Simple RNAi vectors for stable and transient suppression of gene function in rice
Simple RNAi vectors for stable and transient suppression of gene function in rice
复制标题
DOI：
10.1093/pcp/pch048
复制
发表时间：
2004-01-01
影响因子：
4.9
通讯作者：
Shimamoto, K
中科院分区：
文献类型：
--
作者：
Miki, D;Shimamoto, K
自从水稻基因组测序成功以来，水稻基因的功能鉴定变得越来越重要。已经产生了各种标记系;然而，可用的标记基因的数量不足以进行基因功能的广泛研究。为了帮助鉴定水稻基因的功能，我们开发了一个Gateway载体pANDA，用于水稻基因的RNA干扰。该载体可用于水稻的农杆菌转化，并允许简单和快速构建有效的RNAi载体。在该构建体中，源自给定基因的发夹RNA从强玉米泛素启动子转录，并且内含子位于反向重复序列的5'上游以增强RNA表达。使用该载体对水稻基因的分析表明，在90%以上的转基因植物中观察到mRNA表达的抑制，并且在每个沉默的植物中检测到指示RNA沉默的短干扰RNA。还开发了类似的载体pANDA-mini，用于直接转移到叶细胞或原生质体中。该载体可用于水稻基因功能的瞬时抑制。这些载体将有助于鉴定目前尚未标记突变体的水稻基因的功能，并补充现有的水稻功能基因组学方法。
Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.