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马铃薯(Solanum tuberosum)中编码核酮糖-1,5-二磷酸羧化酶/加氧酶激活酶基因的器官特异性和光诱导型启动子的分离与鉴定

Isolation and characterization of the organ-specific and light-inducible promoter of the gene encoding rubisco activase in potato (Solanum tuberosum).

作者信息

Qu D, Song Y, Li W M, Pei X W, Wang Z X, Jia S R, Zhang Y Q

机构信息

Biotechnology Research Institute, National Key Facility for Gene Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing, PR China.

出版信息

Genet Mol Res. 2011 Apr 12;10(2):621-31. doi: 10.4238/vol10-2gmr1088.

DOI:10.4238/vol10-2gmr1088
PMID:21491372
Abstract

Constitutive promoters have been widely used in crop biotechnology applications. Tissue-specific or inducible promoters, however, have advantages in some cases. We isolated the 731-bp 5' flanking sequence of a potato (Solanum tuberosum) gene, encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase (RCA), which was isolated by genome walking. By using GUS as a reporter and with Northern blot analysis, the 702-bp fragment (referred to as StRCAp), ranging from nt -731 to -30 relative to the initiation code of the RCA gene, was analyzed in transgenic tobacco plants. The activity of StRCAp in leaves was 0.4-fold less than that of cauliflower mosaic virus 35S promoter, and was expressed throughout the green part of the light-grown transgenic T(1) seedlings, including cytoledons, leaves and young stems, but not roots. Further deletion analysis revealed that a shorter fragment (nt -249 to -30, StRCAp2) retained light-inducible features in cytoledons and leaves, but showed no detectable activity in young stems and roots. Although the activity of StRCAp2 in leaves was reduced significantly compared with that of StRCAp, the overall data indicated that cis-elements sufficient to regulate organ-specific and light-inducible transcription are within the 220-bp fragment. There is potential for application of StRCAp in plant genetic engineering.

摘要

组成型启动子已广泛应用于作物生物技术领域。然而,组织特异性或诱导型启动子在某些情况下具有优势。我们通过基因组步移技术分离出了马铃薯(Solanum tuberosum)基因的731 bp 5'侧翼序列,该基因编码核酮糖-1,5-二磷酸羧化酶/加氧酶(rubisco)激活酶(RCA)。以GUS作为报告基因,并通过Northern杂交分析,对相对于RCA基因起始密码子从nt -731至-30的702 bp片段(称为StRCAp)在转基因烟草植株中进行了分析。StRCAp在叶片中的活性比花椰菜花叶病毒35S启动子低0.4倍,并且在光照培养的转基因T(1)幼苗的绿色部分,包括子叶、叶片和幼茎中均有表达,但在根中不表达。进一步的缺失分析表明,较短的片段(nt -249至-30,StRCAp2)在子叶和叶片中保留了光诱导特性,但在幼茎和根中未检测到活性。尽管StRCAp2在叶片中的活性与StRCAp相比显著降低,但总体数据表明,足以调控器官特异性和光诱导转录的顺式元件位于220 bp片段内。StRCAp在植物基因工程中具有应用潜力。

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