Identification of Rice Blast Resistance Mutant lmm326 and Preliminary Analysis of Its Regulatory Pathway

doi:10.16819/j.1001-7216.2017.7007 335

Abstract

Abstract:

【Objective】Rice lesion mimic mutants often have a certain resistance to rice blast. To understand the genetic mechanisms behind its blast resistance, we identified the rice lesion mimic mutant lmm326 and analyzed its regulatory pathway. 【Method】 lmm326 was obtained from an EMS-induced Zhonghua 11 mutant bank, and population derived from lmm326/ Zhonghua 11 and lmm326/Dular were used for genetic and fine mapping. 【Result】At 5-leaf stage, there were brown lesions on lower leaves of lmm326 initially. Compared with the wild type(WT), photosynthetic pigment contents, net photosynthetic rate, plant height, seed setting rate, tiller number, and 1000-grain weight of lmm326 were significantly reduced. Evans blue and DAB staining assay indicated that leaves of lmm326 accumulated more dead cells and H2O2. Additionally, lmm326 displayed higher resistance to 4 races of rice blast compared with WT. According to genetic analysis, the phenotype of mutant was controlled by a single recessive gene. Here, we employed map-based cloning approach to finely map LMM326 through F2 population derived from a cross between lmm326 and Dular, and the gene was narrowed to a 38-kb region on the long arm of chr. 1, which harbored six ORFs. Sequence analysis revealed that a single base substitution at position C433T in Os01g0919900 CDS resulted in a substitution of leucine for phenylalanine at position 145. Real-time PCR showed that expression levels of defense-related genes were significantly higher in lmm326 than in WT.【Conclusion】These results demonstrated that the target gene is allelic to OsSSI2, and LMM326 is likely to participate in salicylic acid-signaling pathway, and its mutation activates the defense response.

Key words: rice, rice blast, defense response, lesion mimic

摘要： 目的

绝大多数水稻类病斑突变体中免疫系统被激活可以有效提升其对稻瘟病的抗性,为进一步解析类病斑突变体抗病的分子机制,对类病斑突变体lmm326进行了研究。

方法

lmm326是粳稻中花11通过EMS辐射诱变,经多代自交和回交获得的一个类病斑突变体,并将突变体分别与中花11和Dular杂交获得的F2群体用于遗传分析,采用图位克隆的方法对目的基因进行精细定位。

结果

5叶期时,该突变体下部叶片表面开始出现类病斑表型;与野生型相比,突变体叶片光合色素含量、净光合速率、株高、结实率、单株有效分蘖数、千粒重等显著下降;突变体带病斑叶片中死亡细胞数量及过氧化氢的积累量明显多于野生型;突变体相较于野生型对4个已鉴定的稻瘟病生理小种的抗性明显提高。遗传分析表明该性状由一对单隐性核基因控制。采用图位克隆方法将该基因定位在第1染色体长臂端38 kb的区间内,其中包含6个开放阅读框。测序发现基因Os01g0919900第2外显子上对应CDS的第433位碱基由C突变成T,最终导致其编码蛋白的第145个氨基酸由苯丙氨酸(F)变为亮氨酸(L)。qRT-PCR结果显示,与野生型相比,lmm326中参与水杨酸信号通路的防卫基因显著上调表达。

结论

LMM326与OsSSI2互为等位基因,该基因可能参与负调控水杨酸信号转导途径,其突变激活了水稻体内的防卫反应。

关键词: 水稻, 稻瘟病, 防卫反应, 类病斑

Tingting XU, Ning YU, Yingxin ZHANG, Zhenzhen BI, Weixun WU, Yongrun CAO, Beifang WANG, Yue ZHANG, Dandan XUAN, Daibo CHEN, Xiaodeng ZHAN, Shihua CHENG, Liyong CAO. Identification of Rice Blast Resistance Mutant lmm326 and Preliminary Analysis of Its Regulatory Pathway[J]. Chinese Journal OF Rice Science, 2017, 31(4): 335-344.

徐婷婷, 余宁, 张迎信, 毕真真, 吴玮勋, 曹永润, 王备芳, 张越, 轩丹丹, 陈代波, 占小登, 程式华, 曹立勇. 水稻抗稻瘟病突变体lmm326的鉴定与调控通路初步分析[J]. 中国水稻科学, 2017, 31(4): 335-344.

