Cloning and Activity Identification of High Efficiency <i>Ubiquitin</i> Promoters from Sorghum
Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2022, Vol. 43 ›› Issue (12): 2443-2452.DOI: 10.3969/j.issn.1000-2561.2022.12.006

• Omics & Biotechnology • Previous Articles     Next Articles

Cloning and Activity Identification of High Efficiency Ubiquitin Promoters from Sorghum

XIA Qiyu1,2, HE Pingping1,2, ZHANG Lili1,2, ZHANG Yuliang1,2, XIAO Susheng1,2, ZHAO Hui1,2,3,*()   

  1. 1. Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan 572024, China
    2. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs / Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Haikou, Hainan 571101, China
    3. Hainan Institute for Tropical Agricultural Resources, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China
  • Received:2022-04-23 Revised:2022-05-18 Online:2022-12-25 Published:2023-01-12
  • Contact: ZHAO Hui

Abstract:

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.

Key words: sorghum, ubiquitin gene, promoter, clone, activity identification

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