利用CRISPR/cas9技术研究铁皮石斛Csl4基因的功能

Study on the Function of Csl4 Gene Using CRISPR/Cas9 Technology in Dendrobium officinale

为了研究铁皮石斛类纤维素合成酶基因的功能和利用类纤维素合成基因培育低纤维素新品种提供基础,本研究通过从铁皮石斛转录组中选取茎中表达量较高的类纤维素合成酶DeCsl4基因,利用CRISPR/Cas9技术构建敲除载体,获得了Csl4基因突变株。测序结果表明,目的基因靶位点序列发现有7个碱基的缺失,其中2个碱基因缺失发生在外显子上。突变植株纤维素含量的测定结果表明,平均纤维素含量(12.95 mg/g)显著低于正常植株(32.16 mg/g),说明DeCsl4基因的突变能降低铁皮石斛茎干中纤维素的合成。本研究结果首次表明CIRISPR/Cas9技术能成功的编辑兰科植物,这为以后研究兰科植物基因功能运用提供技术支撑。

To study the function of cellulose synthetase-like gene and provide a basis for breeding new varieties with lower cellulose. In this study, cellulose synthetase-like Csl4 gene with high expression in stem was selected from the transcriptome of Dendrobium officinale as target gene to construct the knockout vector by CRISPR/Cas9.Mutant plants of Csl4 gene were obtained. Sequencing revealed that there were deletion of seven bases between two target gene sites, which of them, two bases deletion happened in extron sequence. The results of the determi nation of cellulose content in mutant plants showed that the average cellulose content(12.95 mg/g) was significantly lower than that of normal plants(32.16 mg/g). These research results provide that the Csl4 gene plays an important role in cellulose synthesis in stem of Dendrobium candidum. The results of this study show for the first time that CIRISPR/Cas9 technology can successfully edit orchid, which provides technical support for future studies on the application of gene function in it.