玉米抗禾谷镰刀菌茎腐病主效QTL基因ZmCCT的克

发布时间：2018-03-16 16:27

  本文选题：玉米茎腐病　切入点：ZmCCT　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：玉米茎腐病是一种普遍发生的危害严重的土传性病害,在世界各玉米产区均有发生。近几年来,我国玉米茎腐病亦呈蔓延趋势,危害逐年加剧,迫切需要培育一批抗茎腐病的新品种。深入了解玉米茎腐病的抗性遗传规律,克隆相应的抗病基因,剖析其抗病机理,可为玉米抗病育种提供理论依据。本实验室前期将玉米抗禾谷镰刀菌茎腐病主效QTL-qRfg1定位在10号染色体bin 10.04区域,两侧分子标记为SNP551和CCT11,物理距离大约170Kb。通过BAC序列比较及功能注释,确定ZmCCT为QTL-qRfg1的候选基因。本研究在此基础上,开展以下工作:1、QTL-qRfg1的定位区段含有二个转座子(TE1和TE2)插入/缺失的变异,其中TE1位于候选基因ZmCCT启动子上游～2.4 kb位置,而TE2距ZmCCT约91kb。利用47份玉米材料分析了二个转座子插入/缺失与田间抗性表现的关联,结果表明TE1的等位变异与抗性的密切相关。2、通过转基因手段将抗病自交系1145来源的ZmCCT基因导入到感病的HiII受体中,对4个独立转基因事件的T2、T4及T6代进行田间抗性鉴定,结果显示转基因阳性植株比非转基因对照抗病率提高8.2-37.5%。对2个独立的RNAi干扰转基因事件的T2BC2代进行田间抗性鉴定,结果表明阳性转基因植株比非转基因对照抗病率降低17.35-18.51%。转基因功能互补和RNAi干扰试验证实了 ZmCCT是QTL-qRfg1的抗病基因,调控玉米禾谷镰刀菌茎腐病的抗性。3、利用高世代回交后代的重组个体培育了遗传背景为Y3 31的近等基因系材料:即Y331-ATE (缺失TE1 的Y331-ZmCCT等位基因)和Y331-qRfg1 (包含完整 1145-ZmCCT等位基因)。禾谷镰刀菌接种后,Y331-ATE和Y331-qRfg1的ZmCCT基因的表达量迅速升高,在侵染后3小时达到峰值,随后又快速下降,在侵染6小时后回复到接种前水平。而Y331中含有转座子TE1插入的ZmCCT等位基因对病原菌侵染反应迟钝,表达量略有升高。这一结果表明TE1转座子插入与否直接影响到ZmCCT向应病原菌侵染的表达模式。4、利用近等基因系材料Y331-ATE和Y331检测ZmCCT启动子DNA甲基化修饰以及对病原菌的响应模式。结果发现无TE1转座子插入时,其下游ZmCCT启动子区域的DNA甲基化程度较低,在病原菌侵染后3小时后,其DNA甲基化水平迅速升高。相比较而言,含有转座子TE1插入的ZmCCT启动子区域DNA甲基化程度变化不大。这说明转座子TE1改变了ZmCCT启动子区域DNA的甲基化修饰以及其对于病原菌侵染的响应。5、利用近等基因系Y331-ATE和Y331检测ZmCCT启动子组蛋白甲基化修饰以及对病原菌的响应模式。发现无TE1插入时,ZmCCT启动子的组蛋白呈二价甲基化修饰,富含转录激活(H3K4me3)和转录抑制(H3K9me3/H3K27me3)组蛋白修饰。当TE1插入后,ZmCCT启动子选择性的去除了转录激活组蛋白修饰H3K4me3。病原菌侵染后,无TE1插入的ZmCCT启动子转录激活的H3K4me3逐渐下降,而转录抑制的H3K9me3/H3K27me3短时间内迅速下降,随后又上升。而含有转座子TE1插入的ZmCCT启动子组蛋白的甲基化修饰几乎没有任何变化。6、利用互补转基因材料验证了ZmCCT于玉米开花期及相关性状的影响。通过检测近等基因系材料Y331-△TE和Y331叶片中ZmCCT的光周期表达模式及ZmCCT启动子DNA甲基化水平,证明了TE1插入/缺失的变异可能通过改变ZmCCT启动子DNA甲基化状态,来影响叶片中ZmCCT的光周期诱导表达模式,从而改变了玉米的开花期及相关性状。7、利用近等基因系材料Y331-ATE和Y331鉴定ZmCCT对低氮胁迫、盐胁迫的作用。Y331 -ATE在胁迫条件下比Y331展现出更好的生长势。8、利用互补转基因材料研究ZmCCT的光周期敏感性与茎腐病抗性之间的内在关联。结果表明在玉米叶片中ZmCCT具有非常强烈的光周期敏感性,而根中的ZmCCT仅仅受到病原菌侵染的诱导,并不被光周期所调控,在不同的光周期条件下表现出稳定的抗性表现。
[Abstract]:Corn stalk rot is a kind of harm the widespread occurrence of serious soil borne diseases occur in the world, the corn producing areas were. In recent years, corn stalk rot in China was also spread trend, harm increasingly urgent need to cultivate a batch of new varieties of resistance to stem rot disease. Understanding the inheritance of resistance corn stalk rot resistance gene cloning, accordingly, analysis of its resistance mechanism, which can provide the theoretical basis for maize breeding. Ourprevious corn anti Fusarium stalk rot of major QTL-qRfg1 located in chromosome 10 bin 10.04 area, on both sides of molecular markers for SNP551 and CCT11, the physical distance of about 170Kb. by BAC sequence analysis and functional annotation, ZmCCT was identified as the candidate gene of QTL-qRfg1. On this basis, carry out the following work: 1, locate QTL-qRfg1 containing two transposons (TE1 and TE2) insertion / deletion mutation, TE1 Located in the candidate gene ZmCCT promoter upstream