Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing
Elucidation of the functions of genes central to diterpene metabolism in tobacco trichomes using posttranscriptional gene silencing
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DOI：
10.1007/s00425-002-0904-4
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发表时间：
2003-02-01
期刊：
影响因子：
4.3
通讯作者：
Wagner, GJ
中科院分区：
文献类型：
--
作者：
Wang, EM;Wagner, GJ
使用不同的转录后基因沉默策略（PTGS）评估两个关键的毛状体表达基因的功能。高效RNA干扰（RNAi）揭示了负责将香叶基香叶基焦磷酸转化为CBT-ols的西柏三烯醇（CBT-ol）环化酶基因的功能，并验证了负责将CBT-ols转化为CBT-二醇的P450基因的功能。 CBT-二醇是丰富的二萜，分别占烟草 (Nicotiana tabacum, T.I.) 毛状体分泌物重量和叶子干重的约 60% 和 10%。 1068. 比较了这两个基因使用反义 (AS)、有义共抑制 (S) 和 RNAi 的相对效率和抑制水平。对于 P450 基因的部分 cDNA，抑制效率（具有高 CBT-ol/CBT-二醇的初级转化体的百分比）较低，AS 植物为 3.3%，S 植物为 0%。相比之下，使用部分基因序列的RNAi，实现了约45%的敲低效率。对于CBT-ol环化酶基因，使用AS或S方向的部分cDNA没有观察到抑制，而具有部分基因序列的RNAi产生了约64%的效率。使用AS和S方向两个基因的全长编码区进行基因沉默的效率小于或等于20%。我们的结果鉴定了 CBT-ol 环化酶基因的功能，并证明了 RNAi 在评估两个毛状体特异性基因的功能方面的功效和优越性，这两个基因编码具有广泛不同功能的酶。
The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratrieneol (CBT-ol) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-ols, and verified the function of a P450 gene responsible for conversion of CBT-ols to CBT-diols. CBT-diols are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense cosuppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-ols/CBT-diols) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-ol cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding reigons of both genes in AS and S orientations were less than or equal to20%. Our results identify the function of a CBT-ol cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.