Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic rice seeds
Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic rice seeds
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DOI：
10.1023/a:1006194326804
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发表时间：
1999-04-01
影响因子：
5.1
通讯作者：
Gautier, MF
中科院分区：
文献类型：
--
作者：
Digeon, JF;Guiderdoni, E;Gautier, MF
通过反向PCR分离出含有对应于普通小麦嘌呤吲哚啉-b cDNA的5'上游序列和部分开放阅读框的基因组DNA片段。延伸至翻译起始ATG密码子上游-1068、-388、-210或-124的启动子片段和编码嘌呤吲哚啉-b的前13个氨基酸的序列，被翻译融合至编码β-葡萄糖醛酸酶的uidA报告基因，并通过粒子轰击介导的转化转移至水稻愈伤组织。 1068 bp 和 124 bp 启动子也与 uidA 报告基因转录融合。从转化的水稻愈伤组织再生的 196 株植物中，有 118 株结籽。在整合了嘌呤吲哚啉-b启动子或其缺失的转基因水稻的茎、根、叶或花粉中未检测到GUS活性；仅在种子中检测到 GUS 活性，整合了 124 bp 启动子的种子除外。在种子内，组织学定位显示 GUS 活性仅限于胚乳、糊粉细胞和果皮细胞层；胚胎轴中未检测到 GUS 活性。对5'启动子缺失的分析确定-388和-210之间的区域是胚乳表达所必需的，-210和-124之间的区域是盾片上皮中表达所必需的。在翻译和转录融合基因之间没有观察到表达差异。
A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to -1068, -388, -210 or -124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding beta-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5' promoter deletions identified the region between -388 and -210 as essential for endosperm expression, and the region between -210 and -124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes.