positive plants	突变率 Mutant ratio/%	纯合突变率 Homozygote ratio	杂合突变率Heterozygote ratio	双等位突变率 Bi-allele ratio	嵌合突变率 Chimera ratio
靶点1 Target 1	12	66.7	12.5	12.5	75.0	0.0
靶点2 Target 2	12	75.0	22.2	22.2	44.4	11.1

图1 利用CRISPR/Cas9技术定向突变OsAFP1基因 A-敲除靶点设计; B-CRISPR/Cas9敲除载体,箭头代表扩增引物。

Fig. 1. Directed mutation of OsAFP1 using CRISPR/Cas9 technology. A, Design of mutated sites; B, Schematic diagram of CRISPR/Cas9 construction; Arrows represent amplification primers.

图2 突变体特征分析

Fig. 2. Analysis of mutation type.

图3 T2代纯合敲除突变体的突变类型长方形代表推测蛋白;红色区域为突变蛋白序列,下方为相应蛋白序列;倒三角形和蓝色区域均表示靶位点。WT-野生型;KO1~KO6代表6个不含外源基因的纯合突变株系。

Fig. 3. Mutated types of T2 homozygous mutants. Rectangles are putative mutated proteins; Red shades indicate mutated protein regions and letters below are putative protein sequences;Inverted triangle and blue area represent target sites.WT, Wild type; KO1-KO6 indicate six homozygous mutants without exogenous genes.

图 4 afp1敲除突变体和野生型的表型比较 A-整体株型;B-穗部性状;C-株高;D-穗长;E-有效分蘖数;F-结实率;G-单株产量;均值±标准误,n=3;*和**分别表示突变体与野生型的差异达0.05和0.01显著水平（t检验）。HZ代表野生型华占,KO代表敲除突变体。

Fig. 4. Phenotypic comparisons of afp1 knockdown mutants and the wildtype. A, Plant architectures; B, Panicle traits; C, Plant height; D, Panicle length; E, Effecttivepanicle per plant; F, Seed setting rate; G, Yield per plant;Mean±SE, n=3; * and ** represent significant difference between the mutant and the wild type at the 0.05 and 0.01 levels by t-test, respectively.HZ, Wild type Huazhan; KO, afp1knockdown mutants.

图5 野生型和突变体ABA敏感性分析 A-3μmol/L ABA处理后的幼苗; B-ABA处理后幼苗的株高和根长; C-ABA处理后幼苗根和地上部分的鲜质量; D-3μmol/L ABA处理后种子的萌发率;均值±标准误,n=3;*和**表示在0.05和0.01水平上的差异（t检验）。

Fig. 5. ABA sensitivity of WT and mutants. A, Phenotype of seedlingsafter 3μmol/L ABA treatment; B, Length of root and shootafter 3μmol/L ABA treatment; C, Fresh weight of seedlings after 3μmol/L ABA treatment; D, Germination rate of seeds after 3μmol/L ABA treatment;Mean±SE, n=3; * and ** represent significant difference at the 0.05 and 0.01 levels by t-test, respectively.

图6 耐旱性和耐渗透性鉴定 A-干旱处理后幼苗表型;B-干旱处理后存活率;C-渗透胁迫处理后幼苗表型;D-水稻离体叶片水分散失率;E-渗透胁迫处理后幼苗鲜重;F-渗透胁迫处理后幼苗株高和根长;均值±标准误,n=3;*和**分别表示在0.05和0.01水平上的差异（t检验）。

Fig. 6. Identification of drought and osmotic stress tolerances. A, Phenotype of seedlings after drought treatment; B, Survival rate of seedlings after drought treatment; C, Phenotype of seedlings after osmotic stress treatment;D, Water loss rate of rice detached leaves;E, Fresh weight of seedlings after osmotic stress treatment; F, Length of root and shoot after osmotic stress treatment;Mean±SE, n=3; * and ** represent significant difference at the 0.05 and 0.01 levels by t-test, respectively.

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