Payame Noor University Crop Biotechnology 2252-0783 9 2 2020 02 20 Designing and construction of a plant expression vector containing the hygromycin antibiotic resistance marker gene Designing and construction of a plant expression vector containing the hygromycin antibiotic resistance marker gene 1 9 6522 10.30473/cb.2020.49245.1788 FA Ali Saeedpour Ph.D. Student, Faculty of Agricultural Sciences & Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran. Soodabeh Jahanbakhsh Associate Professor, Faculty of Agricultural Sciences & Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran. Tahmineh Lohrasebi Assistant Professor, Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. Kasra Esfahani Assistant Professor, Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. 0000-0003-3079-7009 Ali Hatef Salmanian Professor, Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Khadijeh Razavi Assistant Professor, Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. Journal Article 2019 10 23 Conventional pBI121-based binary vectors are widely used in transformation of higher plants mediated by Agrobacterium but they are useless in transformation of some monocots because of inefficiency of kanamycin in selection, while, hygromycin resistance gene is an important selectable marker that usually used in transformation of several plants, especially monocots. The aim of this study was to improve the pBI121 vector for transformation of monocot plants. For this purpose, the hygromycin resistance gene with the 35S terminator were isolated from p6-ubi-rnai plasmid and cloned into pBlueScript intermediate vector via SmaІ and NotI restriction enzyme digestion. The CaMV 35 promoter was isolated from pBI121 vector by using SmaI and HindIII enzymes and cloned upstream of the gene. By using HindIII and Eco53KI enzymes, the complete hygromycin resistance gene cassette was replaced the kanamycin resistance gene cassette (which digested by HindIII and MssI) of pBI121 vector. Construction of this vector was confirmed by PCR method, digestion pattern analysis, and sequencing. Due to the popularity of pBI121-based vectors than other binary vectors and the researchers' familiarity with their manipulation, the vector which is introduced in this study could be used in gene transfer studies of monocot plants. Conventional pBI121-based binary vectors are widely used in transformation of higher plants mediated by Agrobacterium but they are useless in transformation of some monocots because of inefficiency of kanamycin in selection, while, hygromycin resistance gene is an important selectable marker that usually used in transformation of several plants, especially monocots. The aim of this study was to improve the pBI121 vector for transformation of monocot plants. For this purpose, the hygromycin resistance gene with the 35S terminator were isolated from p6-ubi-rnai plasmid and cloned into pBlueScript intermediate vector via SmaІ and NotI restriction enzyme digestion. The CaMV 35 promoter was isolated from pBI121 vector by using SmaI and HindIII enzymes and cloned upstream of the gene. By using HindIII and Eco53KI enzymes, the complete hygromycin resistance gene cassette was replaced the kanamycin resistance gene cassette (which digested by HindIII and MssI) of pBI121 vector. Construction of this vector was confirmed by PCR method, digestion pattern analysis, and sequencing. Due to the popularity of pBI121-based vectors than other binary vectors and the researchers' familiarity with their manipulation, the vector which is introduced in this study could be used in gene transfer studies of monocot plants. https://cropbiotech.journals.pnu.ac.ir/article_6522_d91166b732b637ded44764773bd0091d.pdf