利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性

doi:10.16819/j.1001-7216.2019.9043

摘要/Abstract

摘要：

【目的】CRISPR/Cas9基因编辑技术是作物遗传改良的有效工具。本研究通过对水稻Pita、Pi21和ERF922稻瘟病相关基因进行定点编辑,以期获得能够稳定遗传的抗稻瘟病水稻材料。【方法】利用CRISPR/Cas9基因编辑技术,以Pita、Pi21和ERF922为靶基因,构建共编辑载体pC1300-2×35S::Cas9-g^Pita-g^Pi21-g^ERF922 ,用农杆菌转化长粒粳稻恢复系L1014,筛选获得稳定遗传的纯合突变体用于稻瘟病抗性鉴定。【结果】在T₀代转基因株系中,Pita、Pi21和ERF922突变频率分别为75%、85%和65%,突变基因型多为双等位突变。筛选到的不含T-DNA成分的T₁代能够稳定遗传给T₂代,并从中获得Pi21单突变纯合株系及Pita、Pi21和ERF922的三突变纯合株系。稻瘟病抗性鉴定结果表明,与野生型相比,突变株系的抗性显著提高。同时,接种后纯合突变体株系内水杨酸、茉莉酸和乙烯等信号转导途径相关基因的表达量均上调。据此,我们推测纯合突变株系对稻瘟病的抗性增强可能与其对稻瘟病菌的响应被激活有关。【结论】利用CRISPR/Cas9技术获得了能够稳定遗传和具有较高稻瘟病抗性的纯合突变株系,为水稻稻瘟病抗性改良提供了良好的材料。

关键词: 水稻, CRISPR/Cas9, Pita, Pi21, ERF922, 稻瘟病抗性

Abstract:

【Objective】 CRISPR/Cas9 gene editing is an effective biotechnology for crop genetic improvement. This study aims to edit Pita, Pi21 and ERF922 genes for obtaining valuable and stable genetic materials with resistance to rice blast. 【Method】The target genes Pita, Pi21 and ERF922 were selected for constructing the pC1300-2×35S::Cas9-g^Pita-g^Pi21-g^ERF922 expression vector by CRISPR/Cas9 gene editing technology, and transformed by Agrobacterium-mediated method into long-grain japonica rice restore lines L1014. Stable genetic mutant lines were selected and their resistance to rice blast was identified. 【Result】 In the T₀ transgenic lines, the mutation rates of Pita, Pi21 and ERF922 were 75%, 85% and 65%, respectively, and most of the mutant lines are biallelic mutations. The homozygous mutation lines without T-DNA components within the screened T₁ were selected and transferred to T₂ stably. Evaluation analysis of resistance to rice blast suggested that the mutant lines showed significantly higher resistance to M. oryzae compared with the wild type. The expression level of defense-related genes involved in signaling pathways of salicylic acid, jasmonic acid and ethylene metabolisms was up-regulated in the mutant lines compared to the wild type after inoculation of the physiological races of M. oryzae. Therefore, we hypothesized that the increased resistance of homozygous mutant lines to rice blast might be related to the activation of the response to M. oryzae. 【Conclusion】The homozygous mutant lines with stable inheritance and high blast resistance were obtained by CRISPR/Cas9 system, which would provide ideal materials for improving blast resistance in rice.

Key words: rice, CRISPR/Cas9, Pita, Pi21, ERF922, blast resistance

中图分类号:

徐鹏, 王宏, 涂燃冉, 刘群恩, 吴玮勋, 傅秀民, 曹立勇, 沈希宏. 利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性[J]. 中国水稻科学, 2019, 33(4): 313-322.

Peng XU, Hong WANG, Ranran TU, Qunen LIU, Weixun WU, Xiumin FU, Liyong CAO, Xihong SHEN. Orientation Improvement of Blast Resistance in Rice via CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2019, 33(4): 313-322.

