Characterization and Gene Mapping of Lesion Mimic Mutant wy3 in Rice

doi:10.16819/j.1001-7216.2016.5176

Abstract

Abstract:

A lesion mimic mutant wy3 was obtained from the progeny of a japonica variety Wuyujing 3 by natural mutation. The lesions were first observed on the leaves at the seedling stage, then spread gradually to the whole leaf at the initial tillering stage. Compared with the wild type(WT), the plant height, the number of effective tiller, panicle length, grain number per panicle and seed setting rate of wy3 were reduced significantly. The shading assay showed that the lesions on leaves of wy3 were induced by natural light. Compared with the wild type, the photosynthetic pigment and the net photosynthetic rate of wy3 were significantly reduced, but the SOD activity, POD activity, CAT activity and MDA content of wy3 were significantly higher than those of WT. Trypan blue staining experiments showed that there were a large number of dead cells in the mutant lesion. Genetic analysis suggested that the phenotype of wy3 was controlled by a single recessive nuclear locus, and the target gene was mapped between markers W2-17 and W2-18 with the physical distance of 28 kb on chromosome 2. Sequence analysis revealed that the mutated gene in wy3 had a single nucleotide deletion at the 375th in CDS of LOC_Os02g02000, resulting in a premature termination of translation of the target gene, which is an new allele of OsHPL3.

Key words: rice, lesion mimic mutant, gene mapping

摘要：

在粳稻品种武育粳3号栽培群体中获得一个类病斑突变体wy3。该突变体类病斑出现于苗期,分蘖期扩散至整张叶片,属于扩散型类病斑突变体。相比野生型,突变体wy3的株高明显降低,有效分蘖数减少,穗长、每穗粒数、结实率均显著降低。遮光处理表明,突变体wy3类病斑的产生受自然光诱导。台盼蓝染色结果表明,类病斑部位有大量的死亡细胞。突变体wy3的光合色素含量和净光合速率较野生型显著降低,SOD、POD、CAT活性和MDA含量均显著高于野生型。遗传分析表明突变体表型受单隐性核基因控制,采用BSA将该基因初步定位在第2染色体短臂端粒附近。采用F₂群体中1099株类病斑单株将基因定位在标记W2-17和W2-18之间28kb的物理距离内。测序结果表明,突变体wy3中的LOC_Os02g02000编码区(CDS)第375位碱基C缺失,导致翻译提前终止,突变体中该候选基因为OsHPL3的一个新等位基因。

关键词: 水稻, 类病斑突变体, 基因定位

CLC Number:

Q343.5
Q754

Hong-Gen ZHANG, Mao-yu WANG, Li-jia ZHANG, Ya HU, Jia-qi MA, Yi-fan ZHANG, Shu-zhu TANG, Guo-hua LIANG, Ming-hong GU. Characterization and Gene Mapping of Lesion Mimic Mutant wy3 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(3): 239-246.

张宏根, 王茂宇, 张丽佳, 胡雅, 马佳琦, 张翼帆, 汤述翥, 梁国华, 顾铭洪. 水稻类病斑突变体wy3的鉴定和基因定位[J]. 中国水稻科学, 2016, 30(3): 239-246.

