Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance

Lesion mimic mutants(LMMs) are plants that spontaneously form lesions without pathogen infection or external stimulus and exhibit resistance to pathogens. Here, a rice LMM was created by ethyl methane sulfonate mutagenesis, named as hpil(hydrogen peroxide induced lesion). Diaminobenzidine and trypan blue staining showed that large amounts of H_2O_2 were produced and cell death was occurred at and around the parts of lesion mimic in the rice leaves. The phenotype of hpil is controlled by a single recessive gene, localized at a 2 Mb interval on chromosome 2. The data suggested that hpil is a novel LMM with enhanced bacterial and fungal disease resistance, and multiple pathogenesis-related proteins(PRs) were up-regulated. The proteomes of leaves at three positions(different degrees of lesion mimic severity) were characterized in hpil compared with its wild type plant. Differentially expressed proteins were detected by two dimensional difference gel electrophoresis and 274 proteins were identified by MALDI TOF/TOF~(TM). These proteins were related to metabolic process, cellular process and response to stimulus, with mostly down-regulated in hpil leaves. Many of these proteins were related to the Calvin cycle, photosynthetic electron transport chain, glycolysis/gluconeogenesis and phosphonates pathways. Some resistance-related proteins including 14-3-3 proteins, OsPR10 and antioxidases such as peroxidase, superoxide dismutase and ascorbate peroxidase were up-regulated in leaves with lesion mimic. These results provide the foundation for cloning of the target gene and shed light on the mechanism involved in autoimmunity of rice.     

Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice

A lesion mimic stripe mutant,designated as lms1(lesion mimic stripe 1),was obtained from the M2 progeny of a 60Co γ-radiation treated japonica rice variety Jiahua 1.The lms1 mutant displayed propagation type lesions across the whole growth and developmental stages.Physiology and histochemistry analysis showed that the mutant exhibited a phenotype of white stripe when grown under high temperature(30 ℃),and the lesion mimic caused by programmed cell death under low temperature(20 ℃).The genetic analysis indicated that this lesion-mimic phenotype is controlled by a single locus recessive nuclear gene.Furthermore,by using simple sequence repeat markers and an F2 segregating population derived from two crosses of lms1 × 93-11 and lms1 × Pei'ai 64S,the lms1 gene was mapped between markers Indel1 and MM0112-4 with a physical distance of 400 kb on chromosome 6 in rice.     

一个水稻类病条纹斑突变体的鉴定和遗传定位

 粳稻品种嘉花1号经60Co γ射线辐射诱变后获得一个水稻类病斑突变体MR07, 暂命名为lms1。该突变体的病斑在全生育期均表现,属于扩散型类病斑突变体。生理和组织化学分析表明,该突变体在高温条件下（30℃）培养时只表现为白色条斑,低温条件下（20℃）培养时表现出细胞自主性死亡的坏死病斑。遗传分析表明,该突变体受1对隐性核基因控制。以lms1突变体与籼稻9311、培矮64S杂交的两个F2分离群体作为定位群体,利用SSR标记和Indel标记将该基因定位在第6染色体上,位于标记InDel1和MM0112-4之间,其物理距离为400 kb。     

水稻类病斑突变体wy3的鉴定和基因定位

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。     

水稻类病斑突变体lm8015-2的鉴定与基因的精细定位

[目的]为进一步解析水稻抗病分子机制,对类病斑突变体lm8015-2进行了鉴定.[方法]利用EMS诱变籼稻中恢8015,从其后代中鉴定出一个类病斑突变体lm8015-2.对突变体lm8015-2及其野生型的叶片进行超氧化物歧化酶活性、过氧化氢酶活性、过氧化物酶活性、过氧化氢含量、丙二醛含量、可溶性蛋白含量、光合色素含量...     

