TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.).
TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.).
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DOI：
10.1186/1471-2229-11-81
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发表时间：
2011-05-12
影响因子：
5.3
通讯作者：
Ozias-Akins P
中科院分区：
文献类型：
--
作者：
Knoll JE;Ramos ML;Zeng Y;Holbrook CC;Chow M;Chen S;Maleki S;Bhattacharya A;Ozias-Akins P
花生对花生的过敏反应(Arachim subgaea L.)可能会导致严重的症状，在某些情况下可能是致命的，但由于花生衍生产品在加工食品中的普遍存在，很难避免。降低花生过敏性的一个策略是通过突变改变或消除过敏蛋白。其他种子品质性状可以通过改变生物合成酶活性来改善。利用定向诱导基因组局部损伤(TILLING)这一反向遗传学方法来鉴定影响花生种子性状的突变。在四倍体花生中发现了两个类似的主要过敏原基因Arah1拷贝，每个亚基因组中一个。由于异源四倍体花生的同源基因很难区分，采用了套式聚合酶链式反应，用未标记的引物扩增了两个基因的拷贝。随后是使用基因特异性标记引物的第二次聚合酶链式反应、异源双链形成、CEL1核酸酶酶切和标记片段的电泳法检测。以甲烷磺酸乙酯(EMS)为诱变剂，在3420名M2个体中观察到1SNP/967kb的突变频率。发现的最显著的突变是Ara h 2.02中的起始密码子中断和Ara h 1.02中的过早终止密码子。每一种突变的纯合子个体在随后的世代中都被恢复，并且证实了Ara h2.02蛋白的消除。二维凝胶分析显示有几种Arah1蛋白亚型被消除或还原。TILLING还被用来鉴定脂肪酸去饱和酶AhFAD2(也存在于两个拷贝中)的突变，该基因控制种子中油酸和亚油酸的比例。发现了一个移码突变，导致AhFAD2B蛋白截断和失活。AhFAD2A的一个突变被预测可以恢复正常失活的酶的功能。这项工作代表了朝着创造一种降低过敏性的花生品种的目标迈出的第一步。这项研究表明，花生中的耕作可以扩展到几乎任何基因，并可以用来修改其他特征，如种子的营养特性。
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.
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影响因子：
8
作者：
Li, Jinxi;Shefcheck, Kevin;Fenselau, Catherine
通讯作者：
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影响因子：
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