拟南芥RPW8.1介导的细胞死亡增强突变体的筛选和突变相关基因的图位克隆
发布时间：2018-11-15 12:36
【摘要】：拟南芥广谱抗病基因RPW8 (RESISTANCE TO POWDERY MILDEW LOCUS 8, RPW8)包含两个紧密连锁的基因：RPW8.1和RPW8.2,其中RPW8.2受白粉菌侵染诱导表达,特异性锚定到白粉菌的吸器外质膜(Extra-Haustorial Membrane, EHM)上从而启动广谱抗病性。RPW8.1与RPW8.2的氨基酸序列具有一定程度的相似性,并且同样对多种白粉病具有广谱抗性。然而,RPW8.1蛋白在细胞中的定位及表达方式与RPW82存在较大程度的不同,针对RPW8.2的研究得出的结论并不适用于RPW8.1,这就引起了对RPW8.1抗病机理的关注和讨论,从而产生了这样一个问题：定位于叶绿体周围的RPW8.1如何对寄生在表皮细胞中的白粉病菌产生抗性?为了回答这一问题,我们用一个对白粉病菌有抗性的、稳定表达RPW8.1-YFP的转基因系R1Y4作为基础材料,以0.5%的甲基磺酸乙酯(EMS)进行诱变处理。从M1代植株中筛选获得100余份在叶形、叶色以及叶片细胞死亡表型上和R1Y4存在差异的突变体。经观察比较发现,b6-3、b10-6和b9-1三个突变体呈现出叶片皱缩形态逐渐加重的趋势,b6-3最轻,b10-6次之,b9-1叶片皱缩性状最为严重,并伴有细胞死亡。此外,三个突变体材料都没有表达出R1Y4所特有的“凹坑”表型。同一时期种植的突变体和R1Y4相比,突变体植株抽薹时间提前且花轴与茎秆的比例明显大于R1Y4。选取突变性状最明显的b9-1作为代表性突变体材料进行图位克隆实验。将b9-1与拟南芥Landsberg生态型杂交,构建F2分离群体。统计F2代分离比例,结果显示b9-1突变性状受单隐性基因控制。以F2群体中分离的20株突变表型单株作为图位克隆初定位群体,在拟南芥每条染色体上选4个SSLP标记,将b9-1突变相关基因定位在2号染色体的SSLP标记T8018的外端、与F3G5共分离。遂根据Col-gl与Ler在F3G5左右的多态性设计了T1B8、T20F21、F10I1、T1J8、T2N18、 F16M14-1、F16M14-2、T6A23、T7F6、F12L6、T28M21、F27I1、T3K9、F4I1和F11C10等15个SSLP标记,除了T20F21、T28M21、T3K9存在多态性较差及扩增无条带问题外,其余分子标记在突变体与Ler间均具有多态性；同时,从F2群体中继续筛选获得具有突变体表型的单株共133株,将b9-1突变相关基因定位在T2N18和F16M14之间(物理距离0.4 Mb)。根据这两个标记之间的已报道基因的情况,结合突变体表型,选择两个基因进行测序分析,发现在b9-1突变体的At2g37630(编码ASYMMETRIC LEAVES1, AS1)位点有一个碱基的替换,导致At2g37630编码的AS1基因在b9-1突变体中存在一个G到A的单核苷酸突变,形成一个终止码。另外两个突变体与b9-1属于同一基因位点不同碱基的突变,在b10-6中同样形成-个终止码,而在b6-3中引起了一个氨基酸的置换。蛋白分析表明三个突变体的突变位点位于AS1基因的不同区域,表型存在渐变差别的原因可能是因为三个突变区域对AS1功能的影响程度不同。并且横向比较发现突变的区域在不同植物中均较为保守。之前的报道显示,AS1基因能编码MYB区域的转录因子,抑制KNOX基因在叶原基部位的表达,而KNOX基因在控制顶端分生组织形成过程中扮演重要的角色,这就不难推断AS1在拟南芥叶形发育中的重要作用了。下一步将继续进行AS1基因相关功能研究,探索其与RPW8.1的关系。
[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，
本文编号：2333318
[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，
本文编号：2333318
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