REPRESSION OF ACETOLACTATE SYNTHASE ACTIVITY THROUGH ANTISENSE INHIBITION - MOLECULAR-ANALYSIS AND BIOCHEMICAL-ANALYSIS OF TRANSGENIC POTATO (SOLANUM-TUBEROSUM L CV DESIREE) PLANTS
REPRESSION OF ACETOLACTATE SYNTHASE ACTIVITY THROUGH ANTISENSE INHIBITION - MOLECULAR-ANALYSIS AND BIOCHEMICAL-ANALYSIS OF TRANSGENIC POTATO (SOLANUM-TUBEROSUM L CV DESIREE) PLANTS
复制标题
DOI：
10.1104/pp.107.2.469
复制
发表时间：
1995-02-01
期刊：
影响因子：
7.4
通讯作者：
POHLENZ, HD
中科院分区：
文献类型：
--
作者：
HOFGEN, R;LABER, B;POHLENZ, HD
乙酰乳酸合成酶（ALS）是亮氨酸、缬氨酸和异亮氨酸生物合成途径中的第一个酶，是各种除草剂的生化靶标。为了研究反义基因表达对ALS活性的抑制作用，我们从马铃薯中克隆了ALS基因。cv Desiree），构建了花椰菜花叶病毒35 S启动子控制下的嵌合反义基因，并通过根癌农杆菌介导的基因转移获得了转基因马铃薯植株。两个再生显示严重的生长迟缓和强烈的表型效应类似ALS抑制除草剂引起的。反义基因表达降低了这些植物中ALS mRNA的稳态水平，并诱导ALS活性相应降低高达85%。这种减少足以产生在没有氨基酸补充的情况下几乎不能存活的植物。在ALS反义和除草剂处理的植物中，我们可以排除2-氧代丁酸和/或2-氨基丁酸的积累作为所观察到的有害作用的原因，但是我们检测到游离氨基酸水平升高和它们的相对比例失衡。因此，ALS的反义抑制产生除草剂作用的体内模型。此外，针对感兴趣的酶的反义RNA的表达提供了用于验证潜在除草剂靶标的通用方法。
Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L. cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions, Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.