Reviewer
Kangquan Yin
  • Faculty, Beijing Forestry University
Research fields
  • Plant science
A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
Authors:  Kang Li, Yuhui Wang and Chuanying Fang, date: 04/05/2021, view: 4494, Q&A: 0

CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.

Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana
Authors:  Teruhiko Kuroyanagi, Maurizio Camagna and Daigo Takemoto, date: 08/05/2018, view: 4888, Q&A: 0
Plant species produce a wide variety of antimicrobial metabolites to protect themselves against potential pathogens in natural environments. Phytoalexins are low molecular weight compounds produced by plants in response to attempted attacks of pathogens. Accumulation of phytoalexins in attacked plant tissues can inhibit the growth of penetrating pathogens. Thus phytoalexins play a major role in post-invasion defense against pathogens. Major phytoalexins produced by Solanaceous plants are sesquiterpenoids such as capsidiol produced by Nicotiana and Capsicum species, and rishitin produced by Solanum species, which are synthesized in the cytosol and secreted into the intercellular space of plant tissues. We previously reported that deficiency in capsidiol secretion causes enhanced susceptibility of Nicotiana benthamiana to potato late blight pathogen, Phytophthora infestans. Here, we describe a practical protocol to measure the secreted capsidiol in N. benthamiana.
We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.