Figures/Tables 10

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标记	前引物	后引物
Marker	Forward primer	Reverse primer
RD0115	GTTGTAGATGTGATTGGAGAA	GACTATGTATGGCACTGTTGA
RM5310	TAGACAAAGCAACGGGTTCC	CGGAAGCAGGAGAATCGTAG
RM3362	AAGTTGAAGCAGTCGCCAAC	GAATTGCGTGGGATATGGAC
ZH4 ZH7	TCCACGAACAAAGACGAG GACTCCGAGCCAGCAAAA	ACGGCACATTATCAACAACA GTCTCCGTGCCCTTGTGC
ZH8	ATGGAGTCGCCTTGTTGA	AAATGTGGCTGGCTGATC
ZH11	TTCGTCTCATTAGCAGCAT	CATTTATTCACTTGCCACAT
Qpr10	GTCCGGGCACCATCTACACC	CAAGCTTCGTCTCCGTCGAGT
Qpal1	TTCAACGCCGACACCT	GTAGAGCGGATACGACCTG
Qaos2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
WRKY45	GCCGACGACCAGCACGATCACC	ACGAGCCGACGCCGCCCTC
PR1a	CGTGTCGGCGTGGGTGT	GGCGAGTAGTTGCAGGTGATG
PR1b Actin	TACGCCAGCCAGAGGAGC CAGGCCGTCCTCTCTCTGTA	GCCGAACCCCAGAAGAGG AAGGATAGCATGGGGGAGAG

标记	前引物	后引物
Marker	Forward primer	Reverse primer
RD0115	GTTGTAGATGTGATTGGAGAA	GACTATGTATGGCACTGTTGA
RM5310	TAGACAAAGCAACGGGTTCC	CGGAAGCAGGAGAATCGTAG
RM3362	AAGTTGAAGCAGTCGCCAAC	GAATTGCGTGGGATATGGAC
ZH4 ZH7	TCCACGAACAAAGACGAG GACTCCGAGCCAGCAAAA	ACGGCACATTATCAACAACA GTCTCCGTGCCCTTGTGC
ZH8	ATGGAGTCGCCTTGTTGA	AAATGTGGCTGGCTGATC
ZH11	TTCGTCTCATTAGCAGCAT	CATTTATTCACTTGCCACAT
Qpr10	GTCCGGGCACCATCTACACC	CAAGCTTCGTCTCCGTCGAGT
Qpal1	TTCAACGCCGACACCT	GTAGAGCGGATACGACCTG
Qaos2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
WRKY45	GCCGACGACCAGCACGATCACC	ACGAGCCGACGCCGCCCTC
PR1a	CGTGTCGGCGTGGGTGT	GGCGAGTAGTTGCAGGTGATG
PR1b Actin	TACGCCAGCCAGAGGAGC CAGGCCGTCCTCTCTCTGTA	GCCGAACCCCAGAAGAGG AAGGATAGCATGGGGGAGAG

性状 Trait	野生型 Wild type	突变体 lmm326
株高Plant height/cm	101.6&#x000b1;1.8	63.1&#x000b1;4.0^**
每株有效分蘖数No. of productive panicles per plant	11.7&#x000b1;2.4	3.1&#x000b1;0.4^**
每穗总粒数No. of spikelets per panicle	147.1&#x000b1;17.0	46.9&#x000b1;9.1^**
结实率Seed-setting rate/%	85.5&#x000b1;0.0	77.4&#x000b1;0.0^*
千粒重1000-grain weight/g	23.7&#x000b1;0.9	18.7&#x000b1;0.7^**

性状 Trait	野生型 Wild type	突变体 lmm326
株高Plant height/cm	101.6&#x000b1;1.8	63.1&#x000b1;4.0^**
每株有效分蘖数No. of productive panicles per plant	11.7&#x000b1;2.4	3.1&#x000b1;0.4^**
每穗总粒数No. of spikelets per panicle	147.1&#x000b1;17.0	46.9&#x000b1;9.1^**
结实率Seed-setting rate/%	85.5&#x000b1;0.0	77.4&#x000b1;0.0^*
千粒重1000-grain weight/g	23.7&#x000b1;0.9	18.7&#x000b1;0.7^**

编号 Code	野生型 Wild type	突变体 Mutant
12-26-49-2	4.26&#x000b1;0.08	0.79&#x000b1;0.07^**
12-128-12-1	3.89&#x000b1;0.05	0.82&#x000b1;0.08^**
12-901-48-1	4.45&#x000b1;0.14	0.83&#x000b1;0.08^**
13-600-33-2	4.56&#x000b1;0.08	0.81&#x000b1;0.03^**