【摘要】：擬南芥廣譜抗病基因RPW8 (RESISTANCE TO POWDERY MILDEW LOCUS 8, RPW8)包含兩個緊密連鎖的基因：RPW8.1和RPW8.2,其中RPW8.2受白粉菌侵染誘導(dǎo)表達,特異性錨定到白粉菌的吸器外質(zhì)膜(Extra-Haustorial Membrane, EHM)上從而啟動廣譜抗病性。RPW8.1與RPW8.2的氨基酸序列具有一定程度的相似性,并且同樣對多種白粉病具有廣譜抗性。然而,RPW8.1蛋白在細胞中的定位及表達方式與RPW82存在較大程度的不同,針對RPW8.2的研究得出的結(jié)論并不適用于RPW8.1,這就引起了對RPW8.1抗病機理的關(guān)注和討論,從而產(chǎn)生了這樣一個問題：定位于葉綠體周圍的RPW8.1如何對寄生在表皮細胞中的白粉病菌產(chǎn)生抗性?為了回答這一問題,我們用一個對白粉病菌有抗性的、穩(wěn)定表達RPW8.1-YFP的轉(zhuǎn)基因系R1Y4作為基礎(chǔ)材料,以0.5%的甲基磺酸乙酯(EMS)進行誘變處理。從M1代植株中篩選獲得100余份在葉形、葉色以及葉片細胞死亡表型上和R1Y4存在差異的突變體。經(jīng)觀察比較發(fā)現(xiàn),b6-3、b10-6和b9-1三個突變體呈現(xiàn)出葉片皺縮形態(tài)逐漸加重的趨勢,b6-3最輕,b10-6次之,b9-1葉片皺縮性狀最為嚴重,并伴有細胞死亡。此外,三個突變體材料都沒有表達出R1Y4所特有的“凹坑”表型。同一時期種植的突變體和R1Y4相比,突變體植株抽薹時間提前且花軸與莖稈的比例明顯大于R1Y4。選取突變性狀最明顯的b9-1作為代表性突變體材料進行圖位克隆實驗。將b9-1與擬南芥Landsberg生態(tài)型雜交,構(gòu)建F2分離群體。統(tǒng)計F2代分離比例,結(jié)果顯示b9-1突變性狀受單隱性基因控制。以F2群體中分離的20株突變表型單株作為圖位克隆初定位群體,在擬南芥每條染色體上選4個SSLP標記,將b9-1突變相關(guān)基因定位在2號染色體的SSLP標記T8018的外端、與F3G5共分離。遂根據(jù)Col-gl與Ler在F3G5左右的多態(tài)性設(shè)計了T1B8、T20F21、F10I1、T1J8、T2N18、 F16M14-1、F16M14-2、T6A23、T7F6、F12L6、T28M21、F27I1、T3K9、F4I1和F11C10等15個SSLP標記,除了T20F21、T28M21、T3K9存在多態(tài)性較差及擴增無條帶問題外,其余分子標記在突變體與Ler間均具有多態(tài)性；同時,從F2群體中繼續(xù)篩選獲得具有突變體表型的單株共133株,將b9-1突變相關(guān)基因定位在T2N18和F16M14之間(物理距離0.4 Mb)。根據(jù)這兩個標記之間的已報道基因的情況,結(jié)合突變體表型,選擇兩個基因進行測序分析,發(fā)現(xiàn)在b9-1突變體的At2g37630(編碼ASYMMETRIC LEAVES1, AS1)位點有一個堿基的替換,導(dǎo)致At2g37630編碼的AS1基因在b9-1突變體中存在一個G到A的單核苷酸突變,形成一個終止碼。另外兩個突變體與b9-1屬于同一基因位點不同堿基的突變,在b10-6中同樣形成-個終止碼,而在b6-3中引起了一個氨基酸的置換。蛋白分析表明三個突變體的突變位點位于AS1基因的不同區(qū)域,表型存在漸變差別的原因可能是因為三個突變區(qū)域?qū)S1功能的影響程度不同。并且橫向比較發(fā)現(xiàn)突變的區(qū)域在不同植物中均較為保守。之前的報道顯示,AS1基因能編碼MYB區(qū)域的轉(zhuǎn)錄因子,抑制KNOX基因在葉原基部位的表達,而KNOX基因在控制頂端分生組織形成過程中扮演重要的角色,這就不難推斷AS1在擬南芥葉形發(fā)育中的重要作用了。下一步將繼續(xù)進行AS1基因相關(guān)功能研究,探索其與RPW8.1的關(guān)系。
[Abstract]:The Arabidopsis thaliana broad-spectrum anti-disease gene RPW8 (RESISTANCE TO-DERY MILDEW LOCUS 8, RPW8) contains two closely linked genes: RPW8.1 and RPW8.2, wherein the RPW8.2 is induced by the infection of the powdery mildew, and is specifically anchored to the external membrane of the suction device of the powdery mildew (Extra-Hausory Member, EHM) so as to activate the broad-spectrum disease resistance. The amino acid sequence of RPW8.1 and RPW8.2 has a certain degree of similarity, and also has broad-spectrum resistance to various powdery mildew. however, that position and expression of the RPW8. 1 protein in the cell are different from that of the RPW82, and the conclusion for the RPW8. 2 study is not applicable to the RPW8.1, which results in a concern and discussion of the resistance mechanism of the RPW8. 1, thus creating a problem: How can RPW8.1 located around the chloroplast resistance to powdery mildew in the epidermal cells? In order to answer this problem, we used a transgenic line R1Y4, which is resistant to powdery mildew, to stably express the RPW8.1-YFP as a base material, and was subjected to a mutagenesis treatment with 0. 5% of ethyl sulfonate (EMS). A mutant with a difference in leaf shape, leaf color and leaf cell death phenotype and R1Y4 was selected from the M1 generation plant. It was found that the three mutants of b6-3, b10-6 and b9-1 showed a trend of gradual increase of the shape of the leaves, b6-3 was the most light, b10-6 and b9-1 were the most severe, and the b9-1 was the most severe and accompanied by cell death. In addition, none of the three mutant materials expressed the 鈥減it鈥，

本文編號：2333318