聚合R基因<i>Pigm</i>和非R基因<i>bsr-d1</i>改良水稻稻瘟病抗性

doi:10.7668/hbnxb.20193133

摘要/Abstract

摘要：

综合利用CRISPR/Cas9基因编辑技术和基因芯片技术,聚合主效R基因Pigm和非R基因bsr-d1,以改良优良食味水稻品种水晶3号的稻瘟病抗性。首先利用CRISPR/Cas9基因编辑系统,以Bsr-d1为靶基因构建重组表达载体并通过农杆菌介导法转化水晶3号,筛选获得无T-DNA元件的bsr-d1纯合突变体,包括T插入、G插入、GA缺失、CGCA缺失和CGCAGA缺失5种突变类型。以无T-DNA成分的bsr-d1纯合突变体为母本、以携带Pigm基因的金玉1号为父本杂交、回交、自交,并利用分子育种芯片同时进行Pigm基因和背景辅助选择,最终获得抗病基因(同时携带bsr-d1和Pigm基因)纯合,且背景回复率均在96%以上的水晶3号改良株系(SJ3-G1、SJ3-G2、SJ3-G3、SJ3-G4、SJ3-G5)。稻瘟病抗性鉴定结果表明,各改良株系对稻瘟病生理小种GUY11的叶瘟抗性与野生型相比均显著提高;接种稻瘟病菌后,改良株系叶片中POD活性显著低于野生型对照,H₂O₂含量则显著高于野生型对照。通过CRISPR/Cas9基因编辑技术结合基因芯片技术获得了同时携带bsr-d1和Pigm基因的抗稻瘟病水晶3号改良系。

关键词: 水稻, Pigm, bsr-d1, 稻瘟病抗性, CRISPR/Cas9

Abstract:

In order to improve the blast resistance of Shuijing 3,an excellent food-flavor rice variety,CRISPR/Cas9 gene editing technology combined with gene chip technology were used to pyramid the R gene Pigm and the non-R gene bsr-d1 into Shuijing 3.Firstly,Bsr-d1 was selected as the target gene to construct a recombinant expression vector using the CRISPR/Cas9 gene editing system,and transformed into the excellent food-flavor rice Shuijing 3 by Agrobacterium-mediated method.The homozygous bsr-d1 mutant lines without T-DNA elements,including five mutation types as T insertion,G insertion,GA deletion,CGCA deletion and CGCAGA deletion,were screened out.The japonica line Jinyu 1 containing a broad-spectrum blast resistance gene Pigm was used as the gene donor parent to cross with the homozygous bsr-d1 mutant lines without transgenic components.The Pigm gene was introduced into bsr-d1 mutant lines by cross,backcross and self-cross combing molecular breeding chip to simultaneously perform Pigm gene and background-assisted selection.The improved lines SJ3-G1,SJ3-G2,SJ3-G3,SJ3-G4,SJ3-G5,which were homozygous for the disease resistance genes(carrying both bsr-d1 and Pigm genes)and whose background recovery rates were all above 96%,were finally obtained.The improved strains of Shuijing 3 displayed enhanced leaf blast resistance compared with the wild type in inoculated identification test using Magnaporthe grisea strain GUY11.After inoculation with M.oryzae,the POD activities in the improved strains of Shuijing 3 were significantly lower than that of the wild-type control,while the H₂O₂ contents were significantly higher than that of the wild-type control.The improved Shuijing 3 lines with blast resistance carrying both bsr-d1 and Pigm genes are obtained by CRISPR/Cas9 gene editing technology combined with gene chip technology.

Key words: Rice, Pigm, bsr-d1, Blast resistance, CRISPR/Cas9

王亚, 王越涛, 申关望, 王付华, 王生轩, 白涛, 尹海庆. 聚合R基因Pigm和非R基因bsr-d1改良水稻稻瘟病抗性[J]. 华北农学报, 2022, 37(5): 157-165. doi: 10.7668/hbnxb.20193133.

