本文選題：玉米莖腐病　切入點(diǎn)：ZmCCT　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：玉米莖腐病是一種普遍發(fā)生的危害嚴(yán)重的土傳性病害,在世界各玉米產(chǎn)區(qū)均有發(fā)生。近幾年來,我國玉米莖腐病亦呈蔓延趨勢,危害逐年加劇,迫切需要培育一批抗莖腐病的新品種。深入了解玉米莖腐病的抗性遺傳規(guī)律,克隆相應(yīng)的抗病基因,剖析其抗病機(jī)理,可為玉米抗病育種提供理論依據(jù)。本實(shí)驗(yàn)室前期將玉米抗禾谷鐮刀菌莖腐病主效QTL-qRfg1定位在10號染色體bin 10.04區(qū)域,兩側(cè)分子標(biāo)記為SNP551和CCT11,物理距離大約170Kb。通過BAC序列比較及功能注釋,確定ZmCCT為QTL-qRfg1的候選基因。本研究在此基礎(chǔ)上,開展以下工作:1、QTL-qRfg1的定位區(qū)段含有二個轉(zhuǎn)座子(TE1和TE2)插入/缺失的變異,其中TE1位于候選基因ZmCCT啟動子上游～2.4 kb位置,而TE2距ZmCCT約91kb。利用47份玉米材料分析了二個轉(zhuǎn)座子插入/缺失與田間抗性表現(xiàn)的關(guān)聯(lián),結(jié)果表明TE1的等位變異與抗性的密切相關(guān)。2、通過轉(zhuǎn)基因手段將抗病自交系1145來源的ZmCCT基因?qū)氲礁胁〉腍iII受體中,對4個獨(dú)立轉(zhuǎn)基因事件的T2、T4及T6代進(jìn)行田間抗性鑒定,結(jié)果顯示轉(zhuǎn)基因陽性植株比非轉(zhuǎn)基因?qū)φ湛共÷侍岣?.2-37.5%。對2個獨(dú)立的RNAi干擾轉(zhuǎn)基因事件的T2BC2代進(jìn)行田間抗性鑒定,結(jié)果表明陽性轉(zhuǎn)基因植株比非轉(zhuǎn)基因?qū)φ湛共÷式档?7.35-18.51%。轉(zhuǎn)基因功能互補(bǔ)和RNAi干擾試驗(yàn)證實(shí)了 ZmCCT是QTL-qRfg1的抗病基因,調(diào)控玉米禾谷鐮刀菌莖腐病的抗性。3、利用高世代回交后代的重組個體培育了遺傳背景為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)。禾谷鐮刀菌接種后,Y331-ATE和Y331-qRfg1的ZmCCT基因的表達(dá)量迅速升高,在侵染后3小時(shí)達(dá)到峰值,隨后又快速下降,在侵染6小時(shí)后回復(fù)到接種前水平。而Y331中含有轉(zhuǎn)座子TE1插入的ZmCCT等位基因?qū)Σ≡秩痉磻?yīng)遲鈍,表達(dá)量略有升高。這一結(jié)果表明TE1轉(zhuǎn)座子插入與否直接影響到ZmCCT向應(yīng)病原菌侵染的表達(dá)模式。4、利用近等基因系材料Y331-ATE和Y331檢測ZmCCT啟動子DNA甲基化修飾以及對病原菌的響應(yīng)模式。結(jié)果發(fā)現(xiàn)無TE1轉(zhuǎn)座子插入時(shí),其下游ZmCCT啟動子區(qū)域的DNA甲基化程度較低,在病原菌侵染后3小時(shí)后,其DNA甲基化水平迅速升高。相比較而言,含有轉(zhuǎn)座子TE1插入的ZmCCT啟動子區(qū)域DNA甲基化程度變化不大。這說明轉(zhuǎn)座子TE1改變了ZmCCT啟動子區(qū)域DNA的甲基化修飾以及其對于病原菌侵染的響應(yīng)。5、利用近等基因系Y331-ATE和Y331檢測ZmCCT啟動子組蛋白甲基化修飾以及對病原菌的響應(yīng)模式。發(fā)現(xiàn)無TE1插入時(shí),ZmCCT啟動子的組蛋白呈二價(jià)甲基化修飾,富含轉(zhuǎn)錄激活(H3K4me3)和轉(zhuǎn)錄抑制(H3K9me3/H3K27me3)組蛋白修飾。當(dāng)TE1插入后,ZmCCT啟動子選擇性的去除了轉(zhuǎn)錄激活組蛋白修飾H3K4me3。病原菌侵染后,無TE1插入的ZmCCT啟動子轉(zhuǎn)錄激活的H3K4me3逐漸下降,而轉(zhuǎn)錄抑制的H3K9me3/H3K27me3短時(shí)間內(nèi)迅速下降,隨后又上升。而含有轉(zhuǎn)座子TE1插入的ZmCCT啟動子組蛋白的甲基化修飾幾乎沒有任何變化。6、利用互補(bǔ)轉(zhuǎn)基因材料驗(yàn)證了ZmCCT于玉米開花期及相關(guān)性狀的影響。通過檢測近等基因系材料Y331-△TE和Y331葉片中ZmCCT的光周期表達(dá)模式及ZmCCT啟動子DNA甲基化水平,證明了TE1插入/缺失的變異可能通過改變ZmCCT啟動子DNA甲基化狀態(tài),來影響葉片中ZmCCT的光周期誘導(dǎo)表達(dá)模式,從而改變了玉米的開花期及相關(guān)性狀。7、利用近等基因系材料Y331-ATE和Y331鑒定ZmCCT對低氮脅迫、鹽脅迫的作用。Y331 -ATE在脅迫條件下比Y331展現(xiàn)出更好的生長勢。8、利用互補(bǔ)轉(zhuǎn)基因材料研究ZmCCT的光周期敏感性與莖腐病抗性之間的內(nèi)在關(guān)聯(lián)。結(jié)果表明在玉米葉片中ZmCCT具有非常強(qiáng)烈的光周期敏感性,而根中的ZmCCT僅僅受到病原菌侵染的誘導(dǎo),并不被光周期所調(diào)控,在不同的光周期條件下表現(xiàn)出穩(wěn)定的抗性表現(xiàn)。
[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,鑰岃漿褰曟姂鍒剁殑H3K9me3/H3K27me3鐭椂闂村唴榪呴，

本文編號：1620719