Figures/Tables 9

References 37

[1]	王忠华. 植物类病变突变体的诱发与突变机制. 细胞生物学杂志, 2006, 27(5): 530-534.
	Wang Z H.Induction and mutation mechanism of plant lesion mimic mutants.Chin J Cell Biol, 2005, 27: 530-534 (in Chinese with English abstract)
[2]	Dietrich R A, Delaney T P, Uknes S J, et al.Arabidopsis mutants simulating disease resistance response.Cell, 1994, 77(4): 565-577.
[3]	Büschges R, Hollricher K, Panstruga R, et al.The barley Mlo gene: A novel control element of plant pathogen resistance.Cell, 1997, 88(5): 695-705.
[4]	Gray J, Close P S, Briggs S P, et al.A novel suppressor of cell death in plants encoded by the Lls1 gene of maize.Cell, 1997, 89(1): 25-31.
[5]	Badigannavar A M, Kale D M, Eapen S, et al.Inheritance of disease lesion mimic leaf trait in groundnut.J Hered, 2002, 93(1): 50-52.
[6]	Malamy J, Carr J P, Klessig D F, et al.Salicylic acid: A likely endogenous signal in the resistance response of tobacco to viral infection.Science, 1990, 250(4983): 1002-1004.
[7]	Takahashi A, Kawasaki T, Henmi K, et al.Lesion mimic mutants of rice with alterations in early signaling events of defense.Plant J, 1999, 17(5): 535-545.
[8]	Dangl J L, Dietrich R A, Richberg M H.Death don’t have no mercy: Cell death programs in plant-microbe interactions.Plant Cell, 1996, 8(10): 1793.
[9]	Neuffer M G, Calvert O H.Dominant disease lesion mimics in maize.J Hered, 1975, 66(5): 265-270.
[10]	Shirasu K, Schulze-Lefert P.Regulators of cell death in disease resistance.Plant Mol Biol, 2000, 44(3): 371-385.
[11]	Dietrich R A, Richberg M H, Schmidt R, et al.A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death.Cell, 1997, 88(5): 685-694.
[12]	Ryerson D E, Heath M C.Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or by abiotic treatments.Plant Cell, 1996, 8(3): 393-402.
[13]	Lamb C, Dixon R A.The oxidative burst in plant disease resistance.Annu Rev Plant Biol, 1997, 48(1): 251-275.
[14]	王建军, 朱旭东, 王林友, 等. 水稻类病变突变体 lrd40 的抗病性与细胞学分析. 中国水稻科学, 2005, 19(2): 111-116.
	Wang J J, Zhu X D, Wang L Y, et al.Disease resistance and cytological analyses on lesion resembling disease mutant lrd40 inOryza sativa. Chin J Rice Sci, 2005, 19: 111-116. (in Chinese with English abstract)
[15]	陈析丰, 金杨, 马伯军. 水稻类病变突变体及抗病性的研究进展. 植物病理学报, 2011, 41(1): 1-9.
	Chen X F, Jin Y, Ma B J.Progress on the studies of rice lesion mimics and their resistant mechanism to the pathogens.Acta Phytopathol Sin, 2011, 41: 1-9. (in Chinese with English abstract)
[16]	Qiao Y, Jiang W, Lee J H, et al.SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit μ1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa).New Phytol, 2010, 185(1): 258-274.
[17]	Wu C, Bordeos A, Madamba M R S, et al. Rice lesion mimic mutants with enhanced resistance to diseases.Mol Genet Genom, 2008, 279(6): 605-619.
[18]	Yin Z, Chen J, Zeng L, et al.Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight.Mol Plant-Microbe Interac, 2000, 13(8): 869-876.
[19]	李小红, 施勇烽, 张晓波, 等. 水稻斑点叶突变体 hm197 的鉴定及其基因定位. 中国水稻科学, 2015, 29(5): 447-456.
	Li X H, Shi Y F, Zhang X B,et al.Identification and gene mapping of a spotted-leaf mutant hm197 in rice.Chin J Rice Sci, 2015, 29(5): 447-456. (in Chinese with English abstract)
[20]	Chen X, Hao L, Pan J, et al.spl5, a cell death and defense-related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice.Mol Breeding, 2012, 30(2): 939-949.
[21]	Yamanouchi U, Yano M, Lin H, et al.A rice spotted leaf gene, spl7, encodes a heat stress transcription factor protein.PNAS, 2002, 99(11): 7530-7535.
[22]	Zeng L R, Qu S, Bordeos A, et al.Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity.Plant Cell, 2004, 16(10): 2795-2808.
[23]	Mori M, Tomita C, Sugimoto K, et al.Isolation and molecular characterization of a spotted leaf 18 mutant by modified activation-tagging in rice.Plant Mol Biol, 2007, 63(6): 847-860.
[24]	Takahashi A, Agrawal G K, Yamazaki M, et al.Rice Pti1a negatively regulates RAR1-dependent defense responses.Plant Cell, 2007, 19(9): 2940-2951.
[25]	Yin Z, Chen J, Zeng L, et al.Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight.Mol Plant-Microbe Interac, 2000, 13(8): 869-876.
[26]	Lichtenthaler H K.Chlorophylls and carotenoids: Pigments of photosynthetic bio membranes.Methods Enzymol, 1987 (148C): 350-382.
[27]	张志良,瞿伟菁. 植物生理学实验指导. 北京: 高等教育出版社, 1990.
	Zhang Z L, Qu W J.Plant Physiology Experimental Guidance. 3rd edn. Beijing: Higher Education Press, 2003. (in Chinese)
[28]	王丹. 水稻分蘖调控基因TE的功能分析和类病变突变体lms1的图位克隆. 北京:中国农业科学院, 2012.
	Wang D.Functional analysis of a key tillering regulator TE and map-based cloning of gene lms1 in rice (Orzya sativa L.). Beijing: CAAS, 2012. (in Chinese with English abstract)
[29]	邱结华, 马宁, 蒋汉伟, 等. 水稻类病斑突变体 lmm4 的鉴定及其基因定位. 中国水稻科学, 2014, 28(4): 367-376.
	Qiu J H, Ma N, Jiang H W, et al.Identification and gene mapping of a lesion mimic mutant lmm4 in rice.Chin J Rice Sci, 2014, 28(4):367-376. (in Chinese with English abstract)
[30]	钟振泉, 罗文龙, 刘永柱, 等. 一份新的水稻斑点叶突变体 spl32 的鉴定和基因定位. 作物学报, 2015, 41(6): 861-871.
	Zhong Z Q, Luo W L, Liu Y Z, et al.Characterization of a novel spotted leaf mutant spl32 and mapping of spl32(t) gene in rice (Oryza sativa).Acta Agron Sin, 2015, 41(6): 861-871. (in Chinese with English abstract)
[31]	刘林. 水稻类病变突变体g340的鉴定和基因定位.北京:中国农业科学院, 2014.
	Liu L.Identification and gene mapping of a rice lesion mimic mutant g340. Beijing: CAAS, 2014 (in Chinese with English abstract)
[32]	代高猛, 朱小燕, 李云峰, 等. 水稻类病斑突变体spl31的遗传分析与基因定位. 作物学报, 2013, 39(7): 1223-1230.
	Dai G M, Zhu X Y, Li Y F, et al.Genetic analysis and fine mapping of a lesion mimic mutant spl31 in rice.Acta Agron Sin, 2013, 39(7): 1223-1230. (in Chinese with English abstract)
[33]	Samuilov V D, Kiselevsky D B, Sinitsyn S V, et al.H2O2 intensifies CN--induced apoptosis in pea leaves.Biochem (Moscow), 2006, 71(4): 384-394.
[34]	Wang L, Pei Z Y, He C.OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation.Mol Plant-Microbe Interac, 2005, 18(5):375-384.
[35]	Kojo K, Yaeno T, Kusumi K, et al.Regulatory mechanisms of ROI generation are affected by rice spl mutations.Plant Cell Physiol, 2006, 47(8): 1035-1044.
[36]	Liu X, Li F, Tang J, et al.Activation of the jasmonic acid pathway by depletion of the hydro peroxide lyase OsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice.PLoS ONE,2012,7(11):1-14.
[37]	Tong X, Qi J, Zhu X, et al.The rice hydro peroxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway.Plant J, 2012, 71(5): 763-775.

材料 Material	株高 Plant height /cm	分蘖数 No. of tillers	穗长 Panicle length /cm	结实率 Seed-setting rate/%	每穗粒数 Grain number per panicle	粒长 Grain length /mm	粒宽 Grain width /mm	千粒重 1000-grain weight/g
wy3	56.9±0.6^**	3.5±0.6^**	14.8±1.2^**	79.0±0.1^**	89.6±6.8^**	7.5±2.2	3.4±1.4	26.54±0.56
WT	87.5±0.5	10.0±1.7	16.6±0.8	98.3±0.1	123.6±11.0	7.3±2.8	3.3±0.9	26.85±0.62

材料 Material	株高 Plant height /cm	分蘖数 No. of tillers	穗长 Panicle length /cm	结实率 Seed-setting rate/%	每穗粒数 Grain number per panicle	粒长 Grain length /mm	粒宽 Grain width /mm	千粒重 1000-grain weight/g
wy3	56.9±0.6^**	3.5±0.6^**	14.8±1.2^**	79.0±0.1^**	89.6±6.8^**	7.5±2.2	3.4±1.4	26.54±0.56
WT	87.5±0.5	10.0±1.7	16.6±0.8	98.3±0.1	123.6±11.0	7.3±2.8	3.3±0.9	26.85±0.62

材料 Material	叶绿素a含量 Chlorophyll a content /(mg·g^-1)	叶绿素b含量 Chlorophyll b content /(mg·g^-1)	叶绿素a/b Chlorophyll a/b	类胡萝卜素含量 Carotenoid content /(mg·g^-1)
wy3	1.28±0.09^**	0.27±0.11^**	4.68±0.13^**	0.15±0.11^**
WT	2.28±0.03	0.99±0.08	2.31±0.07	0.59±0.04

材料 Material	叶绿素a含量 Chlorophyll a content /(mg·g^-1)	叶绿素b含量 Chlorophyll b content /(mg·g^-1)	叶绿素a/b Chlorophyll a/b	类胡萝卜素含量 Carotenoid content /(mg·g^-1)
wy3	1.28±0.09^**	0.27±0.11^**	4.68±0.13^**	0.15±0.11^**
WT	2.28±0.03	0.99±0.08	2.31±0.07	0.59±0.04

材料 Material	净光合速率 P_n /(mmol·m^-2 s^-1)	蒸腾速率 T_r /(mmol·m^-2 s^-1)	气孔导度 G_s /(mol·m^-2 s^-1)	胞间CO₂浓度 C_i /(mmol·mol^-1)
wy3	14.16±0.27^**	193.27±10.10^**	0.13±0.02^**	2.76±0.23^**
WT	22.46±0.29	276.96±6.30	0.66±0.08	8.93±0.43