稻瘟病抗性基因Pi-d2、Pi-d3和Pigm不同敲除突变体的抗性评价

张柱坚; 陈子强; 顾建强; 田大刚

doi:10.19303/j.issn.1008-0384.2018.12.001

稻瘟病抗性基因Pi-d2、Pi-d3和Pigm不同敲除突变体的抗性评价

doi: 10.19303/j.issn.1008-0384.2018.12.001

福建省农业遗传工程重点实验室/福建省农业科学院生物技术研究所, 福建福州 350003

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2017R1019-10

福建省科技计划项目——省属公益类科研院所基本科研专项 2018R1019-9

详细信息

作者简介:
张柱坚(1994-), 男, 硕士研究生, 研究方向:分子植物育种(E-mail:723926289@qq.com)

通讯作者:
田大刚(1978-), 男, 副研究员, 研究方向:水稻分子免疫和抗病育种(E-mail:tdg@fjage.org)

中图分类号: S511
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出版历程
- 收稿日期: 2018-11-13
- 修回日期: 2018-12-10
- 刊出日期: 2018-12-28

Resistance on Rice Blast of Knockout Mutants of Pi-d2, Pi-d3 and Pigm

Fujian Key Laboratory of Genetic Engineering for Ariculture/Biotechnology Institute of Fujian Academy of Agricutural Sciences, Fuzhou, Fujian 350003, China

摘要

摘要: 谷丰B是一个广谱高抗稻瘟病水稻保持系，含有稻瘟病抗性基因Pi-d2，Pi-d3和Pigm。为了进一步明确3个抗性基因在谷丰B中的作用，本研究利用CRISPR/Cas9多基因编辑系统构建了共敲除Pi-d2+Pi-d3+Pigm基因载体。通过遗传转化试验以及DNA测序，T₀代植株获得多种突变体组合类型。对其中的Pi-d2、Pigm、Pi-d2+Pi-d3、Pi-d2+Pigm、Pi-d2+Pi-d3+Pigm等5种纯合突变体的T₁代株系进行稻瘟病室内接种鉴定，研究结果表明，Pi-d2、Pi-d3和Pigm分别对86/501-3、KJ201/CHE86061和86/CHE86061/501-3的菌株存在显著的抗性效应，Pi-d2+Pi-d3，Pi-d2+Pigm和Pi-d2+Pi-d3+Pigm的敲除株系的抗性不完全等同于单个基因的简单叠加，其中Pi-d2+Pi-d3+Pigm的突变体对菌株CHL768表现完全不同于其他类型突变体的感病性。上述结果为解析广谱高抗稻瘟病水稻材料谷丰B的抗性遗传机理提供重要信息，也为水稻稻瘟病抗病育种研究提供参考。
- CRISPR/Cas9 /
- 水稻 /
- 稻瘟病 /
- 抗性基因
Abstract: Gufeng B is a broad-spectrum, high resistance maintainer line to rice blast. Its blast resistance genes, Pi-d2, Pi-d3 and Pigm, were identified using the molecular marker technology. To study and verify the role of these genes, their multiple sites editing vectors were constructed by the high-efficiency and designated-editing CRISPR/Cas9 system. The vectors were subsequently transformed to Gufeng B by means of the agrobacterium-mediated method. Through the mutant analysis targeting mutation types, a variety of combinations in the T₀ plants was found. The indoor inoculation of the T₁ generation of 5 homozygous mutants indicated that Pi-d2, Pi-d3 and Pigm were significantly resistant to 86/501-3, KJ201/CHE86061 and 86/CHE86061/501-3. The resistance of the knockout lines of Pi-d2+Pi-d3, Pi-d2+Pigm and Pi-d2+Pi-d3+Pigm showed a simple superposition that was not completely equivalent to a single gene. The mutant of Pi-d2+Pi-d3+Pigm had a completely different sensitivity to CHL768 from the other mutants. This study unveiled genetic information on the blast resistance of Gufeng B of value for rice breeding.
- CRISPR/Cas9 /
- rice /
- rice blast /
- resistant gene

