Gachotte D, Meens R, Benveniste P
Département d'Enzymologie Moléculaire et Cellulaire, UPR 406 du CNRS, Strasbourg, France.
Plant J. 1995 Sep;8(3):407-16. doi: 10.1046/j.1365-313x.1995.08030407.x.
The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22,000 M2 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 (1H-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.
通过筛选经甲基磺酸乙酯（EMS）诱变的拟南芥群体分离出突变体STE 1，该群体由22000株M2植株组成，通过对从小叶片样品中提取的甾醇进行气相色谱分析，将其分为1100个池，每个池20株植物。STE 1的特征是三种δ7-甾醇积累，同时三种相应的δ5-甾醇减少，而δ5-甾醇是野生型叶片中甾醇途径的终产物。这些δ7-甾醇的结构在经过两步高效液相色谱（HPLC）纯化后，通过气相色谱-质谱联用（GC-MS）和质子核磁共振光谱（1H-NMR）确定。δ7-甾醇的积累表明该突变体在δ7-甾醇-C-5-去饱和酶活性方面存在缺陷。遗传分析表明，δ7-甾醇的积累是由于单个隐性核突变所致。突变体品系STE 1与野生型回交了四次。所得的STE 1植株具有野生型形态且正常结籽，这表明STE 1中的δ7-甾醇是生理活性δ5-甾醇的良好替代物，以维持正常发育。在花椰菜花叶病毒（CaMV）35S启动子的控制下，用编码δ7-甾醇-C-5-去饱和酶的酿酒酵母ERG 3基因转化STE 1根。与未转化的STE 1根来源的愈伤组织相比，七个转基因STE 1根来源的愈伤组织中δ5-甾醇增加，同时δ7-甾醇减少。使用ERG 3探针的Northern印迹分析表明，七个转基因愈伤组织中的三个中ERG 3有强烈表达。这些结果表明，STE 1突变体中δ7-甾醇的积累是由于植物甾醇生物合成途径中δ7-甾醇-C-5-去饱和步骤的缺陷所致。
