TRANSIENT TRANSFORMATION OF ARABIDOPSIS LEAF PROTOPLASTS - A VERSATILE EXPERIMENTAL SYSTEM TO STUDY GENE-EXPRESSION
TRANSIENT TRANSFORMATION OF ARABIDOPSIS LEAF PROTOPLASTS - A VERSATILE EXPERIMENTAL SYSTEM TO STUDY GENE-EXPRESSION
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DOI：
10.1111/j.1365-313x.1994.00421.x
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发表时间：
1994-03-01
期刊：
影响因子：
7.2
通讯作者：
THEOLOGIS, A
中科院分区：
文献类型：
--
作者：
ABEL, S;THEOLOGIS, A
报道了一种改进的拟南芥叶肉原生质体分离和瞬时转化方法。在花椰菜花叶病毒（CaMV）35 S启动子的控制下，转染的叶原生质体支持编码β-葡萄糖醛酸酶（GUS）的细菌报告基因的高水平表达。GUS活性的瞬时表达进行了监测，并达到最大值聚乙二醇（PEG）介导的DNA摄取后18和48小时之间。GUS活性的组织化学染色显示，可重复的转化频率在40%和60%之间，根据存活的原生质体的数量。为了证明瞬时表达系统的适用性，在转染的叶肉原生质体中研究了用不同核多肽标记的GUS蛋白的亚细胞定位，揭示了嵌合GUS酶的核区室化。此外，拟南芥叶肉原生质体支持生长素介导的氯霉素乙酰转移酶（CAT）活性的诱导时，转染的CAT报告基因和早期生长素诱导的PS-IAA 4/5启动子之间的转录融合。因此，该方法允许在同源转化系统中对融合蛋白的启动子活性和亚细胞定位进行体内分析。
An-improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for beta-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethyleneglycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyltransferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.