HTML全文

图 1 Pigm、Pi-d2和Pi-d3多基因编辑载体构建

注：(A)Pigm、Pi-d2和Pi-d3多基因编辑载体结构。(B)gRNA表达盒电泳检测。M为DL 2 000 Plus DNA Marker；1为u6b-Pigm-gRNA表达盒；2为u6a-Pi-d2-gRNA表达盒；3为u6b-Pi-d2-gRNA表达盒；4为u6a-Pi-d3-gRNA表达盒；5为u6b-Pi-d2-gRNA表达盒。(C)Pigm、Pi-d2和Pi-d3多基因编辑载体菌落PCR检测。M为DL 2 000 Plus DNA Marker；1为不含模板的空白对照；2~11为单菌落PCR产物。

Figure 1. Construction of Pigm, Pi-d2 and Pi-d3 multi-gene editing vectors

下载: 全尺寸图片幻灯片

图 2 靶位点突变序列分析

注：(A)PCR鉴定转基因阳性株系。M为DL 2 000 Plus DNA Marker；1为阳性对照；2为不含模板的空白对照；3为以野生型谷丰B植株DNA为模板的PCR产物；4~23为以阳性苗DNA为模板的PCR产物。(B)T₀代纯合突变类型分析结果。下划线部分为PAM区；横线为碱基缺失，箭头为碱基插入。(C)T₁代植株突变类型测序结果。下划线部分为PAM区。

Figure 2. Analysis on mutation sequences of target spots

下载: 全尺寸图片幻灯片

表 1 本研究所用的引物

Table 1. Primers applied

引物名称	引物序列	用途
Pigm-u6b-F	GTTGGATACGGATAGCGATCCGG	构建Pigm敲除载体
Pigm-u6b-R	AAACCCGGATCGCTATCCGTATC
Pi-d2-u6a-F	GCCGAAGTCATCTGAATGGGTGAC	构建Pi-d2敲除载体
Pi-d2-u6a-R	AAACGTCACCCATTCAGATGACTT
Pi-d2-u6b-F	GTTGAATGCCTTCCTGCAGTCAGG	构建Pi-d2敲除载体
Pi-d2-u6b-R	AAACCCTGACTGCAGGAAGGCATT
Pi-d3-u6a-F	GCCGCCTCTTTTCTGAGATCCGGG	构建Pi-d3敲除载体
Pi-d3-u6a-R	AAACCCCGGATCTCAGAAAAGAGG
Pi-d3-u6b-F	GTTGCCGCGTTCGTCAAGCAGGTG	构建Pi-d3敲除载体
Pi-d3-u6b-R	AAACCACCTGCTTGACGAACGCGG
Cas9-Pigm-F	GAAAGACGAACTATTAAAGG	Pigm敲除载体测序鉴定
Cas9-Pigm-R	TGATCCAGAGAACTGGGGCC
Cas9-Pi-d2-F	GCTCTGTCTTTGGCTTTGGT	Pi-d2敲除载体测序鉴定
Cas9-Pi-d2-R	GCTCTGTCTTTGGCTTTGGT
Cas9-Pi-d3-F	GTGTTGTGGGCTCACTAATC	Pi-d3敲除载体测序鉴定
Cas9-Pi-d3-R	CAGCAAATCCCTCTTCTTCT
HPT-F	AAGCTGCATCATCGAAATTG	潮霉素鉴定
HPT-R	TCGTTATGTTTATCGGCACT

下载: 导出CSV

表 2 突变株系与对照株系的稻瘟病抗性鉴定结果

Table 2. Blast-resistance of mutant and control lines as evaluated by fungal inoculation

材料抗性等级	菌株
材料抗性等级	KJ201	86	CHE86061	501-3	CHL768	CHL2110
谷丰B	0	0	0	0	0	4
GFB234-5(Pi-d2)	0	3	0	5	0	4
GFB234-3(Pigm)	0	4	5	5	0	4
GFB234-11(Pi-d2+ Pi-d3)	2	3	4	5	0	4
GFB234-8(Pigm+ Pi-d3)	1	4	4	5	0	4
GFB234-15(Pigm+Pi-d2+ Pi-d3)	4	4	4	5	3	4

下载: 导出CSV

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