of ~ 2.4 KB and TE2 from ZmCCT, about 91kb. using 47 maize accessions analyzed two transposon insertion / deletion and field resistance performance, the results showed that TE1 alleles and resistance is closely related to the.2, the ZmCCT gene resistant inbred line 1145 source to susceptible HiII receptor by means of transgene of 4 independent transgenic events of T2, T4 and T6 generation of field resistance identification results showed that transgenic resistance than non transgenic control rate is increased by 8.2-37.5%. for 2 independent transgenic events RNAi interference T2BC2 generation field resistance identification, the results showed that the positive transgenic plants than transgenic resistance decreased 17.35-18.51%. transgenic function complementation and RNAi interference experiments confirmed that ZmCCT is the QTL-qRfg1 gene, the regulation of corn stalk rot Fusarium graminearum The resistance of.3, cultivate the genetic background for near isogenic lines Y3 31 recombinant individual high generation backcross generations: Y331-ATE (deletion of the TE1 allele of Y331-ZmCCT (Y331-qRfg1) and contains the complete 1145-ZmCCT allele) of Fusarium graminearum. After inoculation, ZmCCT gene expression of Y331-ATE and Y331-qRfg1 increased rapidly in 3 hours after infection, reached the peak, then decreased rapidly, return to the level before inoculation in 6 hours after infection. Y331 contains slow transposon TE1 insertion of the ZmCCT allele in response to pathogen infection, the expression increased slightly. The results show that the TE1 transposon insertion directly affects ZmCCT to be the expression pattern of.4 infection of the pathogen, using near isogenic lines Y331-ATE and Y331 detection of ZmCCT promoter DNA methylation and response patterns to the pathogens. It was found that there was no TE1 transposon inserted into, Downstream of the ZmCCT promoter methylation of DNA promoter region is relatively low, at 3 hours after infection of the pathogen, the methylation level of DNA increased rapidly. Compared with the transposon TE1 insertion ZmCCT start little change in the promoter region of DNA methylation level. This shows that the transposon TE1 methylation of ZmCCT promoter changed the promoter region of DNA and.5 in response to pathogen infection, using near isogenic lines Y331-ATE and Y331 detection of ZmCCT promoter methylation and histone response patterns to pathogens. No TE1 is inserted, the ZmCCT promoter of histone methylation was two price, rich activator (H3K4me3) transcription inhibition (H3K9me3/H3K27me3) and histone modifications. When TE1 is inserted after the selective removal of ZmCCT promoter transcription activation of histone modification H3K4me3. after infection of the pathogen, TE1 insertion of the transcriptional activity of ZmCCT promoter H3K4me3 閫愭笎涓嬮檷,鑰岃浆褰曟姂鍒剁殑H3K9me3/H3K27me3鐭�鏃堕棿鍐呰繀椋�

本文编号：1620719