An Optimized Protocol for Detecting Guard Cell–specific Gene Expression by in situ RT-PCR in Brassica rapa
Yingying Song,
Xinlei Guo,
Jian Wu,
Jianli Liang,
Runmao Lin,
Zifu Yan,
Xiaowu Wang
Affiliations
Yingying Song
College of Horticulture, Henan Agricultural University, Beijing, ChinaInstitute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Xinlei Guo
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Jian Wu
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Jianli Liang
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Runmao Lin
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Zifu Yan
College of Horticulture, Henan Agricultural University, Zhengzhou, China
Xiaowu Wang
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Since the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell–specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.