WANG Ya, WANG Yuetao, SHEN Guanwang, WANG Fuhua, WANG Shengxuan, BAI Tao, YIN Haiqing. Improvement of Rice Blast Resistance by Pyramiding the R Gene Pigm and the Non-R Gene bsr-d1[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 157-165. doi: 10.7668/hbnxb.20193133.

http://www.hbnxb.net/CN/Y2022/V37/I5/157

图/表 7

图1 不同水稻品种中Bsr-d1基因启动子的功能位点分析(A)及CRISPR-Cas9-Bsr-d1重组载体信息(B)

Fig.1 Functional locus of Bsr-d1 gene promoter in different rice varieties(A) and schematic information of the CRISPR-Cas9-Bsr-d1 recombinant vector(B)

图2 34个T₀独立转化体中Hyg基因和Bsr-d1基因的 PCR 检测 Marker.2000 bp DNA标准分子质量;+.Hyg基因的阳性对照;-.双蒸水;1—34.34个T₀ 独立转化体。

Fig.2 PCR detection of Hyg and Bsr-d1 in 34 independent transformants in T₀ generation Marker.2000 bp DNA Marker;+.Positive control of Hyg;-.ddH₂O; 1—34.34 independent transformants in T₀ generation.

图3 水晶3号野生型和突变体的DNA /氨基酸序列比对 A.T₀转基因水晶3号的5 种纯合突变体类型;B.Bsr-d1基因野生型及5种纯合突变类型的氨基酸序列分析:SJ3.野生型,SJ3+T.T碱基插入,SJ3+G.G碱基插入,SJ3-2.GA碱基缺失,SJ3-4.CGCA碱基缺失,SJ3-6.CGCAGA碱基缺失。

Fig.3 DNA/amino acid sequences alignment of wild type and mutants of Shuijing 3 A.Five types of homozygous mutants of Shuijing 3 in T₀ generation;B.Bsr-d1 gene amino acid sequences in the wild type and five homozygous mutant lines: SJ3.Wild type,SJ3+T.T base insertion,SJ3+G.G base insertion,SJ3-2.GA bases deletion,SJ3-4.CGCA bases deletion,SJ3-6.CGCAGA bases deletion.

图4 bsr-d1纯合突变株T₁部分单株中Hyg基因的 PCR 检测 Marker.2000 bp DNA标准分子质量;1—24.24个T₁ 单株。

Fig.4 PCR detection of Hyg in partial bsr-d1 homozygous mutant plants in T₁ generation Marker.2000 bp DNA Marker;1—24.24 independent plants in T₁ generation.

图5 携带Pigm基因的水晶3号bsr-d1纯合突变体(SJ3+T)的遗传背景回复情况蓝色.杂合基因位点;红色.纯合Pigm基因位点。

Fig.5 Genetic background recovery of Shuijing 3 bsr-d1 homozygous mutant(SJ3+T)carrying Pigm gene Blue.Heterozygous gene locus;Red.Homozygous Pigm locus.

图6 水晶3号改良株系及其野生型对照的稻瘟病菌接种鉴定结果

Fig.6 Identification of improved strains of Shuijing 3 and the wild-type control after inoculation with M.coryza

图7 水晶3号改良株系及其野生型接种稻瘟病菌后不同时期叶片的POD活性和H₂O₂含量柱上不同小写字母表示野生型和各改良株系在接种后同一时期0.05水平上差异显著(t测试)。

Fig.7 POD activities and H₂O₂contents in leaves of improved strains of Shuijing 3 and the wild-type control inoculated with M.oryzae at different stages Different lowercase letters above the bars indicate significant difference between the wild type and the improved lines at the 0.05 levels by t-test at the same stage.

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聚合R基因Pigm和非R基因bsr-d1改良水稻稻瘟病抗性

Improvement of Rice Blast Resistance by Pyramiding the R Gene Pigm and the Non-R Gene bsr-d1

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聚合R基因Pigm和非R基因bsr-d1改良水稻稻瘟病抗性

Improvement of Rice Blast Resistance by Pyramiding the R Gene Pigm and the Non-R Gene bsr-d1

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