图/表 12

图1 Pita、Pi21和ERF922靶点位置带下划线的字母为起始密码子,加粗字母为PAM序列。

Fig. 1. Schematic diagram of the targeted sites in Pita, Pi21 and ERF922. The underlined letters are the initiation codons. The bold letters are the protospacer adjacent motif (PAM) sequences.

表1 本研究所用引物

Table 1 Primers used in this study.

引物名称 Primer name	引物序列 Sequence(5'-3')	引物名称 Primer name	引物序列 Sequence(5'-3')
Pita-g-F	GGCACCATGGCGCCGGCGGTCATTGC	Pi21-F	CTGAATTCCACGGGAATTGC
Pita-g-R	AAACGCAATGACCGCCGGCGCCATGG	Pi21-R	CTCCTCTGCGTTCATATTC
Pi21-g-F	GGCAGGTATATTGGTCATCTTGGTGG	ERF922-F	GCGACAACATCACGCATCGCC
Pi21-g-R	AAACCCACCAAGATGACCAATATACC	ERF922-R	TCAGGCCAAGTGCCTACTGCAA
ERF922-g-F	GGCAGTGGACGTGTCTCTGTCGCTGG	Hyg-F	GCTGTTATGCGGCCATTGTC
ERF922-g-R	AAACCCAGCGACAGAGACACGTCCAC	Hyg-R	GACGTCTGTCGAGAAGTTTC
Pita-F	CAGCCTCTCTGCATGATTTC	Cas9-F	ACCAGACACGAGACGACTAA
Pita-R	TCTAGCAGGTGTCGGAGATC	Cas9-R	ATCGGTGCGGGCCTCTTC

图2 表达载体构建流程

Fig. 2. Flow diagram of the expression vector construction.

表2 对水稻防卫相关基因进行qRT-PCR分析所用的引物

Table 2 Primers used for the qRT-PCR of rice defense-related genes.

基因 Gene	前引物 Forward primer	后引物 Reverse primer
OsActin	GTGGACGTACTACTGGTATTGT	GAATCAGTCAGATCCCTACCAG
OsPR1a	GGCCAATCTCCCTACTGATTAA	GCATAAACACGTAGCATAGCAT
OsPR10	CAGTGGTCAGTAGAGTGATCAG	GGGTTAAGCTTCATGGTGTAGA
OsAOS2	CAATACGTGTACTGGTCGAATG	CTTATTGCATATGCGTAGGACG
OsPAL1	GACCCTGTATTTTCTTCGTTCG	AGTAGCAATACTTTCACCCCAA
OsRaP2.6	AGAGAAAACCAGAAAAACGCTG	TGCTTTGGGGATGGAATATGTA
β-1,3-glucanase	GGATCGGAGGAGTGTAGATAGA	GCAGAAAAGAAGGCAAAGTATACG
qPita	CAGCAACTAACGAGGCATAATC	AGGTAGTAGTCATCTAGCAGGT
qPi21	GGCAAGATCATCAAGGAGATCC	CTTGGGCTTCTCGCAGTGA
qERF922	TGGACGTGTCTCTGTCGCT	GACGTCGAAGAGGACCATGTC

表3 T0突变体突变基因型和突变类型频率

Table 3 Ratios of mutant genotype and mutation types in T0 plants.

基因 Gene	株数 No. of plants	突变基因型比率Ratio of mutation genotypes / %			突变类型比率Ratio of mutation types / %
基因 Gene	株数 No. of plants	纯合突变率 Homozygous	杂合突变率 Heterozygous	双等位突变率 Bi-allele	碱基插入 Insert	碱基缺失 Deletion	碱基替换 Substitution	替换和缺失 Substitution and deletion
Pita	17	41.2(7/17)	0.0(0/17)	47.1(8/17)	73.5(25/34)	14.7(5/34)	0.0(0/34)	0.0(0/34)
Pi21	17	35.3(6/17)	5.9(1/17)	58.8(10/17)	8.8(3/34)	85.3(29/34)	2.9(1/34)	0.0(0/34)
ERF922	17	5.9(1/17)	17.7(3/17)	64.7(11/17)	17.6(6/34)	50.0(17/34)	8.8(3/34)	2.9(1/34)

