【作者】 史利玉；

【导师】 潘光堂； 张世煌；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 玉米粗缩病是由玉米粗缩病病毒引起的一种世界性病害,也是我国黄淮海玉米产区的重要病害之一。感病植株显著矮化、不抽雄、雌穗不结实或籽粒少、产量严重损失甚至绝收。开展玉米粗缩病抗性遗传研究,选育抗病品种,是防治此病的经济有效途径之一。然而,现有种质资源中抗性材料较少。因此,寻找抗玉米粗缩病种质资源,探索其抗性遗传规律,发掘优异抗病等位基因,为玉米抗病分子育种实践提供基础。本论文就玉米粗缩病抗源鉴定、功能标记开发、QTL分析及候选基因功能分析做了相关研究,主要结果如下：1.采用网箱集团接种结合移栽的方法对200份国内外玉米自交系进行粗缩病抗性鉴定。结果表明,抗源较少,仅占鉴定材料的10%。筛选出的抗源有沈137、多黄29、金黄96B、中自01、海9-21、P138、CA339、齐319、丹3130、9046、835、黄野四、齐318、R18、SH15、CA335、X178、辽68、金黄59等,为玉米粗缩病抗性遗传研究以及抗病育种提供了材料基础。2.利用抗病自交系(沈137、多黄29、中自01、P138、CA339、齐319、丹3130、齐318、CA335、X178)和感病自交系(掖107、掖478、吉4112、803、K22、鲁原92、掖3189、辽5114、U8112、B73)分别构建抗、感基因组DNA池,结合AFLP标记筛选多态AFLP扩增片段,将稳定多态AFLP扩增片段转化为SCAR标记并进行相关验证。研究获得2个与玉米粗缩病抗性显著相关的SCAR标记SCAR69和SCAR74,分别位于染色体bin 2.07和5.04区；2个SCAR标记可应用于抗玉米粗缩病毒分子标记辅助选择。3.以现有X178×B73以及黄早四×掖107衍生的重组自交系群体为材料,分别构建高密度分子标记连锁图谱。其中,X178×B73 RIL群体的图谱包括643个分子标记(SSR, SNP和SCAR),覆盖玉米10条染色体,平均图距为1.59cM；黄早四×掖107 RIL群体构建的连锁图谱包括540个分子标记(SSR,SNP和SCAR),覆盖玉米10条染色体,平均图距为2.38 cM；选用3个抗病衡量指标(病株率—ID,病情指数—SI,校正病情指数—CSI)对玉米粗缩病抗性进行多年多点的QTL分析。结果表明,在X178×B73 RIL群体,检测到玉米第8染色体8.03处存在1个主效抗病QTL,其抗性基因来源为亲本X178,可解释28%的表型方差。在黄早四×掖107 RIL群体,于染色体2、3、4、6、7、8、10均检测到抗病QTL,其效应较小。通过比较定位分析以及抗病基因簇集分布,发现在玉米第3染色体3.04(SNP610-SNP1438)、第4染色体4.03(SNP1287—SNP581)、第6染色体(SNP1518-SNP408)、第7染色体7.02/03(SNP637—SNP686)、第8染色体8.06(SNP619—SNP68)存在玉米粗缩病抗性位点。4.真核翻译起始因子4E (Eukaryotic Initiation Factor 4E, eIF4E)是RNA病毒成功感染植物的重要决定因素。以玉米抗病自交系X178和感病自交系掖478为材料：1)分离eIF4E同源基因ZmeIF4E,研究基因表达模式。结果表明ZmeIF4E有5个外显子和4个内含子组成,编码218个氨基酸的蛋白；荧光定量PCR显示,该基因受植物激素——乙烯(ETH)、水杨酸(SA)和茉莉酸甲酯(JA)的调控,且在X178和掖478两自交系中的表达模式不同。2)ZmeIF4E序列比对(X178和掖478)和启动子分析表明,顺式作用元件DOFCOREZM、EECCRCAH1、GT1GAMSCAM4和GT1CONSENSUS存在的差异是ZmeIF4E基因表达模式发生变化的主要原因。3)关联分析表明,顺式作用元件EECCRCAH1与玉米粗缩病抗性显著相关；同时还发现顺式作用元件GT-1与玉米粗缩病抗性也显著相关。此外,这两个顺式作用元件在ZmeIF4E基因表达中存在互补或协同效应。综上可得ZmeIF4E基因的活性在玉米与粗缩病毒互作中发挥重要作用。更多还原

【Abstract】 Maize Rough Dwarf Disease (MRDD) caused by maize rough dwarf virus (MRDV) is a worldwide disease. It is one of the most serious diseases in maize-growing area of China. Developing and cultivating resistant hybrids is an effective approach to control MRDD. However, a few resistance germplasm are identified so far. In this study, identification of resistant materials, development of molecular markers and QTL analysis were investigated. The results as follows:1. 200 maize inbred lines for MRDD resistance was evaluated by inoculating small brown plantthopper into net-boxes and the inbred lines of Shen137, Duohuang29, Jinhuang96B, Zhongzi01, Hai9-21, P138, CA339, Qi319, Dan3130,9046,835, Huangye4, Qi318, R18, SH15, CA335, X178, Liao68, Jinhuang59, Zixuanxi were identified.2. Resistant (Shen137, Duohuang29, Zhongzi01, P138, CA339, Qi319, Dan3130, Qi318, CA335, X178) and susceptible(Ye107, Ye478, Ji4112,803, K22, Luyuan92, Ye3189, Liao5114, U8112, B73) DNA bulks were composed by using genomic DNAs of 