水稻类病斑突变体spl34的遗传分析与精细定位

发布时间：2018-01-18 16:21

本文关键词：水稻类病斑突变体spl34的遗传分析与精细定位　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：类病斑突变体(Disease lesion mimic mutants)是指植物在未受逆境胁迫或病原菌侵害的情况下,在其叶片或叶鞘等部位自发产生类似于过敏反应时所形成的坏死病斑的一类突变体。在类病斑的形成过程中通常会伴随着细胞程序性死亡,活性氧的积累,防卫相关基因的表达量上调以及水杨酸、茉莉酸和乙烯等信号分子的合成量增加。在大量已报道的类病斑突变体中,很多还表现出抗性增强的特性,因此类病斑突变体可作为人类研究植物抗病机理和细胞程序性死亡的良好材料。在水稻中,由于很多类病斑突变体对稻瘟病和白叶枯病表现出抗性增强的特性,因此,类病斑突变体能够为人类培育抗性优良的品种提供丰富的种质资源。本研究利用化学诱变剂EMS(ethyl methane sulfonate)处理水稻籼型恢复系“缙恢10号”从其后代中筛选到一个遗传稳定的类病斑突变体spl34。以野生型“缙恢10号”和突变体spl34为材料,进行了形态学鉴定、农艺性状考察、光合色素含量测定、叶绿体的荧光显微镜观察、组织化学分析、生理生化指标测定等方面的研究,并以表型正常的西农1A与突变体spl34杂交构建群体,F1和F2代用于遗传分析与基因定位,主要研究结果如下。1.形态学鉴定突变体spl34从分蘖后期开始在下部叶片的叶鞘上出现褐色的类病斑,随后褐斑沿着中脉扩散然后分布于整个叶片。至成熟期,褐色类病斑扩散至整个植株。遮光实验表明突变体类病斑的形成受光诱导。2.农艺性状考察成熟期分别对野生型和突变体的株高、穗长、穗粒数、结实率和千粒重等农艺性状进行考察。结果显示,突变体的株高和穗长均较野生型降低,达到了显著差异水平,而千粒重、穗粒数和结实率的降低程度则达到了极显著差异水平。3.光合色素含量测定抽穗期分别对野生型和突变体spl34的光合色素含量进行测定。结果显示,突变体spl34的倒一叶光合色素含量均略高于野生型但未达到显著差异水平,而倒二叶和倒三叶光合色素含量则均较野生型低且分别达到了显著差异水平和极显著差异水平。4.叶绿体的荧光显微镜观察抽穗期取野生型和突变体spl34的叶片用于荧光显微镜观察发现,突变体spl34的叶绿体产生的红色荧光现象较野生型的弱,且突变体中出现类病斑的部位叶绿体产生的红色荧光也较未出现类病斑的部位弱。5.组织化学分析台盼蓝染色和DAB染色结果显示,突变体在类病斑形成部位发生了细胞程序性死亡和过氧化氢沉积。6.生理生化指标测定抽穗期对野生型和突变体spl34的各项生理指标进行检测。结果显示,突变体中CAT、POD、SOD等抗氧化酶活性均较野生型的低,而且突变体中产生类病斑的部位H_2O_2和O_2~-的含量均比野生型高,·OH的含量则与野生型无明显差异。7.稻瘟病抗谱测定与抗性鉴定稻瘟病抗谱测定结果显示,突变体spl34对ZA生理群的抗病频率比野生型高,而对ZB生理群的抗病频率比野生型低,对ZC、ZD、ZE和ZG生理群的抗病频率则与野生型相同。抗性鉴定结果显示,突变体苗瘟、叶瘟病情指数和穗颈瘟的病穗率均比野生型略高,但差异并不显著。8.遗传分析和基因定位遗传分析结果表明,突变体spl34的表型受一对隐性核基因控制。该基因定位于第4染色体的LR49和LR52两个分子标记之间,物理距离为200Kb。测序分析发现该区间内的候选基因:LOC_Os04g56480的第3449位碱基发生突变(G3449T),导致氨基酸替换由色氨酸突变为半胱氨酸。该基因与已报道的HM47为等位突变基因。9.目的基因与防卫相关基因的表达q RT-PCR结果表明,与野生型相比,目的基因在突变体中的表达量下降;而用于进行表达分析的病程相关基因和稻瘟病抗性基因中,除了PR1a、PR1b和NPR1三个病程相关基因在突变体中的相对表达量显著升高外,其余的病程相关基因和稻瘟病抗性基因的相对表达量均未出现上升。
[Abstract]:A lesion mimic mutant (Disease lesion mimic mutants) is a plant without stress or pathogen attack under the condition of spontaneous generation of a class of mutants with similar necrotic lesion formed in the allergic reaction when the leaf sheath or other parts. In the forming process of the lesions usually accompanied by programmed cell death and the accumulation of active oxygen and defense related gene expression and synthesis of salicylic acid, jasmonic acid and ethylene signaling molecules increased. The lesion has been reported in a large number of mutants, many also exhibit characteristics of enhanced resistance, so the lesion mimic mutants can be used as good materials of human research on the mechanism of plant resistance and cell program death. In rice, because a lot of lesion mimic mutants of rice blast and bacterial blight showed enhanced resistance characteristics, therefore, to cultivate a lesion mimic mutant for man The excellent resistant cultivars provide rich germplasm resources. This study uses chemical mutagen EMS (ethyl methane sulfonate) processing of Indica Rice Restorer Line Jinhui 10 "from the progeny of screening to a stable genetic lesion mimic mutant spl34. with wild type" Jinhui 10 "and mutant spl34 as material for the morphological, agronomic traits, photosynthetic pigment content, chloroplast fluorescence microscopy, histochemical analysis of physiological and biochemical indexes and other aspects, and with normal phenotype of Xinong 1A and mutant spl34 hybrid construction group, F1 and F2 for genetic analysis and gene mapping. The main results are as follows.1. morphological identification of mutant spl34 from late tillering began Brown lesion in leaf sheath of lower leaves, then brown midrib and then spread throughout the diffusion along the blade. To maturity Brown, the lesions spread to the whole plant. Experiments show that