图3 PCR筛选T1代无转基因成分突变株系 M-DNA标记; 1-双蒸水; 2-野生型; 3-阳性对照; 4~24-T1突变单株; P1-引物Hyg-F和Hyg-R; P2-引物Cas9-F和Cas9-R。

Fig. 3. Screening of transgenic-free mutant plants by PCR in T1. M, DNA marker; 1, ddH2O; 2, Wild type; 3, Positive control; Lines 4 to 24, T1 mutation plants; P1, Primer Hyg-F and Hyg-R; P2, Primer Cas9-F and Cas9-R.

图4 T2代的3种纯合突变类型蓝色字母表示PAM序列;红色字母表示碱基插入;红色连字符表示碱基缺失;下划线表示起始密码子。D2101和D2102是Pi21单基因发生突变,分别为缺失1 bp和4 bp,D0301是Pita、Pi21和ERF922三基因共突变,Pita和Pi21突变类型是插入1 bp,ERF922的突变类型是缺失23 bp。

Fig. 4. Three mutation types of homozygotes in T2. The PAM sequence is highlighted in blue and insertions are represented by red letters. The deletions are shown by red hyphens. The initiation codon is highlighted by underline in black. D2101 and D2102 are monogenic mutation of Pi21, mutations with 1 bp and 4 bp deletions, respectively. D0301 is co-mutation of Pita, Pi21 and ERF922, mutations with 1 bp insertion in Pita and Pi21, respectively, and the mutation with 23 bp deletion in ERF922.

图5 Pita、Pi21和ERF922在纯合突变系的表达量分析*和**分别表示野生型与纯合突变体的差异达0.05和0.01显著水平(t检验)。

Fig. 5. Expression analyses of Pita, Pi21 and ERF922 in homozygous mutant plants. * and ** represent significant difference between the wild type and the homozygous lines at the 0.05 and 0.01 levels, respectively(t-test).

图6 Pita、Pi21和ERF922纯合突变系及野生型的氨基酸序列分析 A为D2101和D2102纯合突变株系中Pi21氨基酸序列的比对;B, C和D分别为D0301纯合突变株系中Pita、Pi21和ERF922的氨基酸序列比对。

Fig. 6. Pita, Pi21 and ERF922 gene amino acid sequences in homozygous mutant lines and the wild type. A indicate the alignment of Pi21 gene amino acid sequences in homozygous mutant lines D2101 and D2102; B, C and D show the alignment of Pita, Pi21 and ERF922 gene amino acid sequences in homozygous mutant line D0301.

图7 纯合突变体与对照株的接种鉴定结果 A-纯和突变株系和野生型在苗期的接种表型;B-野生型和纯合突变株系的相对病斑面积(均值±标准误,n=3)。柱上不同小写字母表示野生型与纯合株的差异达0.01显著水平(t检验)。LTH-丽江新团黑谷;WT-L1014野生型;D2101和D2102-Pi21纯合株系;D0301-Pita、Pi21和ERF922纯合株系。

Fig. 7. Identification of homozygous mutant lines and the control varieties after inoculation. A, Homozygous mutant lines and wild type inoculated at the seedling stage. B, Statistical lesion area of wild type and homozygous mutant lines(Mean±SE, n=3). Different lowercase letters above the bars indicate significant difference between the mutant and the wild type at the 0.01 level(t-test). LTH, Lijiangxintuanheigu; WT, Wild type; D2101 and D2102, Homozygous lines of Pi21; D0301, Homozygous lines of Pita, Pi21 and ERF922.

图8 接种稻瘟菌后不同时期野生型(L1014, WT)和纯合突变株中防卫相关基因的相对表达量分析(均值±标准误,n=3) *和**表示野生型和纯合株系在接种后同一时期0.05和0.01水平上的差异。

Fig. 8. Relative expression levels of defense-related genes in the wild type (L1014, WT) and the homozygous mutant lines inoculated with M. coryza (Mean±SE, n=3). * and ** represent significant difference between the wild type and the homozygous lines at the 0.05 and 0.01 levels by t-test with the same time point.

图9 野生型(L1014, WT)和纯合突变株(D0301)在接种稻瘟菌后不同时期中防卫相关基因的相对表达量(均值±标准误,n=3) *和**表示在接种后同一时期野生型与纯合株系的差异达0.05和0.01显著水平。

Fig. 9. Relative expression level of defense-related genes in the wild type(L1014) and the homozygous mutant line D0301, inoculated with M. coryza (Mean±SE, n=3). *and** represent significant difference between the wild type and the homozygous lines at the 0.05 and 0.01 levels by t-test at the same stage, respectively.

参考文献 35

[1]	Liu J, Wang X, Mitchell T, Hu Y, Liu X, Dai L, Wang G L.Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction.Mol Plant Pathol, 2010, 11(3): 419-427.
[2]	Borrelli V, Brambilla V, Rogowsky P, Marocco A, Lanubile A.The enhancement of plant disease resistance using CRISPR/Cas9 technology. Front Plant Sci, 2018, 9: 1245.
[3]	Wilson R A, Talbot N J.Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.Nat Rev Microbiol, 2009, 7(3): 185-195.
[4]	Xie K, Yang Y.RNA-guided genome editing in plants using a CRISPR-Cas system.Mol Plant, 2013, 6(6): 1975-1983.
[5]	Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol, 2013, 31(8): 686-688.
[6]	Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F.Multiplex genome engineering using CRISPR/Cas systems.Science, 2013, 339(6121): 819-823
[7]	Islam W.CRISPR-Cas9: An efficient tool for precise plant genome editing.Mol Cell Probes, 2018, 39: 47-52.
[8]	Vriend L E M, Prakash R, Chen C, Vanoli F, Cavallo F, Zhang Y, Jasin M, Krawczyk P M. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks.Nucleic Acids Res, 2016, 44(11): 5204-5217.
[9]	Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant, 2013, 6(6): 2008-2011.
[10]	Shan Q, Wang Y, Li J, Gao C.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Protoc, 2014, 9(10): 2395-2410.
[11]	Zhang J, Zhang H, Botella J R, Zhu J K.Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. J Integr Plant Biol, 2018, 60(5): 369-375.
[12]	Jiang W, Zhou H, Bi H, Fromm M, Yang B Weeks D P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.Nucleic Acids Res, 2013, 41(20): e188.
[13]	Fauser F, Schiml S, Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J, 2014, 79(2): 348-359.
[14]	Jiang W, Yang B Weeks D P. Efficient CRISPR/Cas9- mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations.PLoS One, 2014, 9(6): e99225.
[15]	Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol, 2014, 32(9): 947-951.
[16]	Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D.Simultaneous modification of three homologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat.Plant J, 2017, 91(4): 714-724.
[17]	Shen L, Hua Y, Fu Y, Li J, Liu Q, Jiao X, Xin G, Wang J, Wang X, Yan C, Wang K.Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice.Sci China Life Sci, 2017, 60(5): 506-515.
[18]	Fei Y Y, Yang J, Wang F Q, Fan F J, Li W Q, Wang J, Xu Y, Zhu J Y, Zhong W G.Production of two elite glutinous rice varieties by editing Wx gene.Rice Sci, 2019, 26(2): 118-124.