10 resistant and 10 susceptible inbred lines, respectively. Polymorphic AFLP markers were screened between two bulks and then transformed into SCAR (sequence characterized amplified region) markers. These SCAR markers associated with MRDD resistance was analyzed with disease incidence of 152 inbred lines. Results showed that SCAR69 and SCAR74 were validated to be highly associated with MRDD and could be used for MAS of MRDD resistance in maize.3. Two recombined inbred line populations (RIL) derived from Ye 107×Huangzao4, X178×B73, were evalutated for MRDV resistance in replicated field trials in different sites. Three resistance evaluating index (ID, SI, and CSI) were used for QTL analysis. Linkage maps were constructed using SSRs, SNPs and SCARs, and resistant QTL analyzed using MIM in MapQTL software. Results showed that in X178×B73 RIL population, a major resistance QTL was detected on chromosome bin 8.03, accounting for about 28% of the phenotypic variance. In Huangzao4×Ye 107 RIL population,7 resistant QTLs were detected on chromosome bin 3.04 (SNP610-SNP1438), bin 4.03 (SNP1287-SNP581), bin 6.05, bin7.02/03 (SNP637-SNP686), bin 8.06 (SNP619-SNP68). These QTLs accounted for lower than 10% of the phenotypic variance.4. Translation initiation factors, particularly the eukaryotic initiation factor 4E (eIF4E), were found to be essential determinants of the outcome of plant infections by RNA viruses. In this study, we isolated an eIF4E orthologue and analyzed its expression patterns in two maize inbred lines X178 and Ye478. ZmeIF4E contains five exons encoding a protein with 218 amino acid residues. Quantitative RT-PCR showed that the ZmeIF4E gene was regulated in response to three plant hormones, namely ethylene, salicylic acid and jasmonates and its gene expression varied widely in two inbred lines. Furthermore, sequence analysis of the ZmeIF4E promoter region showed that base substitutions and insertion/deletion polymorphisms were present in four cis-acting elements, including DOFCOREZM, EECCRCAH1, GT1GAMSCAM4, and GT1CONSENSUS. These cis-acting elements may be responsible for diverse gene-expression patterns in two different inbred lines. Association analysis revealed that one SNP polymorphism in EECCRCAH1 was significantly associated with maize rough dwarf virus disease index obtained in 2007 and 2008 in Hebei, China. In addition, one SNP polymorphism in the GT-1 motif was found to affect MRDV resistance in 2007 in Hebei, China. We found that these two SNP polymorphism sites have complementary or synergistic effects on ZmeIF4E gene expression. Collectively, these results imply that these two regulatory motifs in the ZmeIF4E promoter are involved in MRDV resistance, indicating that eIF4E activity plays vital roles in pathogen infections and control of recessive resistance.更多还原