AN ARABIDOPSIS MUTANT DEFICIENT IN STEROL BIOSYNTHESIS - HETEROLOGOUS COMPLEMENTATION BY ERG-3 ENCODING A DELTA(7)-STEROL-C-5-DESATURASE FROM YEAST
AN ARABIDOPSIS MUTANT DEFICIENT IN STEROL BIOSYNTHESIS - HETEROLOGOUS COMPLEMENTATION BY ERG-3 ENCODING A DELTA(7)-STEROL-C-5-DESATURASE FROM YEAST
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DOI：
10.1046/j.1365-313x.1995.08030407.x
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发表时间：
1995-09-01
期刊：
影响因子：
7.2
通讯作者：
BENVENISTE, P
中科院分区：
文献类型：
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作者：
GACHOTTE, D;MEENS, R;BENVENISTE, P
通过对拟南芥的22 000株M(2)植株，分成20株1100株植物的小叶样品进行气相色谱分析，筛选出突变株STE 1。STE 1的特征是在野生型叶片中积累3个Delta(7)-甾醇，同时相应的3个Delta(5)-甾醇减少。用气相色谱-质谱仪(GC-MS)和质子核磁共振波谱(H-1-核磁共振)对化合物进行了结构鉴定。Delta(7)-甾醇的积累表明该突变体缺乏Delta(7)-甾醇-C-5-去饱和酶的活性。遗传分析表明，Delta(7)-甾醇的积累是由单个隐性核突变引起的。突变系STE-1与野生型回交4次。STE-1植株形态呈野生型，结实正常，表明STE-1中的Delta(7)-甾醇是维持正常发育所需的具有生理活性的Delta(5)-甾醇的良好替代品。与未转化的STE-1根愈伤组织相比，7个转基因STE-1根愈伤组织的Delta(5)-甾醇含量增加，Delta(7)-甾醇含量降低。用ERG3探针进行Northern杂交分析表明，在7个转基因愈伤组织中有3个表达了ERG3。这些结果表明，STE 1突变体中Delta(7)-甾醇的积累是由于植物甾醇生物合成途径中Delta(7)-甾醇-C-5-去饱和步骤的缺失。
The mutant STE 1 was isolated by screening an ethyl-methane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22 000 M(2) plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples.STE 1 was characterized by the accumulation of three Delta(7)-sterols concomitantly with the decrease of the three corresponding Delta(5)-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these Delta(7)-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (H-1-NMR). The accumulation of Delta(7)-sterols suggested that the mutant is deficient in the activity of the Delta(7)-sterol-C-5-desaturase.Genetic analysis showed that the accumulation of Delta(7)-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the Delta(7)-sterols in STE 1 are good surrogates of physiologically active Delta(5)-sterols to sustain normal development.STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Delta(7)-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in Delta(5)-sterols and a concomitant decrease in Delta(7)-sterols in comparison with STE 1 untransformed root derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Delta(7)-sterols in the STE 1 mutant is due to a deficiency of the Delta(7)-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.