[19]	Fukuoka S, Okuno K.QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice.Theor Appl Genet, 2001, 103(2-3): 185-190.
[20]	Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M.Loss of function of a proline-containing protein confers durable disease resistance in rice. Science, 2009, 325(5943): 998-1001.
[21]	Liu D, Chen X, Liu J, Ye J, Guo Z.The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.J Exp Bot, 2012, 63(10): 3899-3911.
[22]	Bryan G T, Wu K S, Farrall L, Jia Y, Hershey H P, McAdams S A, Faulk K N, Donaldson G K, Tarchini R, Valent B. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell, 2000, 12(11): 2033-2046.
[23]	Jia Y, Martin R.Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance.Mol Plant Microbe Interact, 2008, 21(4): 396-403.
[24]	Tu Z, Yang W, Yan S, Guo X, Li X.CRISPR/Cas9: A powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases.Mol Neurod, 2015, 10: 35.
[25]	Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B.Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.Development, 2013, 140(24): 4982-4987.
[26]	Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.Plant Biotechnol J, 2018, 16(1): 176-185.
[27]	Yu Z, Chen Q, Chen W, Zhang X, Mei F, Zhang P, Zhao M, Wang X, Shi N, Jackson S, Hong Y.Multigene editing via CRISPR/Cas9 guided by a single-sgRNA seed in Arabidopsis.J Integr Plant Biol, 2018, 60(5): 376-381.
[28]	Jakočiu̅nas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, Jendresen C B, Skjødt M L, Nielsen A T, Borodina I, Jensen M K, Keasling J D. CasEMBLR: Cas9-facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.ACS Synthetic Biol, 2015, 4(11): 1226-1234.
[29]	沈兰. 基于CRISPR/Cas9的水稻多基因编辑及其在育种中的应用. 扬州: 扬州大学, 2017.
	Shen L.CRISPR/Cas9-mediated multiples genome editing in rice (Oryza sativa) and the application in rice breeding. Yangzhou: Yangzhou University, 2017. (in Chinese with English abstract)
[30]	王芳权, 范方军, 李文奇, 朱金燕, 王军. 利用CRISPR/Cas9技术敲除水稻Pi21基因的效率分析. 中国水稻科学, 2016, 30(5): 469-478.
	Wang F Q, Fan F J, Li W Q, Zhu J Y, Wang J.Knock-out efficiency analysis of Pi21 gene using CRISPR/Cas9 in rice.Chin J Rice Sci, 2016, 30(5): 469-478. (in Chinese with English abstract)
[31]	杨海河, 毕冬玲, 张玉, 邹小维, 高晓庆, 袁正杰, 曲海艳, 何海燕, 瞿绍洪. 基于CRISPR/Cas9技术的水稻pi21基因编辑材料的创制及稻瘟病抗性鉴定. 分子植物育种, 2017, 15(11): 4451-4465.
	Yang H H, Bi D L, Zhang Y, Zou X W, Gao X Q, Yuan Z J, Qu H Y, He H Y, Qu S H.Generation of rice pi21 gene editing lines based on CRISPR/Cas9 technology and evaluation of their blast resistance.Mol Plant Breed, 2017, 15(11): 4451-4465. (in Chinese with English abstract)
[32]	Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y, Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922.Plos One, 2016, 11(4): e0154027.
[33]	Zhang Y, Zhao J, Li Y, Yuan Z, He H, Yang H, Qu H, Ma C, Qu S.Transcriptome analysis highlights defense and signaling pathways mediated by rice pi21 gene with partial resistance to Magnaporthe oryzae.Front Plant Sci, 2016, 7: 1834.
[34]	Wamaitha M J, Yamamoto R, Wong H L, Kawasaki T, Kawano Y, Shimamoto K. OsRap2.6 transcription factor contributes to rice innate immunity through its interaction with Receptor for Activated Kinase-C1(RACK1). Rice, 2012, 5: 35.
[35]	Zhao H, Wang X, Jia Y, Minkenberg B, Wheatley M, Fan J, Jia M H, Famoso A, Edwards J D, Wamishe Y, Valent B, Wang G, Yang Y.The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance.Nat Commun, 2018, 9(1): 2039.