Orientation Improvement of Blast Resistance in Rice via CRISPR/Cas9 System

doi:10.16819/j.1001-7216.2019.9043

Abstract

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

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

CLC Number:

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.

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.

Figures/Tables 12

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.

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

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

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

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)

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.

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.

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).

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.

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.

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.

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.

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