INTRONS INCREASE GENE-EXPRESSION IN CULTURED MAIZE CELLS
INTRONS INCREASE GENE-EXPRESSION IN CULTURED MAIZE CELLS
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DOI：
10.1101/gad.1.10.1183
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发表时间：
1987-12-01
影响因子：
10.5
通讯作者：
WALBOT, V
中科院分区：
文献类型：
--
作者：
CALLIS, J;FROMM, M;WALBOT, V
利用电穿孔介导的基因转移技术，将编码玉米慢（S）迁移多肽的基因导入到玉米（Zea mays L.）已经将乙醇脱氢酶-1（Adh 1）酶稳定地和瞬时地引入含有内源性Fast（F）ADH 1电型物的玉米细胞中。在稳定的转化体中，相对于内源性F等位基因，11.5 kb的片段足以编程正常的S表达。在瞬时测定中，缺乏9个Adh 1-S间插序列（内含子）的Adh 1-S基因构建体的表达水平比完整基因低50至100倍;单独存在内含子1将基因表达水平恢复到完整基因的水平。最后两个内含子也刺激Adh 1-S表达，但水平是完整基因的三倍。通过添加含有Adh 1内含子1的序列，利用Adh 1基因的5'启动子和3'聚腺苷酸化区域的嵌合氯霉素乙酰转移酶（CAT）基因的表达增加了100倍。Adh 1内含子1序列位于转录区域外时，不刺激CAT表达。当位于转录区域内时，Adh 1内含子1区域仅当位于启动子和CAT编码区之间时才有效地刺激CAT表达。含有Adh 1内含子1片段的构建体产生的mRNA比含有等效cDNA片段的构建体多40倍。Adh 1内含子1和第二个玉米基因Bronze 1的内含子都刺激了其他启动子（花椰菜花叶病毒35 S和胭脂碱合成酶）和其他编码区（荧光素酶和新霉素磷酸转移酶II）的表达。这些结果表明，内含子增加了Adh 1和嵌合基因在玉米中的表达，并且这种内含子的最佳位置靠近mRNA的5'端。
Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adh1) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an 11.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adh1-S gene constructs lacking the 9 Adh1-S intervening sequences (introns) were expressed at levels 50- to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adh1-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adh1 gene was increased 100-fold by the addition of sequences containing the Adh1 intron 1. The Adh1 intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adh1 intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adh1 intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adh1 intron 1 and the intron from a second maize gene, Bronze1, stimulated expression from other promoters (cauliflower mosaic virus 35S and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adh1 and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.