水稻类病变突变体lrd40的抗病性与细胞学分析

 由中花11经γ射线诱变获得的水稻类病变突变体lrd40，是一个对环境不敏感的全生育期表达的突变体。遗传分析表明，lrd40的类病变性状为1个隐性位点控制，与已报道的类病变突变体lrd38、lrd39、lrd42是非等位的。经多点接种试验，lrd40对水稻白叶枯病2个菌系PXO99A和DY89031都表现抗病。lrd40与野生型杂交F2接种结果证实，lrd40的抗白叶枯病与类病变的表现是一致的。锥虫蓝（trypan blue）染色观察到lrd40的类病变发生可能是一个程序性细胞死亡过程，经二氨基联苯胺（diamino benzidine，DAB）染色检测到lrd40的类病变表达时，叶片上产生H2O2沉淀。细胞学观察结果说明，lrd40的类病变发生类似于植物防卫反应的早期特征，如细胞死亡和过敏反应导致的氧迸发（oxidative burst）。     

水稻胡麻斑类似病危害转Bt稻“克螟稻”初报

 于田间转Bt稻“克螟稻”稻叶上观测到一种病斑，病斑中心和周围均呈褐色，长椭圆形与稻胡麻斑病类似，平板上分离到的病原菌菌丝呈灰褐色，菌丝有多个隔膜，分生孢子梗成丛，但在PDA培养基上，产生的分生孢子特征与稻胡麻斑病不吻合。从为害级别和严重度上分析克螟稻对胡麻斑类似病的感病程度远远超过其亲本秀水11。克螟稻叶片间发病速率增长迅速，而秀水11叶片间发病速率增长缓慢。     

水稻类病斑突变体lmm7的鉴定与基因定位

【目的】对水稻类病斑突变体的研究有助于解析其与植物生长和防御反应的关系。【方法】本研究在粳稻品系FI135胚培养过程中获得了1个类病斑突变体lmm7(lesion mimic mutant 7)。通过对其进行系统的表型鉴定、农艺性状考查、超微结构观察、生理学特性分析,阐明LMM7基因对植物生长的调控。通过病原菌抗性鉴定,明确lmm7对植物防御反应的影响。利用9311B与突变体lmm7杂交所得F₂群体对该突变体进行了遗传分析和基因精细定位。【结果】该突变体苗期表型正常,分蘖初期,植株基部叶片从叶尖开始不断出现褐色斑点,并向整株扩散,且斑点数目随植株生长不断增加。与野生型相比,突变体的株高、穗长、有效穗数、每穗粒数、结实率及剑叶长宽都显著降低,但籽粒性状和抽穗期没有显著性差异。遮光处理表明,突变体lmm7的表型受到光照诱导,抽穗期突变体lmm7叶肉细胞严重失绿,光合色素含量显著降低。组织化学分析表明,突变体病斑处的H₂O₂含量显著升高。透射电镜观察结果表明,突变体lmm7叶肉细胞的叶绿体数目减少,叶绿体类囊体片层结构严重受损,细胞器肿胀解体,并出现大量嗜锇小体,同时病斑内部和周围区域积累了大量的ROS。抗性鉴定结果显示突变体lmm7稻瘟病抗性水平显著高于野生型。遗传分析表明lmm7的突变表型受单个隐性基因控制。利用图位克隆的方法,目的基因被定位于水稻第7染色体短臂两InDel标记7B35和7B43之间,区间范围约260 kb。测序结果表明该区间内候选基因LOC_Os07g0203700第2891位碱基T发生了单碱基缺失,导致后续移码突变及翻译提前终止。【结论】lmm7与spl5互为等位基因,其突变抑制了植株的生长,同时增强了对稻瘟病的抗性。     

水稻抗稻瘟病菌防卫反应的细胞学分析与防卫基因表达

对水稻类病变坏死突变体 lmm1　(LmmKaty)与原亲本及双单倍体YT14和YT16接种稻瘟病菌24 h内的抗病反应、细胞学和分子作用机理进行了研究。人工接种实验表明,与原亲本相比,该突变体对稻瘟病菌具有更强的抗性。突变体接种20~24 h后,自动荧光检测到细胞死亡,而对照则未检测到。表明该突变体通过细胞程序性死亡阻止稻瘟病菌的蔓延,从而提高了抗病性。Northern blotting结果发现,抗病品系YT14中的防卫相关基因、苯丙氨酸解氨酶基因和β 葡聚糖酶基因在人工接种稻瘟病菌6 h后开始表达,16~24 h后上述基因表达量快速增加;而病程相关蛋白基因 PR 1和几丁质酶基因在人工接种稻瘟病菌24 h后才开始表达。与YT14相比,感病品系中上述基因的表达明显延迟。表明在人工接种稻瘟病菌24 h内水稻可启动抗病防卫反应,从而对稻瘟病菌产生抗性。     

水稻类病变突变体性状及分子机理研究进展

类病变突变体是植物在无显著逆境或损伤情况下以及未受到病原物危害时叶片上自然形成的类似病斑的一类突变体。水稻类病变突变体的产生常伴有抗病性的增强,其中涉及到防御相关基因的表达。相关研究主要集中于各种突变体的定位和克隆以及突变体的抗病性和抗病机制。综述了水稻类病变突变基因的性状及抗病性的研究进展,特别是对水稻类病变突变体形成原因、遗传特性、调控途径进行了梳理,并提出对水稻抗病育种的展望,以期为进一步分析类病变突变体的各种机制奠定理论基础,并为水稻育种提供参考。     

水稻抗稻瘟病突变体lmm326的鉴定与调控通路初步分析

【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F_2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38kb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。     

一个水稻类病变黄叶突变体的鉴定和精细定位

从15000多个水稻转基因株系中发现了一个类病变黄叶突变体.该突变体最显著的特征为叶片由下而上依次黄化,同时出现类似病原体感染的病斑.根据突变体表型,将该突变体命名为syll(spotted and yellow leayes 1).遗传学分析表明,该突变性状受1对隐性核基因控制.PCR检测和潮霉索抗性分析显示.该突变...     

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-bi and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region (SCAR) marker tightly linked with Stv-bi and a cleaved amplified polymorphic sequence (CAPS) marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, N...     

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-b by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-bⁱ and a translucent endosperm mutant gene Wx-mq. From F₂ generations, a sequence characterized amplified region (SCAR) marker tightly linked with Stv-bⁱ and a cleaved amplified polymorphic sequence (CAPS) marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.     

分子标记辅助选择聚合水稻暗胚乳突变基因Wx-mq和抗条纹叶枯病基因Stv-b~i

以江苏高产粳稻品种武运粳7号为母本,具有条纹叶枯病抗性基因Stv-bi和暗胚乳突变基因Wx-mq的日本粳稻品种关东194作父本配制杂交组合进行聚合育种。利用与Stv-bi基因共分离的SCAR标记及与暗胚乳突变基因Wx-mq紧密连锁的CAPS功能标记对其分离世代进行标记位点的检测,结合田间多代选育、抗性鉴定和籽粒胚乳外观鉴定,将条纹叶枯病抗性基因Stv-bi和暗胚乳突变基因Wx-mq同时转育到高产品种中,筛选、培育出优质、抗病、高产且农艺性状优良的水稻新品系宁9108。利用与水稻优质及抗病基因紧密连锁的分子标记,不仅初步建立了优质、抗病育种的分子标记辅助选择体系,也为水稻优质、抗病、高产育种提供了一种简单、快捷的选择方法和重要的中间材料。     

水稻斑点叶变异研究进展

大多数水稻斑点叶突变体是研究稻瘟病抗性、白叶枯病抗性以及植物程序性细胞死亡机理的理想材料。已经阐明了49份水稻斑点叶突变体的遗传模式,并成功克隆分离了若干斑点叶基因。综述了这些突变体的来源、遗传模式、基因定位和克隆,基因的功能以及它们对水稻主要病害的反应。     

Resistance to Bacterial Leaf Blight in a Somaclonal Rice Mutant HX-3 at Cellular Level

The interaction between rice host and its pathogen Xanthomonas oryzae pv. oryzae (Xoo) at cellular level was studied by using a resistant somaclonal mutant HX-3 and its susceptable donor Minghui 63. After inoculation with Xoo strain Zhe 173 (Chinese pathotype Ⅳ), the activity of superoxide dismutase (SOD) and peroxidase (POD) in the callus of Minghui 63 was increased dramatically, and the active oxygen(O2 - .) was produced at a higher rate; Meanwhile, the callus grew slowly with the reduction of protein content. Compared to the activity of SOD and POD, the production rate of O2-. and the fresh weight in HX-3 callus varied little after the inoculation. It could be proposed that there were great differences between the resistance of HX-3 and Mighui 63 at cellular level. There was no difference detected concerning resistance to bacterial leaf blight in HX-3 between the plant and the callus.