Department of Horticulture/NESPAL, University of Georgia-Tifton Campus, Tifton, GA 31793, USA.
BMC Plant Biol. 2011 May 12;11:81. doi: 10.1186/1471-2229-11-81.
Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.
Two similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme.
This work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.
花生（Arachis hypogaea L.）过敏反应可导致严重症状，在某些情况下甚至可能致命，但由于花生衍生产品在加工食品中的广泛存在，避免食用非常困难。降低花生致敏性的一种策略是通过突变改变或消除致敏蛋白。通过改变生物合成酶的活性，其他种子质量性状也可以得到改善。靶向诱导基因组局部突变（TILLING）是一种反向遗传学方法，用于鉴定影响花生种子特性的突变。
在四倍体花生中，已鉴定出两个 major allergen 基因 Ara h 1 的相似拷贝，每个亚基因组一个。主要过敏原 Ara h 2 也是如此。由于在异源四倍体花生中区分同源基因具有挑战性，因此使用巢式 PCR，使用未标记的引物扩增两个基因拷贝。然后使用基因特异性标记引物进行第二次 PCR，形成异源双链体，CEL1 核酸酶消化，并检测标记片段的电泳。使用乙基甲烷磺酸（EMS）作为诱变剂，观察到 1 SNP/967 kb 的突变频率（筛选了 3420 个 M2 个体）。鉴定出的最显著突变是 Ara h 2.02 的破坏起始密码子和 Ara h 1.02 的过早终止密码子。这些突变的纯合个体在随后的几代中都有恢复，并且确认了 Ara h 2.02 蛋白的消除。根据 2D 凝胶分析，消除或减少了几种 Ara h 1 蛋白同工型。TILLING 还用于鉴定脂肪酸去饱和酶 AhFAD2（也存在两个拷贝）中的突变，该基因控制种子中油酸与亚油酸的比例。鉴定出一个移码突变，导致 AhFAD2B 蛋白的截断和失活。预测 AhFAD2A 中的一个突变将恢复正常无活性酶的功能。
这项工作代表了朝着创建致敏性降低的花生品种的目标迈出的第一步。TILLING 在花生中的应用可以扩展到几乎任何基因，并可用于修饰其他性状，如种子的营养特性，如本研究所示。
