CRISPR/Cas9-mediatedEditing of AFP1Improves Rice Stress Tolerance

doi:10.16819/j.1001-7216.2021.0503

Abstract

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

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

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.

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.

Figures/Tables 8

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

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

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.

Fig. 2. Analysis of mutation type.

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.

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.

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.

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.

References 22

[1]	Akram R, Fahad S, Masood N, Fahad S, Masood N, Rasool A, Ijaz M, Ihsan M Z, Maqbool M M, Ahmad S, Hussain S, Ahmed M, Kaleem S, Sultana S R, Mubeen M, Saud S, Kamran M, Nasim W.Advances in Rice Research for Abiotic Stress Tolerance[M]. Cambridge: Woodhead Publishing, 2019: 69-85.
[2]	Boyer J S.Plant productivity and environment[J]. Science, 1982, 218: 443-448.
[3]	Pauwels L, Barbero G F, Geerinck J, Tilleman S, Grunewald W, Pérez A C, Chico J M, Bossche R V, Sewell J, Gil E, García-Casado G, Witters E, Inzé D, Long J A, De Jaeger G, Solano R, Goossens A.NINJA connects the co-repressor TOPLESS to jasmonatesignaling[J]. Nature, 2010, 464(7289): 788-791.
[4]	Huang M D, Wu W L.Overexpression of TMAC2, a novel negative regulator of abscisic acid and salinity responses, has pleiotropic effects in Arabidopsis thaliana[J]. Plant Molecular Biology, 2007, 63(4): 557-569.
[5]	Chang G X, Wang C T, Kong X X, Chen Q, Yang Y P, Hu X Y.AFP2 as the novel regulator breaks high-temperature-induced seeds secondary dormancy through ABI5 and SOM in Arabidopsis thaliana[J]. Biochemical and Biophysical Research Communications, 2018, 501(1): 232-238.
[6]	Chang G X, Yang W J, Zhang Q L, Huang J L, Yang Y P, Hu X Y. ABI5-BINDING PROTEIN2coordinates CONSTANS to delay flowering by recruiting the transcriptional corepressor TPR2[J]. Plant Physiology, 2019, 179(2): 477-490.
[7]	安敏敏, 杨立明, 罗玉明. AFP2基因调控拟南芥茉莉酸合成与开花时间的分析[J]. 分子植物育种, 2019, 17(10): 3259-3266.
	An M M, Y L M, Luo Y M. Analysis of the role of AFP2gene in regulating jasmonic acid biosynthesis and flowering time of Arabidopsis[J]. Molecular Plant Breeding, 2019, 17(10): 3259-3266. (in Chinese with English abstract)
[8]	Tang N, Ma S Q, Zong W, Yang N, Lv Y, Yan C, Guo Z, Li J, Li X, Xiang Y, Song H Z, Xiao J H, Li X H, Xiong L Z.MODD mediates deactivation and degradation of OsbZIP46 to negatively regulate ABA signaling and drought resistance in rice[J]. The Plant Cell, 2016, 28(9): 2161-2177.
[9]	Ma S, Tang N, Li X, Xie Y J, Xiang D H, Fu J, Shen J Q, Yang J, Tu H F, Li X H, Hu H H, Xiong L Z.Reversible histone H2BMonoubiquitinationfine-tunes abscisic acid signaling and drought response in rice[J]. Molecular Plant, 2019, 12(2): 263-277.
[10]	Liu X, Baird W V.Identification of a novel gene, HaABRC5, from Helianthus annuus (Asteraceae) that is upregulated in response to drought, salinity, and abscisic acid[J]. American Journal of Botany, 2004, 91(2): 184-191.
[11]	Wu J, Seng S S, Carianopol C, Sui J J, Yang Q Y, Zhang F Q, Jiang H R, He J N, Yi M F.Cloning and characterization of a novel Gladiolus hybridusAFP family gene (GhAFP-like) related to corm dormancy[J]. Biochemical and Biophysical Research Communications, 2016, 471(1): 198-204.
[12]	Miao C B, Xiao L H, Hua K, Zou C S, Zhao Y, Bressan R A, Zhu J K.Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity[J]. Proceedings of the National Academy of Sciences, 2018, 115(23): 6058-6063.
[13]	Lou D J, Wang H P, Liang G, Yu D Q.OsSAPK2 confers abscisic acid sensitivity and tolerance to drought stress in rice[J]. Frontiers in Plant Science, 2017, 8: 993.
[14]	Cui Y T, Hu X M, Liang G H, Feng A H, Wang F M, Ruan S, Dong G J, Shen L, Zhang B, Chen D D,Zhu L,Hu J,Lin Y J,Guo L B,Matsuoka M,Qian Q. Production of novel beneficial alleles of a rice yield-related QTL by CRISPR/Cas9[J]. Plant Biotechnology Journal, 2020, 18(10): 1987-1989.
[15]	Zhou T S, Yu D, Dong H, Sun Z Z, Tan Y N, Sun X W, Sheng X B, Duan M J, Yuan D Y.Genome-Wide Identification and Expression Profile of NINJA and AFP Genes in Rice[J]. International Journal of Advances in Biology, 2020, 23(1): 171-182.
[16]	Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant, 2015, 8(8): 1274-1284.
[17]	Liang G, Zhang H M, Lou D J, Yu D Q.Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing[J]. Scientific Reports, 2016, 6: 21451.
[18]	Wang Y P, Cheng X, Shan Q W, Zhang Y, Liu J X, Gao C X.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J]. Nature Biotechnology, 2014, 32(9): 947-951.
[19]	Li J Y, Sun Y W, Du J L, Zhao Y D, Xia L Q.Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system[J]. Molecular Plant, 2017, 10(3): 526-529.
[20]	王影, 李相敢, 邱丽娟. CRISPR/Cas9基因组定点编辑中脱靶现象的研究进展[J]. 植物学报, 2018, 53(4): 521-541.
	Wang Y, Li X G, Qiu L J.Research progress in off-target in CRISPR/Cas9 genome editing[J]. Chinese Bulletin of Botany, 2018, 53(4): 521-541. (in Chinese with English abstract)
[21]	Garcia M E, Lynch T, Peeters J, Snowden C, Finkelstein R.A small plant-specific protein family of ABI five binding proteins (AFPs) regulates stress response in germinating Arabidopsis seeds and seedlings[J]. Plant Molecular Biology, 2008, 67(6): 643-658.
[22]	Lopez-Molina L, Mongrand S, Kinoshita N, Chua N H.AFP is a novel negative regulator of ABA signaling that promotes ABI5 protein degradation[J]. Genes & Development, 2003, 17(3): 410-418.