高粱高效Ubiquitin启动子的克隆及活性鉴定

Cloning and Activity Identification of High Efficiency Ubiquitin Promoters from Sorghum

在转基因植物的研究中,启动子是影响转基因表达效率的重要因素之一。植物泛素(ubiquitin)基因启动子以启动效率高、甲基化程度相对较低、遗传性状稳定等特点成为单子叶植物中应用较为广泛的启动子。其中,玉米的Ubi-1是最常使用的Ubiquitin启动子之一。本研究旨在克隆高粱高效Ubiquitin启动子,为高粱及其他单子叶植物的遗传转化研究提供新的组成型启动子选择。本研究从NCBI数据库中查找到多个polyubiquitin基因,下载其起始密码子上游3000 bp的序列。根据Ubiquitin启动子的特点,选择含有5′UTR内含子且大小合适的序列,并通过在线启动子预测网站进行启动子分析,最后选择2个基因(LOC8076096、LOC8063786)进行启动子克隆,将其启动子序列分别命名为U1和U5。将这2个启动子片段PCR扩增后,连入含有GUS报告基因的植物表达载体Ubi-GUS,得到重组表达载体U1-GUS和U5-GUS。重组载体通过农杆菌介导法转化水稻和狗尾草,PCR筛选阳性转化苗,对阳性转化苗进行GUS染色分析,鉴定U1和U5的启动子活性。GUS染色观察发现,所有以U1和U5为启动子的水稻和狗尾草阳性转化苗的根、茎和叶均呈现较深的蓝色,比以玉米Ubi-1为启动子的阳性转化苗的蓝色略深,且以U5为启动子的阳性转化苗比以U1为启动子的蓝色更深,而非转基因的水稻和狗尾草小苗则完全染不上蓝色。因此,启动子U1和U5在水稻和狗尾草中均具有组成型强启动子的活性,可作为新的组成型启动子应用于高粱、水稻、狗尾草及其他单子叶植物的遗传转化研究。

In the research of transgenic plants, promoter is one of the important factors affecting the efficiency of transgenic expression. Plant ubiquitin gene promoters have been widely used in monocotyledons because of its high starting efficiency, relatively low methylation degree and stable genetic traits. Among them, the Ubi-1 promoter of maize is one of the most commonly used Ubiquitin promoters. The purpose of this study is to obtain high-efficiency ubiquitin promoters of sorghum and provide a new constitutive promoter selection for the genetic transformation of sorghum and other monocotyledonous plants. In this study, multiple polyubiquitin genes were found from NCBI database, and the sequence of 3000 bp upstream of its starting codon was downloaded. According to the characteristics of Ubiquitin promoter, the sequence with 5′ UTR intron and appropriate size was selected, and the promoter was analyzed through the online promoter prediction website. Finally, two genes LOC8076096 and LOC8063786 were selected for promoter cloning, and their promoter sequences were named U1 and U5 respectively. After PCR amplification, the two promoter fragments were connected to the plant expression vector Ubi-GUS containing Gus reporter gene to obtain recombinant expression vectors U1-GUS and U5-GUS. The recombinant vector was transformed into rice and green bristlegrass by the Agrobacterium-mediated method. The positive transformed seedlings were screened by PCR. GUS staining analysis was carried out on the positive transformed seedlings to identify the promoter activities of U1 and U5. GUS staining showed that the roots, stems and leaves of all positive transformed seedlings of rice and green bristlegrass with U1 and U5 as promoters showed darker blue, which was slightly darker than that of positive transformed seedlings with maize Ubi-1 as promoter, and the blue of positive transformed seedlings with U5 as promoter was deeper than that of U1 as promoter, while non-transgenic rice and green bristlegrass seedlings could not be stained with blue at all. Therefore, the promoters U1 and U5 have the activity of constitutive strong promoters in rice and green bristlegrass, and can be used as new constitutive promoters to study the genetic transformation of sorghum, rice, green bristlegrass and other monocotyledonous plants.