导　　师： 吴先军

学科专业： I0102

授予学位： 硕士

作　　者： ;

机构地区： 四川农业大学

摘　　要： 稻瘟病是世界上最重要的水稻病害之一，每年都造成很大的损失。抗病品种的选育是防治稻瘟病的主要途径。随着dna分子标记的出现与迅猛发展，标记辅助选择(marker-assisted selection，简称mas)技术己成为抗病基因正确选择的有效途径.但由于抗病基因与分子标记间的重组率在不同的群体中有较大的差异，同时构建分子标记的群体往往不是育种材料本身，在实际应用中有一定局限性。另外，由于目前已建立的分子标记不是基因本身，在育种大量群体中可能出现目的基因与分子标记不连锁的情形，导致抗病基因选择的失败。因此，最好的办法是利用抗病基因本身来建立相应的分子标记。抗病基因的克隆为建立这种标记提供了可能。 pib基因是一个已经被克隆的抗稻瘟病主效基因，位于水稻第2染色体长臂末端附近的区域，属于一个很小的基因家族(pib，pibh8，hpibh8-1，hpibh8-2)。pib基因编码由1251个氨基酸残基组成的含一个核苷酸结合位点(nucleotide binding site，nbs)和富含亮氨酸重复(1eucine-rich reports，lrr)的蛋白质。 本研究首先对36个稻瘟病菌株进行生理小种鉴定，发现该群体包括7群23个生理小种，其中zc和za群是优势群，zg1、zcl5和ze3小种是最优势小种。同时分别利用其中8个菌株对106个杂交稻亲本进行抗性鉴定。结果发现，106个亲本的平均抗病频率为25.15％。恢复系亲本总体抗性水平高于保持系亲本。同时发现，c15、b13、d7和b15四个生理小种的致病频率较高，分别为84.9％、83％、80.9％和80％。然后分别利用这36个菌株对两个含pib基因的材料(irblb-b和f-145-2)进行抗性鉴定。结果发现，在36个供试菌株中，iblb-b和f-145-2分别抗26和31个菌株的侵染，抗性频率分别为72.2％和86.1％，表明这两个含pib基因的材料具有较高的抗性。但二者抗性频率相差较大，为13.9％。 本实验还利用建立的感病等位基因p/b显性分子标记，并结合前人报道的抗病等位基因pib显性分子标记，组建一套pib基因分子标记对109个杂交稻亲本或材料进行分子鉴定，同时在温室分别选用稻瘟病菌株05-12(zb13)和05-30(zc15)，采用标准喷雾接种法对上述109个杂交稻亲本进行致病性测试，结果发现在109个杂交稻亲本中，94-4、ha303和05h38等5个杂交稻亲本都抗05-12和05-30，且都含有pib基因，从而为抗性辅助选择(resistance-assisted selection，ras)提供理论依据。同时利用这套水稻抗稻瘟病基因pib显性分子标记对600个杂交f<,2>代单株进行早期筛选，得到185个抗病基因pib纯合的单株，田间抗性调查结果与抗病基因分子检测结果一致。因此该套显性分子标记可应用于水稻抗稻瘟病基因pib的分子标记辅助选育。总之，本研究利用己克隆的pib功能基因序列本身建立了新颖的dna分子标记，并成功地将其应用于育种辅助选择和抗病作用机制的研究。该标记克服了常规分子标记的弊端，为mas提供更有力的技术保障，同时也为已克隆的pib基因在育种中的应用开辟新的途径。 Blast is the world's most serious rice disease and annually causes great losses.Breeding disease-resistant varieties is the main approach for rice blast control. With theemergence and rapid development With DNA molecular markers, Marker-assistedselection /(MAS/) technology has become an effective approach for the correct choice ofresistance genes. However, as resistance gene and the molecular marker differ greatly indifferent groups, while the groups used to construct molecular marker are not usually thebreeding material itself, the practical application is limited. In addition, as the establishedmolecular marker is not the gene itself, breeding in large groups may cause the target genenot linked to the molecular marker, resulting in the choice failure of the resistance gene.Therefore, the best approach is to use disease-resistant gene itself to establish thecorresponding molecular markers. Resistance gene cloning makes it possible to establishsuch markers. Pib, a cloned major gene with blast resistance, is located on the region near the endof he long arm of Chromosome 2 of rice and belongs to a small gene family /(Pib, PibH8,HPibH8-1, HPibH8-2/). Pib gene codes the protein with 1251 amino acid residuecomposing a nucleotide binding site /(NBS/) and leucine-rich repeat /(LRR/) proteins. The physiological races identification of 36 rice blast stains found that there were 7groups, 23 races. ZC and ZA group was the dominant group and ZG1, ZC15 and ZE3raced were the most dominant. Meanwhile, 8 strains were utilized to identify the resistanceof the 106 hybrid rice. The results showed that the resistance frequency on average is25.15/%. Parental restorer had higher level of resistance than the parental maintainer ingeneral. It was also found that the four races of C15, B13, D7 and B15 had higherpathogenic frequency of 84.9/%, 83/%, 80.9/% and 80/%respectively. Then these 36 strainswere utilized to identify the resistance of two Pib gene containing materials /(IRBLb-B andF-145-2/). The results showed that the 36 strains tested. IRBLb-B and F-145-2 wereresistant to the infection of 26 and 31 strains of the total 36 tested strain. The resistancefrequency was 72.2/% and 86.1/%, respectively, suggesting that the two Pib gene containingmaterial had high resistance. However, the difference between the resistance frequencywas big /(13.9/%/). With the established the susceptible allele Pib dominant molecular marker, combinedwith the previously reported resistance alleles Pib dominant molecular marker, a set of Pibgene molecular marker was utilized to molecularly identify 109 hybrid parental rice or materials. Meanwhile, in the greenhouse, rice blast strains of 05-12 /(ZB13/) and 05-30/(ZC15/) was selected for the pathogenic test of the above 109 hybrid rice parents, with thestandard spray inoculation protocol. The results show that the 5 hybrid rice parent of 94-4,HA303, 05H38 and so on, carrying the Pib gene, were all resistant to 05-12 and 05-30,providing the theoretical evidence for Resistance-assisted selection /(RAS/) provide atheoretical basis. Meanwhile, this rice blast resistance gene Pib dominant molecularmarker set was utilized to select among 600 F/_2 plants in the early stage and 185 Pibresistance gene homozygous plants were obtained. Field resistance investigation resultswere in accordance with the results of molecular detection of the resistance gene.Therefore this set of dominant molecular markers could be applied to rice blast resistancegene Pib molecular marker-assisted breeding. In conclusion, this study had established novel DNA molecular markers with thecloned Pib functional gene sequence itself, and had successfully applied to the research ofassisted breeding and disease-resistant mechanism. The markers overcome the drawbacksof the conventional molecular markers, providing powerful technical safeguard for MAS,and opened new path for the application of the cloned Pib gene in breeding.

关 键 词： 水稻 稻瘟病 抗基因 基因 分子标记辅助选择

分 类 号： [S511.03]

领　　域： [农业科学]