the formation of mutant shading lesion induced by light.2. agronomic traits in mature stage of wild type and mutant plant height, panicle length, grain number, seed setting rate and grain weight was investigated and other agronomic traits. The results showed that the mutant plant height and panicle length were compared to the wild type decreased, reached the level of significant difference, and 1000 grain weight, grain number and seed setting rate decreased reached respectively for the wild type and spl34 mutant of photosynthetic pigment content were measured at the heading stage was extremely significant difference in the level of.3. of photosynthetic pigment content. The results showed that the leaf photosynthetic mutant spl34 the pigment content was slightly higher than the wild type, but the difference is not obvious, but the two leaves photosynthetic pigment content were lower than wild type and reached significant level and extremely significant difference in the level of.4. Ye Lv Fluorescence microscope observation body leaves at heading stage from the wild type and mutant spl34 for fluorescence microscopy revealed that the red fluorescence phenomenon of spl34 mutant chloroplasts compared to the wild type weak,.5. histochemical analysis of trypan blue staining and DAB staining showed weak red fluorescence produced lesion and mutant site in the chloroplast also is not part of the lesion, the mutant forms occurs heading determination of programmed cell death and hydrogen peroxide deposition of.6. on physiological and biochemical indexes of wild type and mutant spl34 in various physiological indexes were detected in the lesion. The results showed that the mutant CAT, POD, antioxidant enzyme activity SOD were low compared to the wild type. And, in parts of lesion mimic mutants H_2O_2 and O_2~- were higher than those of the wild type, and the content of OH and.7. had no significant difference between the wild type rice blast disease The spectrum of resistance and resistance identification of rice blast resistance spectrum determination showed that the mutant spl34 of ZA resistance frequency physiological group was higher than in the wild type, while the resistance frequency ZB physiological group lower than wild type of ZC, ZD, ZE and ZG physiological group resistance frequency is the same with the wild type. The resistance identification results showed that the mutant seedling blast disease index, leaf blast and Panicle Blast Disease spike rate were slightly higher than the wild type, but the difference was not significant.8. genetic analysis and gene mapping of genetic analysis showed that the phenotype of the spl34 mutant was controlled by a pair of recessive nuclear genes. The gene is located on chromosome LR49 and fourth LR52 two molecular markers, the physical distance of 200Kb. sequencing analysis identified candidate genes within the interval: LOC_Os04g56480 3449th base mutation (G3449T), resulting in amino acid substitution mutation from tryptophan to cysteine. The gene has been reported with HM47 allele The expression of Q RT-PCR gene mutation results of.9. gene and defense related genes showed that, compared with the wild type gene expression in the mutant decreased; and for the analysis of the expression of pathogenesis related genes and blast resistance gene, in addition to PR1a, the relative expression of course three genes PR1b and NPR1 in the mutant significantly, the relative expression of pathogenesis related genes and rice blast resistance genes of the remaining amount did not rise.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.11

【参考文献】