A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes.
A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes.
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DOI：
10.1186/1471-2229-4-6
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发表时间：
2004-04-16
影响因子：
5.3
通讯作者：
Banks JA
中科院分区：
文献类型：
--
作者：
Rutherford G;Tanurdzic M;Hasebe M;Banks JA
Ceratopteris richardii 是研究植物配子体发育和有性生殖的有用实验系统。然而，该物种几乎没有用于克隆突变基因或破坏基因功能的工具。本研究检验了系统基因沉默作为反向遗传学工具的可行性。将叶绿素生物合成所需的角蕨原卟啉 IX 镁螯合酶 (CrChlI) 基因作为靶标的几种 DNA 构建体通过生物射弹递送分别引入幼配子体中。它们在单个细胞中的瞬时表达导致无色细胞表型，影响成熟配子体的大多数细胞，包括分生组织和配子体。无色表型与内源转录物丰度下降 7 倍相关。虽然发现设计用于促进CrChlI双链、可能形成发夹的RNA瞬时表达的构建体对于系统性沉默内源基因是最有效的，但仅含有CrChlI cDNA插入片段的质粒就足以诱导沉默。受到轰击的无色雌雄配子体在自体受精后产生无色胚胎，这表明沉默信号可以通过配子发生和受精传递。用针对角蕨丝状温度敏感 (CrFtsZ) 和尿卟啉脱氢酶 (CrUrod) 基因的构建体轰击幼配子体也产生了预期的突变表型。描述了一种诱导水蕨配子体中靶基因系统沉默的方法。它提供了一种简单、廉价且快速的方法来测试参与配子体发育的基因的功能，特别是那些参与所有植物共有的细胞过程的基因的功能。
Ceratopteris richardii is a useful experimental system for studying gametophyte development and sexual reproduction in plants. However, few tools for cloning mutant genes or disrupting gene function exist for this species. The feasibility of systemic gene silencing as a reverse genetics tool was examined in this study. Several DNA constructs targeting a Ceratopteris protoporphyrin IX magnesium chelatase (CrChlI) gene that is required for chlorophyll biosynthesis were each introduced into young gametophytes by biolistic delivery. Their transient expression in individual cells resulted in a colorless cell phenotype that affected most cells of the mature gametophyte, including the meristem and gametangia. The colorless phenotype was associated with a 7-fold decrease in the abundance of the endogenous transcript. While a construct designed to promote the transient expression of a CrChlI double stranded, potentially hairpin-forming RNA was found to be the most efficient in systemically silencing the endogenous gene, a plasmid containing the CrChlI cDNA insert alone was sufficient to induce silencing. Bombarded, colorless hermaphroditic gametophytes produced colorless embryos following self-fertilization, demonstrating that the silencing signal could be transmitted through gametogenesis and fertilization. Bombardment of young gametophytes with constructs targeting the Ceratopteris filamentous temperature sensitive (CrFtsZ) and uroporphyrin dehydrogenase (CrUrod) genes also produced the expected mutant phenotypes. A method that induces the systemic silencing of target genes in the Ceratopteris gametophyte is described. It provides a simple, inexpensive and rapid means to test the functions of genes involved in gametophyte development, especially those involved in cellular processes common to all plants.